SELECTIVE  REGULATION  OF  BMP4   SIGNALING  BY  THE  RECEPTOR   TYROSINE  KINASE  MuSK                 ATILGAN  YILMAZ   B.S.  MOLECULAR  BIOLOGY  AND  GENETICS,  BOĞAZİÇİ  UNIVERSITY,  2005                       A  DISSERTATION  SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS   FOR  THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY  IN  THE  DIVISION  OF  BIOLOGY  AND   MEDICINE  AT  BROWN  UNIVERSITY                   PROVIDENCE,  RHODE  ISLAND   May  2013       i                                           Copyright©  2012  by  Atılgan  Yılmaz                                                   ii     This  dissertation  by  Atılgan  Yılmaz  is  accepted  in  its  present  form  by  the  Division  of   Biology  and  Medicine,  Department  of  Molecular  Biology,  Cell  Biology  and   Biochemistry  as  satisfying  the  dissertation  requirement  for  the  degree  of  Doctor  of   Philosophy.       Date_____________                                                                          __________________________________             Dr.  Justin  R.  Fallon,  Advisor       Recommended  to  the  Graduate  Council       Date_____________                                                                          __________________________________             Dr.  Kristi  Wharton,  Reader  (Chairman)         Date_____________                                                                          __________________________________             Dr.  Gilad  Barnea,  Reader         Date_____________                                                                          __________________________________             Dr.  Mark  Zervas,  Reader           Date_____________                                                                          __________________________________             Dr.  Steven  Burden,  Outside  Reader                                                                                                        Approved  by  the  Graduate  Council       Date_____________                                                                          __________________________________             Dr.  Peter  Weber,   Dean  of  the  Graduate  School           iii   ATILGAN  YILMAZ                                                                                            DOB:                                                          November  21,  1982                                                                                  Mailing  Address:    Department  of  Neuroscience,                                                                                                                                                            Brown  University,  Box  G-­‐LN                                                                                                                                                          185  Meeting  Street,  Providence,  RI  02912                                                                                  Phone:                                            401  919  7262                                                                                  E-­mail:                                            atilgan_yilmaz@brown.edu           EDUCATION     Ph.D.  in  Molecular  Biology,  Cellular  Biology  and  Biochemistry              (expected)  Summer  2012   Brown  University,  Providence,  RI   Thesis:  “Selective  regulation  of  BMP4  signaling  by  the  receptor  tyrosine  kinase  MuSK”       B.S.  in  Molecular  Biology  and  Genetics                                                                                                                                                              June  2005   Bogazici  University,  Istanbul,  Turkey,  with  Honors                                                                                                                                   RESEARCH  EXPERIENCE     04/06  –       Graduate  Student,  Brown  University,  Providence,  USA   Selective  regulation  of  BMP4  signaling  by  the  receptor  tyrosine  kinase  MuSK     Advisor:  Prof.  Justin  Fallon,  Department  of  Neuroscience     06/04  –  09/04     Undergraduate  Summer  Research  Trainee.  UCLA,  Los  Angeles,  USA   Mechanism  of  cofilin-­‐mediated  actin  depolymerization.     Advisor:  Prof.  Emil  Reisler,  Department  of  Chemistry  &  Biochemistry     07/03  –  10/03       Undergraduate   Summer   Research   Trainee,   Georg   August   University,   Göttingen,   Germany   Molecular  mechanisms  of  endocytosis  and  synaptic  vesicle  formation  in  neurons.     (Supported  by  the  Science  and  Culture  Ministry  of  Niedersachsen)   Advisor:   Prof.   Volker   Haucke,   Biochemistry   Institute   (relocated   to   Freie   Universität   Berlin)       11/01  –  06/05                   Undergraduate  Research  Assistant.  Bogazici  University,  Istanbul,  Turkey     Role  of  FGF  family  in  development  and  physiopathology  in  vertebrate  retina.   Advisor:  Prof.  Kuyas  Bugra,  Department  of  Molecular  Biology  and  Genetics           iv   PUBLICATIONS       Amenta  AR,  Yilmaz  A,  Bogdanovich  S,  McKechnie  BA,  Abedi  M,  Khurana  TS,  Fallon  JR.  (2011)   Biglycan  recruits  utrophin  to  the  sarcolemma  and  counters  dystrophic  pathology  in  mdx   mice.  PNAS  108(2),  762-­‐767     Bobkov  AA,  Muhlrad  A,  Pavlov  DA,  Kokabi  K,  Yilmaz  A,  Reisler  E.  (2006)  Cooperative  effects   of  cofilin  (ADF)  on  actin  structure  suggest  allosteric  mechanism  of  cofilin  function.  J  Mol   Biol.  356(2),  325-­‐334     Manuscripts  in  Progress:       Yilmaz  A,  Fallon  JR.  (in  preparation)  The  receptor  tyrosine  kinase  MuSK  regulates  BMP4   signaling.         Yilmaz  A,  Fallon  JR.  (in  preparation)  BMP4  induces  acetylcholine  receptor  clustering  in  a   MuSK-­‐dependent  manner.       PATENT  APPLICATIONS      “Biglycan  mutants  and  related  therapeutics  and  methods  of  use”   Inventors:  Amenta  AR,  McKechnie  BA,  Dechene  M,  Yilmaz  A,  Fallon  JR.   U.S.  Patent  Application  No.:  13/109,558            “Therapeutic  and  diagnostic  methods  involving  biglycan  and  utrophin”   Inventors:  Amenta  AR,  Yilmaz  A,  McKechnie  BA,  Fallon  JR.   Provisional  Application  No.:  61/427,468       ABSTRACTS  and  INVITED  LECTURES     Yilmaz  A,  Amenta  A,  McKechnie  B,  Fallon  J.  Biglycan  recruits  utrophin  to  the  muscle   membrane  and  is  a  potential  therapeutic  for  Duchenne  Muscular  Dystrophy.  Poster.  EMBO   Conference  Series:  Molecular  and  cellular  basis  of  regeneration  and  tissue  repair,  Sesimbra,   Portugal.  September  26-­‐30,  2010.     A,  Yilmaz.  Mechanisms  of  action  of  biglycan,  a  potential  therapeutic  for  Duchenne  Muscular   Dystrophy.  Invited  Lecturer.  Department  of  Molecular  Biology  and  Genetics,  Boğaziçi   University,  Istanbul,  Turkey.  July,  2008     TEACHING  EXPERIENCE     Spring  2009  –  Fall  2011  Rotation  student  trainer,  6  rotation  students  and  1  MD-­‐PhD   student     January  2011      Graduate  student  assistant,  Suna  Kıraç  Workshop  on  Neurodegenerative   Disease:  From  Genetic  Models  to  Therapies,  Boğaziçi  University,  Istanbul,  Turkey     Spring  2011        Guest  Lecturer,  Analysis  of  Development,  Axolotl  Embryo  Lab,  Brown   University   v     Spring  2010        Guest  Lecturer,  Analysis  of  Development,  Axolotl  Embryo  Lab,  Brown   University     Spring  2007      Teaching  Assistant,  Analysis  of  Development,  Brown  University       AWARDS  and  HONORS     2011            Graduate  International  Colloquium  Grant,  approved  by  Brown  University  Office  of   International  Affairs           LANGUAGES     Turkish,  English,  German,  French,  Greek                                                               vi                                             To  my  mother,  father  and  sister...                                                   vii     Acknowledgements     I   would   like   to   first   thank   my   advisor,   Dr.   Justin   Fallon.   Without   his   support   and   ideas  this  work  would  not  have  been  possible.  I  am  especially  grateful  to  Dr.  Fallon   for  trusting  my  ideas  and  for  all  our  fruitful  discussions  which  helped  my  training  to   become  an  independent  scientist  tremendously.       I   also   want   to   thank   my   thesis   committee   members,   Dr.   Kristi   Wharton,   Dr.   Gilad   Barnea  and  Dr.  Mark  Zervas  for  their  valuable  ideas  that  helped  me  bring  together   the  work  presented  in  this  thesis.  They  were  always  available  to  discuss  ideas  and   give  me  feedback.  I  would  like  to  specifically  thank  Dr.  Wharton  with  whom  I  had  a   phone   interview   before   I   came   to   Brown   University.   Thank   you   for   giving   me   the   chance  to  be  here,  Dr.  Wharton!     I  am  also  thankful  to  the  past  and  present  members  of  our  laboratory:  Beatrice   Lechner  for  training  me  when  I  joined  Dr.  Fallon’s  laboratory,  Anne  Booker  whom  I   will  mention  below,  Michelle  Dechene  for  her  protein  purifications  that  helped  move   my  project  greatly  and  also  for  advancing  my  baking  skills,  Julia  Najera,  Emily   Stackpole  and  Hanna  Berk  Rauch  for  their  discussions  and  friendship,  Carolyn   Schmiedel  and  Eamon  Quick  for  helping  me  with  cell  cultures,  Beth  McKechnie  and   Sarah  Mentzer  for  making  sure  that  the  laboratory  is  running  efficiently,  Michael   Akins  and  Alison  Amenta  for  their  ideas,  and  all  rotation  students  I  worked  with  for   their  help  in  the  experiments  and  for  their  interest  in  my  thesis  project.     I  would  also  like  to  mention  a  few  people  who  have  great  impacts  on  my  life.  Very   special  thanks  go  to  two  dear  friends  of  mine:  İlker  Öztop,  with  whom  I  shared  both   laughter  and  tears  very  many  times.  I  cannot  imagine  a  life  without  such  a  great   friend  and  I  feel  very  lucky  for  knowing.    İyi  ki  varsın!  And  Anne  Booker,  who  not   only  was  a  great  lab  mate  but  is  also  a  life-­‐long  friend.  Without  her,  so  many  good   memories  would  be  missing  from  my  life.    I  want  to  thank  Konstantinos  Kotakis  for   his  constant  support  and  life  advices,  Gökhan  Demirkan  and  Ahmet  Eken  for  their   company  throughout  the  graduate  school  and  their  friendship.  I  will  always  be   grateful  to  all  of  you.  I  would  like  to  thank  Theocharis  Vadivoulis  for  reviving  me  at   one  of  the  most  hopeless  times  of  my  life  and  for  reminding  me  that  life  is  “plastic”.    I   now  hope  for  iluke-­‐full  of  years.  Ευχαριστώ  πολύ!     All  other  friends  whose  names  I  could  not  mention  here  deserve  many  thanks.     Finally,   I   would   like   to   thank   three   most   special   people   to   whom   I   dedicate   my   thesis:   My   mother   and   father   ,   Fadime   and   Hüseyin   Yılmaz,   as   well   as   my   sister   Nagihan   Yılmaz.   No   matter   how   far   we   are,   you   have   always   been   by   my   side.   Sizi   çok  seviyorum....           viii   Preface     Communication   between   the   extracellular   matrix   and   the   intracellular   environments   of   the   cells   is   crucial   for   multiple   processes   such   as   survival,   proliferation   or   maintenance   of   cellular   states.   Failure   or   mistakes   in   this   communication   leads   to   inability   to   form   subcellular   organizations   or   to   give   appropriate  responses  to  the  changes  in  the  cellular  environment  or  several  disease   states   such   as   cancer.   Therefore   it   is   important   to   understand   how   cells   employ   different   mechanisms   to   relay   information   from   the   extracellular   matrix   to   their   cytoplasm.   One   common   way   of   such   communication   is   established   by   the   use   of   signaling  molecules  that  are  recognized  by  their  receptors  at  the  cell  surface  and  the   signaling   cascades   initiated   by   this   receptor-­‐ligand   interaction.   This   mechanism   is   key   to   both   the   intercellular   communication   and   the   autocrine   regulatory   events.   In   this   thesis   I   discuss   two   examples   of   interactions   between   signaling   pathways,   as   well   as   mechanisms   in   which   extracellular   molecules   regulate   cytosolic   proteins.   The  first  project  details  the  studies  based  on  an  incidental  observation  showing  that   the   receptor   tyrosine   kinase   MuSK   and   the   signaling   molecule   BMP4   bind   to   each   other.   I   also   demonstrate   how   the   muscle-­‐specific   receptor   tyrosine   kinase   MuSK   regulates   BMP4   signaling   in   muscle   cells   (Chapter   2).   The   results   of   these   studies   show  that  MuSK  binds  to  BMP4  and  is  required  for  the  transcription  of  a  subset  of   genes   in   response   to   BMP4.   In   Chapter   3,   I   demonstrate   a   role   for   BMP4   in   the   induction   of   AChR   clusters   in   cultured   muscle   cells.   My   results   suggest   that   this   BMP4  effect  requires  MuSK  and  Wnt11  activities.  In  the  last  data  chapter,  I  present   ix   the   work   detailing   biglycan’s   regulation   of   sarcolemmal   utrophin   expression,   the   therapeutic   effect   of   this   regulation   for   Duchenne   Muscular   Dystrophy   (DMD)   and   the   mechanisms   thereof   (Chapter   4).   Finally,   I   discuss   the   importance   of   these   findings  and  the  future  directions  (Chapter  5).                                                             x   Table  of  Contents       CHAPTER  1:  INTRODUCTION     1.1 BMP  ligands...............................................................................................2   1.2 BMP  pathway............................................................................................3   1.3 Secreted  extracellular  regulators  of  BMPs...................................5   1.4 BMP  regulators/co-­‐receptors  at  the  cell  surface.......................5   1.5 BMP4.............................................................................................................6   1.6 BMP4  in  muscle........................................................................................7   1.7 BMP4  target  genes..................................................................................8   1.7.1 Id1  and  Id2...........................................................................................8   1.7.2 Ptgs2  and  Ptger4...............................................................................9   1.7.3 RGS4.......................................................................................................10   1.7.4 Fabp7.....................................................................................................11   1.7.5 Car3.........................................................................................................12   1.7.6 Myh15....................................................................................................13   1.7.7 Wnt11....................................................................................................13   1.8 Acetylcholine  Receptor  clustering  and  the  neuromuscular  junction  formation   .................................................................................................................................14   1.9 Muscle  Specific  Kinase..........................................................................15   1.10 Muscle  fiber  types  and  fiber-­‐type  switch................................18   Conclusion.........................................................................................................20   xi   References.........................................................................................................21   Figures................................................................................................................39                                                                                   xii   CHAPTER  2:  The  receptor  tyrosine  kinase  MuSK  binds  BMPs  and  selectively   regulates  their  signaling     Abstract..............................................................................................................50   Introduction.....................................................................................................51   Results................................................................................................................55     MuSK  binds  to  BMPs......................................................................55     MuSK  Ig3  domain  is  required  for  BMP4  binding...............56     MuSK  regulates  canonical  BMP4  signaling..........................57     MuSK  selectively  regulates  distinct  sets  of  BMP4-­‐induced  genes  in  myoblasts   and  myotubes..................................................................................................59     MuSK  kinase  activity  is  not  required  for  MuSK  regulation  of  BMP4   signaling.............................................................................................................62   Discussion.........................................................................................................63     MuSK  binding  to  BMPs.................................................................65     MuSK  regulation  on  canonical  BMP4  pathway..................66     MuSK-­‐dependence  of  a  subset  of  BMP4  responses........67     MuSK  kinase  activity  and  BMP4  pathway............................70   Materials  and  Methods................................................................................71     Antibodies  and  materials.............................................................72     Mammalian  cell  culture  and  mice............................................72     Luciferase  reporter  assays..........................................................73     Immunoprecipitation  and  co-­‐immunoprecipitation.......74   xiii     Western  blots....................................................................................75     ELISAs..................................................................................................75     Immunocytochemistry.................................................................76     RNA  extraction,  reverse  transcription  and  quantitative  real  time  polymerase   chain  reaction.................................................................................................77     Microarrays  and  bioinformatics  analysis............................78     Surface  plasmon  resonance.......................................................78     Statistical  analysis..........................................................................79   Figures...............................................................................................................80   References........................................................................................................97   Supplementary  material.............................................................................104                                                   xiv     CHAPTER  3:  BMP4  induces  acetylcholine  receptor  clustering  in  a  MuSK-­  and   Wnt11-­dependent  manner     Abstract................................................................................................................154   Introduction........................................................................................................155   Results...................................................................................................................157     BMP4  induces  AchR  clusters  in  a  MuSK-­‐dependent  fashion   ..................................................................................................................................157     BMP4  induces  Wnt11  expression...............................................158     Wnt11  activity  iis  necessary  for  the  formation  of  BMP4-­‐induced  AchR   clusters..................................................................................................................159   Discussion............................................................................................................159     Future  directions...............................................................................................161   Materials  and  Methods....................................................................................162     Antibodies  and  materials.................................................................162     Mammalian  cell  culture.....................................................................162     Acetylcholine  receptor  clustering  assay....................................163     RNA  extraction,  reverse  transcription  and  quantitative  real  time  polymerase   chain  reaction......................................................................................................163     Statistical  analysis...............................................................................164   Figures.....................................................................................................................165   References..............................................................................................................173     xv   CHAPTER  4:  Biglycan  recruits  utrophin  to  the  sarcolemma  and  counters   dystrophic  pathology  in  mdx  mice     Abstract.....................................................................................................................180   Introduction.............................................................................................................181   Results.........................................................................................................................182     Endogenous  biglycan  regulates  utrophin  expression  in  immature  muscle   .........................................................................................................................................182     rhBGN  treatment  up-­‐regulates  membrane-­‐associated  utrophin  in  cultured   muscle  cells................................................................................................................183     Systemic  delivery  of  rhBGN..................................................................184     rhBGN  up-­‐regulates  utrophin  and  other  DAPC  components  in  mdx  mice   ..........................................................................................................................................185     rhBGN  reduces  dystrophic  pathology  in  mdx  mice....................187     rhBGN  efficacy  is  utrophin  dependent..............................................188     rhBGN  treatment  improves  muscle  function  in  mdx  mice.......188     rhBGN  is  well  tolerated  in  mdx  mice..................................................189       Discussion......................................................................................................................190   Materials  and  Methods.............................................................................................194     Biglycan............................................................................................................194     Animals  and  Injections...............................................................................194     Histology  and  immunohistochemistry................................................194     Quantitative  RT-­‐PCR  and  Western  blot  analysis.............................195   xvi   Muscle  physiology.........................................................................................196   Figures  ..............................................................................................................................197   References........................................................................................................................209   Supplementary  information.....................................................................................213     SI  materials  and  methods...........................................................................213     Figures  and  tables..........................................................................................217                                     xvii   CHAPTER  5:  DISCUSSION     Discussion..............................................................................................................229     References..............................................................................................................236         APPENDIX.............................................................................................................................238                                                                   xviii       List  of  Figures       1.1 BMP  pathway.....................................................................................................................39   1.2 BMP4  inhbitors  and  co-­‐receptors.............................................................................41   1.3 Schematic  representation  of  MuSK..........................................................................43   1.4 NMJ  and  AChR  clusters..................................................................................................44   1.5 Skeletal  muscle  fiber  types..........................................................................................46     2.1  MuSK  ectodomain  binds  to  BMP4.............................................................................80   2.2  SPR  binding  analysis  of  MuSK  to  BMPs...................................................................82   2.3  The  MuSK  Ig3  domain  is  required  for  BMP4  binding........................................84   2.4  MuSK  regulates  the  canonical  BMP4  pathway......................................................86   2.5  MuSK  selectively  regulates  BMP4-­‐induced  expression  of  a  subset  of  genes  in   myoblasts.....................................................................................................................................88   2.6  MuSK  selectively  regulates  BMP4-­‐induced  expression  of  a  subset  of  genes  in   myotubes......................................................................................................................................90   2.7  BMP4  does  not  induce  MuSK  phosphorylation....................................................92   2.8  Differences  between  BMP4-­‐  and  agrin-­‐mediated  signaling  of  MuSK.........94   2.9  Putative  models  for  MuSK  regulation  of  BMP4  signaling.................................95   Supplementary  Figure  2.1  BMP4-­‐induced  RGS4  expression  at  an  earlier  time-­‐point   ........................................................................................................................................................104     xix   3.1  BMP4  induces  AChR  clustering  and  this  activity  requires  MuSK.................165   3.2  BMP4-­‐induced  AChR  clusters  form  between  8  and  16  hours  after  treatment   ...........................................................................................................................................................167   3.3  BMP4  induces  Wnt11  expression  in  a  MuSK-­‐independent  manner............169   3.4  Wnt11  activity  is  required  for  BMP4-­‐induced  AChR  clusters.........................171     4.1  Utrophin  is  reduced  at  the  sarcolemma  of  immature  bgn-­‐/o  mice...............197   4.2  rhBGN  treatment  increases  membrane-­‐associated  utrophin  and  γ-­‐sarcoglycan   protein  in  cultured  myotubes................................................................................................199   4.3  rhBGN  treatment  up-­‐regulates  utrophin  at  the  sarcolemma  of  mdx  mice   .............................................................................................................................................................201   4.4  rhBGN  up-­‐regulates  DAPC  components  at  the  sarcolemma  of  mdx  mice   .............................................................................................................................................................203   4.5  Systemically  administered  rhBGN  counters  dystrophic  pathology  in  mdx  mice   .............................................................................................................................................................205   4.6  Physiological  improvement  of  muscle  in  rhBGN-­‐treated  mdx  mice.  ...........207     Supplementary  Figure  4.1  Systemically  delivered  rhBGN  can  be  detected  in  the   circulation  and  becomes  localized  to  muscle.................................................................217   Supplementary  Figure  4.2  rhBGN  treatment  increases  sarcolemmal  utrophin   expression  in  the  tibialis  anterior  of  mdx  mice.............................................................219   Supplementary  Figure  4.3  Creatine  kinase  levels  in  rhBGN-­‐treated  mdx  mice   ............................................................................................................................................................221   xx   Supplementary  Figure  4.4  rhBGN  fails  to  counter  dystrophic  pathology  in  mdx:utr-­‐/-­‐   double  KO  animals.....................................................................................................................223   Supplementary  Figure  4.5  rhBGN  is  well  tolerated  in  mdx  mice..........................225     Appendix  Figure  1  Car3  and  γ-­‐sarcoglycan  messages  are  upregulated  by  rhBiglycan   in  mdx  mice.  ................................................................................................................................238   Appendix  Figure  2  Non-­‐glycanated  biglycan  exhibits  less  binding  to  BMP4  than   proteoglycan  form  of  biglycan  ............................................................................................240   Appendix  Figure  3  DAPC  regulation  by  MuSK  .............................................................242                               xxi   List  of  Tables     2.1  KD  values  for  the  interaction  of  the  MuSK  with  BMP2,  4  &  7  as  determined  using   SPR.......................................................................................................................................................96   Supplementary  Table  2.1  Transcripts  upregulated  by  BMP4  only  in  wild  type   myoblasts.  ........................................................................................................................................105   Supplementary  Table  2.2  Transcripts  upregulated  by  BMP4  both  in  wild-­‐type  and   MuSK  null  myoblasts  ...................................................................................................................116   Supplementary  Table  2.3  Transcripts  upregulated  by  BMP4  only  in  MuSK  null   myoblasts...........................................................................................................................................121   Supplementary  Table  2.4  Transcripts  upregulated  by  BMP4  only  in  wild  type   myotubes.  ..........................................................................................................................................126   Supplementary  Table  2.5  Transcripts  upregulated  by  BMP4  both  in  wild-­‐type  and   MuSK  null  myotubes......................................................................................................................132   Supplementary  Table  2.6  Transcripts  upregulated  by  BMP4  only  in  MuSK  null   myotubes.............................................................................................................................................138       Supplementary  Table  4.1  Contractile  properties  of  extensor  digitorum  longus  (EDL)   muscles.................................................................................................................................................227     Appendix  Table  1  Genome-­‐wide  gene  expression  analysis  of  non-­‐glycanated   biglycan-­‐treated  biglycan  null  myotubes.  .............................................................................244   xxii   Appendix  Table  2  Genome-­‐wide  gene  expression  analysis  of  proteoglycan  biglycan-­‐ treated  biglycan  null  myotubes.  .................................................................................................248   Appendix   Table   3   Genome-­‐wide   microRNA   expression   analysis   of   non-­‐glycanated   biglycan-­‐treated  biglycan  null  myotubes  ..............................................................................256   Appendix   Table   4   Transcripts   downregulated   by   BMP4   treatment   in   wild   type   myoblasts  ................................................................................................................................................259   Appendix   Table   5     Transcripts   downregulated   by   BMP4   treatment   in   MuSK   null   myoblasts    ...............................................................................................................................................268   Appendix   Table   6   Transcripts   downregulated   by   BMP4   treatment   in   wild-­‐type   myotubes  .................................................................................................................................................275   Appendix   Table   7   Transcripts   downregulated   by   BMP4   treatment   in   MuSK   null   myotubes  ...............................................................................................................................................281                                               xxiii                               CHAPTER  1     INTRODUCTION                                                         1   1.1 BMP  ligands       Bone  Morphogenic  Proteins  (BMPs)  are  phylogenetically  conserved  growth  factors   that  were  first  detected  in  bone  extracts  that  have  the  ability  to  induce  ectopic  bone   formation  (Ferguson  et  al.,  1992;  Ray  et  al.,  1991;  Urist,  1965).  Over  20  members  of   BMPs  have  been  identified  so  far.  This  number  makes  them  the  largest  subgroup  of   Transforming  Growth  Factor  Beta  (TGFβ)  superfamily  (Lavery  et  al.,  2008;   Kawabata  et  al.,  1998).  As  opposed  to  the  tissue  restriction  that  is  implied  by  their   initial  nomenclature,  BMPs  function  during  early  development  throughout  the   embryo  in  both  invertebrates  and  vertebrates  (Ferguson  et  al.,  1992;  Ray  et  al.,   1991;  Urist,  1965).  Furthermore,  this  diverse  group  of  growth  factors  functionally   supports  multiple  processes  such  as  organ  morphogenesis  and  regeneration  in  a   range  of  developing  and  adult  tissues.  Based  on  their  functions  and  sequence   similarity,  BMPs  are  divided  into  at  least  four  subgroups:  BMP2/4,   BMP5/6/7/8a/8b,  BMP9/10  and  BMP12/13/14  (Mazerbourg  et  al.,  2006;  von   Bubnoff  et  al.,  2001).  BMP2,  4,  8b  and  10  are  involved  at  the  earliest  stages  of  the   embryo.  Hence  their  homozygous  nulls  are  embryonic  lethal  (Zhang  et  al.,  1996;   Winnier  et  al.,  1995;  Zhao  et  al.,  1996;  Chen  et  al.,  2004).     BMPs  are  synthesized  as  large  precursors  with  an  N-­‐terminal  signal  peptide,  a   prodomain  and  a  C-­‐terminal  mature  peptide  (Xiao  et  al.,  2007;  Sieber  et  al.,  2009).   Upon  dimerization  mature  proteins  are  proteolytically  cleaved  from  the  prodomain   2   (Nelsen  et  al.,  2009).  BMPs  can  form  biologically  active  heterodimers  and   homodimers.       BMP  activity  is  regulated  at  several  levels,  which  serves  to  regulate  the  ligand   activity  and  increase  the  diversity  of  the  signaling  complexes.  The  expression  of  the   ligand  is  the  first  step  of  regulation.  Another  type  of  regulation  was  shown  recently   for  Glass  bottom  boat  (Gbb),  the  Drosophila  ortholog  of  vertebrate  BMP  5,  6  and  7.   In  this  study,  Gbb  was  shown  to  have  a  second  furin  processing  site  in  the   prodomain,  which  generates  a  larger  active  ligand  with  greater  signaling  activity   and  a  longer  range  (Akiyama  et  al.,  2012).  The  bioavailability  of  the  ligand  in  a   particular  tissue  environment  is  also  regulated  by  extracellular  matrix  and  surface   molecules.  This  class  of  regulation  will  be  discussed  in  the  following  sections.         1.2 BMP  pathway       Biologically  active  BMPs  bind  to  two  different  classes  of  receptors  on  the  cell   surface;  type-­‐1  (Alk2,  4  or  6)  and  type-­‐2  (BMPRII,  ActRIIa)  (ten  Dijke  et  al.,  1994,  de   Sousa  Lopes  et  al.,  2004,  Nohno  et  al.,  1995,  Xia  et  al.,  2007).  For  BMP  ligands  it  has   been  shown  that  the  binding  to  the  type-­‐1  receptor  has  a  higher  affinity  than  the   binding  to  the  type-­‐2  receptor  (Berasi  et  al.,  2011).  Upon  binding  of  the  ligand  to  the   receptor  complex,  the  constitutively  active  type-­‐2  receptor  phosphorylates  the  type-­‐ 1  receptor  (Wrana  et  al.,  1994).  This  phosphorylation  is  required  for  the  activation   3   of  the  type-­‐1  receptor.  The  activated  type-­‐1  receptor  phosphorylates  the  cytosolic   member  of  the  signaling  pathway,  SMAD1/5/8  (Kretzschmar  et  al.,  1997).   Phosphorylated  SMAD1/5/8  forms  a  complex  with  SMAD4,  translocates  to  and   accumulates  in  the  nucleus  where  it  functions  as  part  of  a  transcriptional  activator   or  repressor  complex  for  a  range  of  genes  at  different  stages  of  development  or  in   the  adult  (Lagna  et  al.,  1996,  Liu  et  al.,  1996,  Hoodless  et  al.,  1996)  (Figure  1.1).       BMP  binding  to  the  type-­‐1  receptor  is  regulated  by  multiple  factors  including  ligand   concentration  and  post-­‐translational  modifications.  BMP  binding  to  monomeric   type-­‐1  receptor  requires  high  BMP  concentrations  (Heinecke  et  al.,  2009).  At  low   concentrations,  BMPs  bind  to  dimeric  type-­‐1  receptors  since  they  have  a  higher   affinity  to  the  dimer  than  to  the  mononer.  For  the  signaling  to  occur,  a  final   assembly  of  a  minimum  of  one  type-­‐1  and  one  type-­‐2  receptor  has  to  be  formed  in   complex  with  the  ligand.  For  TGFβ  receptors,  homodimerization  of  receptors  leads   to  formation  of  a  heterohexamerix  complex  that  includes  the  dimeric  ligand  and   homodimers  of  each  receptor  (Gilboa  et  al.,  1998).  However,  for  BMPs  a  final   complex  assembly  has  yet  to  be  shown  clearly.  It  is  possible  that  heterodimerization   of  the  ligand  and  of  both  receptor  types  and  the  formation  of  a  heterohexameric   complex  of  these  heterodimers  is  used  as  a  way  to  increase  the  complexity  of  the   signaling.         The  BMP  pathway  can  also  be  regulated  at  the  downstream  steps,  particularly  at  the   level  of  SMAD1/5/8  phosphorylation.  Fuentealba  et  al.  have  reported  that   4   SMAD1/5/8  that  is  phosphorylated  by  type-­‐1  receptors  can  be  further   phosphorylated  by  GSK3β  and  MAP  kinases  (Fuentealba  et  al.,  2007).  These   phosphorylations  are  required  for  polyubiquitination  of  SMAD1/5/8.         1.3  Secreted  extracellular  regulators  of  BMPs     The  bioavailability  of  the  BMP  ligands  is  regulated  by  various  secreted  extracellular   matrix  proteins.  Among  the  best-­‐characterized  examples  of  such  secreted   antagonists  are  noggin,  chordin  and  follistatin.  These  inhibitors  bind  to  BMPs  and   prevent  them  from  associating  with  their  signaling  receptors  (Zimmerman  et  al.,   1996,  Piccolo  et  al.,  1996,  Fainsod  et  al.,  1997).  One  of  the  most  important  functions   of  these  antagonists  was  observed  in  early  embryos.  At  the  blastula  stage  of  Xenopus   embryos,  chordin  and  noggin  are  secreted  from  Spemann’s  organizer  at  the  dorsal   half  of  the  embryo  where  they  prevent  the  signaling  by  BMP4  that  is  secreted  from   the  ventral  side  (DeRobertis,  2009).  Other  antagonists  such  as  Inhibin  compete  with   BMPs  for  receptor  binding  (Rosen  et  al.,  2006).         1.4  BMP  regulators/co-­receptors  at  the  cell  surface     Another  regulatory  mechanism  of  BMP  signaling  uses  surface  molecules  that  act  as   stimulatory   or   inhibitory   co-­‐receptors.   Dragon   family   co-­‐receptors   are   structurally   5   similar   to   type-­‐1   receptors   in   their   ectodomain   and   interact   with   BMPs   to   recruit   type-­‐2   receptors   that   would   not   ordinarily   be   used   by   a   given   BMP.   These   co-­‐ receptors  intensify  the  BMP  signaling  by  recruitment  of  type-­‐2  receptors  (Halbrooks   et  al.,  2007).  The  transmembrane  protein  BAMBI  inhibits  BMP  signaling  by  acting  as   a   pseudo-­‐type   1   receptor   and   occupying   the   available   type-­‐2   receptors   (Onichtchouk   et   al.,   1999).   Neogenin,   a   receptor   for   netrins   and   proteins   of   the   repulsive   guidance   molecule family,   was   recently   shown   to   bind   BMPs   and   inhibit   Smad   signal   transduction   through   the   activation   of   RhoA   (Hagihara   et   al.,   2011).   Stem   cell   factor   receptor   (c-­‐kit)   was   shown   to   interact   with   type-­‐2   receptor   BMPRII   and  positively  regulate  BMP  signaling  (Hassel  et  al.,  2006).         1.5  BMP4     BMP4  is  one  of  the  most  widely  studied  members  of  BMP  family.  It  has  crucial  roles   both  in  the  embryo  and  in  adult.  Its  fundamental  function  in  the  embryo  is  shown  by   the  embryonic  lethality  of  its  homozygous  null  mutants  (Winnier  et  al.,  1995).  BMP4   is  highly  expressed  in  extraembryonic  ectoderm  and  the  primitive  streak  before  and   during  gastrulation  (Winnier  et  al.,  1995;  Lawson  et  al.,  1999;  Ying  et  al.,  2000;  Ying   et  al.,  2001).  Mutations  in  decapentaplegic  (dpp),  the  Drosophila  ortholog  of  BMP2   and  BMP4,  cause  dorsoventral  patterning  abnormalities  at  the  blastoderm  stage   (Ray  et  al.,  1991).  BMP4  acts  as  a  posterior-­‐ventralizing  factor  in  Xenopus  embryos   (Dale  et  al.,  1992;  Jones  et  al.,  1992).  BMP4  is  also  crucial  factor  for  maintaining  the   6   pluripotency  of  mouse  ES  cells  by  signaling  through  Alk3  to  suppress  p38  MAP   kinase  activity  (Qi  et  al.,  2004;  Ying  et  al.,  2003).  BMP4  signaling  is  also  required  for   normal  induction  of  primordial  germ  cells  (de  Sousa  et  al.,  2004;  Lawson  et  al.,   1999;  Ying  et  al.,  2001).  Finally,  BMP4  also  functions  in  the  mature  organisms  such   as  inhibiting  melanin  synthesis  in  epidermal  melanocytes  and  regulating  adult   hippocampal  neurogenesis  (Singh  et  al.,  2012,  Tang  et  al.,  2009).     Several  BMP4  extracellular  antagonists  have  been  identified.  These  include  Dan,   PRDC,  Gremlin,  Cerberus,  Coco,  Tsg,  Chordin,  Noggin  and  Follistatin  (Sudo  et  al.,   2004;  Khokha  et  al.,  2003;  Zuniga  et  al.,  1999;  Bell  et  al.,  2003;  Pearce  et  al.,  1999;   Nosaka  et  al.,  2003;  Oelgeschlager  et  al.,  2000;  Wardle  et  al.,  1999;  Zhang  et  al.,   2002;  Bachiller  et  al.,  2000;  Brunet  et  al.,  1998;  McMahon  et  al.,  1998;  Gazzerro  et   al.,  1998;  Lin  et  al.,  2006).  Notably,  BMP4  activity  is  also  modulated  by   transmembrane  receptors  such  as  neogenin  (Hagihara  et  al.,  2011)  (Figure  1.2).         1.6  BMP4  in  muscle     Several  studies  have  shown  functions  for  BMP4  in  muscle  tissue  and  myogenic  cells.   In  myoblasts,  Id1,  one  of  the  well-­‐characterized  downstream  genes  of  BMP4,  has   been  shown  to  inhibit  differentiation  by  interfering  with  pro-­‐differentiation  bHLH   transcription  factor  complexes  (Kurabayashi  et  al.,  1994).    More  recently,  BMP4  and   its  antagonist  Gremlin  have  been  implicated  in  regulation  of  myogenic  progenitor   7   proliferation  in  human  fetal  skeletal  muscle  (Frank  et  al.,  2006).  This  study  showed   that  BMP4  secreted  by  skeletal  muscle  side  population  cells,  a  stem-­‐like  cell  type,   inhibited  differentiation  of  the  main  population  mononuclear  muscle-­‐derived  cells.   In  contrast  to  the  previous  studies,  BMP2  and  BMP4  have  been  shown  to  regulate  a   miRNA-­‐mediated  mechanism  that  enhances  myocardial  differentiation  (Wang  et  al.,   2010).  Endogenous  BMP4  in  cultured  C2C12  myoblasts  has  been  also  implicated  in   myotube  formation  (Umemoto  et  al.,  2011).  Finally,  defective  myogenesis  in   Duchenne  Muscular  Dystrophy  patients  has  been  correlated  to  increased  BMP4   expression  in  these  patients  (Sterrenburg  et  al.,  2006).         1.7  BMP4  target  genes     BMP4  induces  the  expression  of  many  genes  in  different  tissues  and  at  different   developmental  stages.  Several  BMP4  target  genes  in  myogenic  cells  are  studied  in   this  thesis.  Among  those,  Id1  and  Id2  are  well-­‐characterized  BMP  targets.  Ptgs2  has   been  previously  shown  to  be  expressed  downstream  of  BMP2.  On  the  other  hand,   several  other  genes  studied  here  were  previously  not  implicated  downstream  of   BMP4.  These  include  Ptger4,  RGS4,  Fabp7,  Car3,  Myh15  and  Wnt11.  These  genes   and  their  products  will  be  discussed  in  the  following  sections.         1.7.1 Id1  and  Id2   8     Id  proteins  are  among  most  important  targets  of  BMP  signaling.  Id1  promoter   activity  is  specifically  increased  by  BMPs  and  this  effect  requires  Smad1  or  Smad5   and  Smad4  (Ogata  et  al.,  1993;  Hollnagel  et  al.,  1999;  Lopez-­‐Rovira  et  al.,  2002;   Korchynskyi  et  al.,  2002).  Id  proteins  were  identified  as  negative  regulators  of  bHLH   transcription  factors  and  have  been  shown  to  interact  with  retinoblastoma  (Rb)  and   Ets  family  members  (Norton  et  al.,  1998;  Yokota  et  al.,  2002).  Id  proteins  are   negative  regulators  of  differentiation  and  positive  regulators  of  proliferation.  They   inhibit  cell  differentiation  by  binding  to  the  ubiquitous  bHLH  transcription  factors   and  inhibiting  their  interaction  with  the  tissue  specific  bHLH  transcription  factors.   For  example,  in  myoblasts  Id1  interacts  with  E2A  protein  and  inhibits  the  formation   of  the  functional  E2A-­‐MyoD  heterodimer,  which  leads  to  inhibition  of  differentiation   and  maintenance  of  undifferentiated  phenotype  of  myoblasts  (Kurabayashi  et  al.,   1994).  Id  proteins  also  regulate  cell  cycle  progression.  Id2  binds  to   hypophosphorylated  active  form  of  Rb  family  proteins  and  inhibit  their   antiproliferative  functions  (Lasorella  et  al.,  2000).         1.7.2 Ptgs2  and  Ptger4       Ptgs2  is  the  gene  encoding  for  the  enzyme  COX2.  COX2  is  one  of  the  two  isoforms  of   COX  enzymes  that  catalyze  the  rate-­‐limiting  step  in  the  synthesis  of  prostaglandins   (PG).  PGs  are  autocrine  and  paracrine  signaling  molecules  that  regulate   9   inflammation  and  are  synthesized  in  response  to  growth  factors,  cytokines  and  cell   injury  (Funk,  2001).  BMP2  induces  COX2  expression  through  a  noncanonical  BMP   pathway  that  requires  the  activity  by  p38  kinase  (Susperregui  et  al.,  2011).  COX2   has  been  shown  to  regulate  stretch-­‐induced  proliferation  of  skeletal  muscle   myoblasts,  smooth  muscle  cells  and  retinal  mesangial  cells  (Otis  et  al.,  2005;  Park  et   al.,  1999;  Akai  et  al.,  1994).  Myofiber  growth  during  skeletal  muscle  regeneration  is   inhibited  in  COX2  null  animals  (Bondesen  et  al.,  2004).  The  COX2  pathway  has  also   been  shown  to  regulate  the  growth  of  atrophied  muscle  by  regulating  myonuclear   addition  and  satellite  cell  proliferation  (Bondesen  et  al.,  2006).       Ptger4  encodes  for  the  receptor  EP4,  one  of  the  four  G-­‐protein-­‐coupled  receptors  for   the  prostaglandin  PGE2,  which  is  in  turn  synthesized  by  COX2  pathway  (Giuliano  et   al.,  2002).  EP4  expression  is  regulated  by  various  stimuli  including   lipopolysaccharide  in  RAW  264.7  murine  macrophage-­‐like  cell  line  (Arakawa  et  al.,   1996).    EP4  expression  is  upregulated  by  gonadotropin  in  ovarian  granulosa  and   cumulus  cells  (Segi  et  al.,  2003).  In  this  thesis,  Ptger4  is  shown  to  be  upregulated   downstream  of  BMP4.     1.7.3 Regulator  of  G-­protein  signaling  4  (RGS4)     RGS4  is  one  of  the  30  known  regulators  of  G  protein  signaling  (RGS)  molecules   identified  to  date  (Willars,  2006).  RGS  molecules  are  GTPase  activating  proteins   (GAP),  a  class  of  proteins  that  promote  GTP  hydrolysis  by  Gα  proteins.  This  GAP   10   activity  by  RGS  molecules  is  very  crucial  as  the  intrinsic  GTPase  activity  of  Gα   proteins  is  weak  and  they  rely  on  GAPs  to  sustain  physiologically  meaningful  rates   of  GTP  hydrolysis.  Thus,  RGS  proteins  are  crucial  modulators  of  G  protein  signaling.   RGS4  expression  is  enriched  in  heart  and  central  nervous  system  (Rogers  et  al.,   2001;  Erdely  et  al.,  2004;  Cifelli  et  al.,  2008).  It  has  been  thought  to  counter-­‐regulate   Gαq-­‐induced  signaling  that  is  triggered  by  hypertrophic  stimuli  in  the  heart  (Rogers   et  al.,  2001;  Tokudome  et  al.,  2008).  On  the  other  hand,  injection  of  RGS4  mRNA  into   Xenopus  embryos  resulted  in  decreased  skeletal  muscle  (Wu  et  al.,  2000).       1.7.4 Fatty  acid  binding  protein  7  (Fabp7)     Fabp7  is  a  member  of  fatty  acid  binding  protein  (Fabp)  family.  Fabps  are   intracellular  proteins  involved  in  lipid  trafficking  and  they  have  key  roles  in  taking   up  fatty  acids  into  cytoplasm  and  transporting  them  to  appropriate  cellular   compartments  (Chmurzynska  et  al.,  2006;  Furuhashi  et  al.,  2008;  Storch  et  al.,   2009).  Fabp7  is  expressed  in  adult  rodent  brain  (Owada  et  al.,  1996;  Owada  et  al.,   2008).    Fabp7  is  also  a  well-­‐established  marker  for  neuronal  stem  cells  and  has  been   shown  to  be  important  for  neurogenesis  (Ming  et  al.,  2011;  Steiner  et  al.,  2006;  Duan   et  al.,  2008).  In  this  thesis  Fabp7  expression  in  myogenic  cells  is  shown  to  be   induced  by  BMP4.       11   1.7.5 Carbonic  Anhydrase  3  (Car3)     Carbonic  anhydrase  3  (Car3)  is  a  member  of  carbonic  anhydrase  family  of  enzymes   that  reversibly  hydrate  carbon  dioxide  and  generate  bicarbonate  and  hydrogen  ions.   This  activity  maintains  a  variety  of  physiological  functions  including  acid-­‐base   balance,  respiration,  urinary  acidification  and  bone  resorption  (Sly  et  al.,  1995;   Spicer  et  al.,  1990;  Stanton  et  al.,  1991;  Tashian  et  al.,  1989).  Car3  shows  different   characteristics  than  the  other  Car  isoforms  in  especially  its  very  low  specific  activity   (Koester  et  al.,  1981;  Koester  et  al.,  1977).    It  is  very  abundant  in  skeletal  muscle  and   adipocytes  and  makes  up  to  8  and  25%  of  the  soluble  fraction  in  these  tissues,   respectively  (Carter  et  al.,  1991;  Spicer  et  al.,  1990).  Car3  was  originally  purified  in   rabbit  muscle  where  it  was  shown  to  constitute  1  to  2%  of  the  total  protein  that  was   extracted  (Blackburn  et  al.,  1972).  In  humans  and  most  large  mammals,  Car3   expression  is  enriched  in  slow  skeletal  muscle  and  is  highly  expressed  in  type  1,   slow  twitch  fibers  (Tashian,  1989;  Wade  et  al.,  1986;  Wistrand  et  al.,  1987;   Brownson  et  al.,  1988).  Therefore,  it  was  suggested  to  be  a  slow-­‐twitch  fiber  marker.   Car3  function  in  muscle  tissue,  however,  is  not  known.  In  Car3  null  animals,  which   develop  normally  and  have  normal  life  spans,  fiber  type  composition  of  slow  muscle   Soleus  is  not  affected.  The  main  contractile  properties  of  the  muscles  are  also  not   changed  in  the  absence  of  Car3  (Kim  et  al.,  2004).  Car3  was  alternatively  suggested   to  decrease  oxidative  stress  (Raisanen  et  al.,  1999).         12   1.7.6 Myosin  Heavy  Chain  15  (Myh15)     Myosin  heavy  chain  15  (Myh15)  is  a  member  of  sarcomeric  myosin  heavy  chain   (MyHC)  family  proteins.  MyHC  isoforms  in  this  family  can  assemble  into  highly   ordered  structures  called  sarcomeres,  which  are  found  in  skeletal  and  cardiac   muscles  (Cripps,  Suggs  and  Bernstain,  1999).  The  developmental  regulation  of  the   expression  of  these  MyHC  isoforms  can  change  the  contractile  properties  of   individual  striated  muscle  fibers  (Barany,  1967;  Schiaffino  and  Reggiani,  1996).   Myh15  has  been  suggested  as  a  slow-­‐contracting  MyHC  isoform  (Desjardins  et  al.,   2002)  with  orthologs  in  Xenopus  and  chicken  but  not  fish  (McGuigan  et  al.,  2004;   Garriock  et  al.,  2005;  Ikeda  et  al.,  2007).  In  a  previous  study,  Myh15  was  not   deteceted  in  two  skeletal  muscles  (slow-­‐twitch  Soleus  and  fast-­‐twitch  Tibialis   Anterior)  or  in  heart  it  was  shown  to  be  absent,  whereas  its  expression  was   enriched  in  extraocular  muscles  (Rossi  et  al.,  2010).  In  contrast  to  those  reports,  in   this  thesis  Myh15  expression  is  shown  to  be  present  both  in  soleus  and  extensor   digitorum  longus  (EDL)  muscles  and  to  be  induced  by  BMP4  (Chapter  2).         1.7.7 Wnt11     Wnt11  is  a  member  of  the  Wnt  family  growth  factors.  It  belongs  to  Wnt5a  subgroup   of  Wnt  proteins  along  with  Wnt  4  and  5a.  Wnt11  is  a  354  amino  acid  protein  with  a   molecular  weight  of  45  kDa  that  is  associated  with  the  extracellular  matrix   13   (Christiansen  et  al.,  1996).    Wnt11  is  most  similar  in  sequence  to  Wnt4,  although   their  functions  may  differ  in  certain  contexts  (Kispert  et  al,  1998,  Elizalde  et  al.   2010).  In  the  embryo,  Wnt11  is  expressed  in  various  tissues  including  somites,  pre-­‐ cardiac  mesoderm,    mesenchyme  of  developing  limb  buds  and  the  apical   ectodermanl  ridge  (Christiansen  et  al,  1996,  Kispert  et  al.  1996).  In  adults,  it  is     expressed  in  heart,  skeletal  muscle,  pancreas  and  liver  (Kirikoshi  et  al.,  2001).   Wnt11  promotes  cardiac  differentiation  and  its  overexpression  leads  to  cardiac   hyperthrophy  (Eisenberg  &  Eisenberg,  1999;  Abdul-­‐Ghani  et  al.,  2011).    Several   factors  activate  Wnt11  expression,  including  Ret/GDNF  in  developing  kidney   (Pepicelli  et  al.,  1997)  and  Wnt3a  in  differentiating  mES  cells  (Ueno  et  al.,  2007).   Recently,  Wnt11  has  been  also  shown  to  bind  to  MuSK  and  induce  AChR  clusters  on   cultured  mouse  myotubes  (Zhang  et  al.,  2012).         1.8  Acetylcholine  Receptor  (AChR)  clustering  and  the  neuromuscular  junction   (NMJ)  formation       Acetylcholine  released  from  motor  neuron  terminals  in  vertebrates  binds  to  and   opens  AChRs  in  the  postsynaptic  domains  of  NMJs  initiating  the  endplate  potential   that  in  turn  is  necessary  to  muscle  contraction.  Generation  of  a  sufficiently  large   endplate  potential  requires  a  high  density  of  AChRs  at  the  NMJ.  Thus  AChR   clustering  is  vital  for  the  efficient  neurotransmission,  hence  the  communication   between  neurons  and  the  muscle  tissue.   14     AChR  clustering  occurs  at  two  distinct  stages  during  development.  Prior  to   innervation  of  the  muscle,  AChRs  are  initially  uniformly  distributed  throughout  the   muscle  fiber.  However,  they  eventually  accumulate  in  the  middle  of  the  fiber  where   innervation  will  occur  (Bevan  et  al.,  1977;  Braithwaite  et  al.,  1979;  Creazzo  et  al.,   1983;  Ziskind-­‐Conhaim  et  al.,  1982).  This  phenomenon  is  called  pre-­‐patterning  and   is  nerve-­‐independent  (Yang  et  al.,  2000).  These  so-­‐called  microclusters  disappear   after  innervation  and  the  bigger  clusters  form  at  the  innervated  sites  (Lin  et  al.,   2001;  Vock  et  al.,  2008,  Yang  et  al.,  2001).  Importantly,  both  the  aneural   prepatterned  and  the  neural  clusters  require  Muscle  Specific  Kinase  (MuSK)  (Zhang   et  al.,  2004).  While  agrin  has  been  shown  to  be  necessary  for  stabilizing  neural   clusters,  Wnt11r  has  been  suggested  to  be  the  inducer  of  aneural  clusters  in   zebrafish  (Jing  et  al.,  2009).    The  subsequent  Dishevelled  (Dvl)-­‐mediated  signaling   has  also  been  implicated  in  guiding  motor  axons  for  NMJ  formation  in  zebrafish  (Jing   et  al.,  2009).         1.9  Muscle  Specific  Kinase  (MuSK)     MuSK  is  a  receptor  tyrosine  kinase  that  is  predominantly  expressed  at  the  NMJs  in   mature   muscle   cells   (Valenzuela   et   al.,   1995).   However,   it   is   also   expressed   at   lower   levels  at  the  extrajunctional  membrane  of  muscle  cells  (Bowen  et  al.,  1998)  and  in   other  tissues  such  as  brain  (Garcia-­‐Osta  et  al.,  2006).     15     The   extracellular   domain   of   MuSK   consists   of   3   immunoglobulin-­‐like   (Ig)   domains   and  a  cysteine-­‐rich  domain  (CRD).  In  its  cytoplasmic  domain  it  has  a  juxtamembrane   domain  (JM),  which  is  followed  by  a  catalytic  tyrosine  kinase  (TK)  domain  (Figure   1.3).  Several  MuSK  isoforms  with  the  presence  or  absence  of  3  short  inserts  (10,  15   and  8  amino  acids)  at  the  ectodomain  of  MuSK  have  been  detected  in  neonatal  and   adult  mice,  as  well  as  in  cultured  C2C12  myotubes  (Kuehn  et  al.,  2005).  In  addition,   another   MuSK   isoform   lacking   the   third   Ig   domain   at   the   ectodomain   has   been   identified   (Hesser   et   al.,   1999).   It’s   been   suggested   that   the   alternative   splicing   of   MuSK  adds  more  complexity  to  the  activities  of  MuSK.       MuSK   also   binds   to   LRP4,   which   is   a   receptor   for   agrin   and   forms   a   complex   with   MuSK.  Binding  of  neural  derived  proteoglycan  agrin  (N-­‐agrin)  to  LRP4  increases  the   association   between   LRP4   and   MuSK   and   also   activates   MuSK   (Kim   et   al.,   2008).   MuSK  is  activated  by  autophosphorylation  and  this  activation  leads  to  a  cascade  of   events   that   is   crucial   for   NMJ   formation,   maturation   and   stability.   MuSK   has   a   master   regulatory   role   for   these   events   (Herbst   et   al.,   2000;   Zhou   et   al.   1999;   Zhu   et   al.   2008;   Zong   et   al.,   2012;   Zang   et   al.,   2011;   DeChiara   et   al.,   1996)   (Figure   1.4).   Mouse   embryos   lacking   MuSK   fail   to   form   NMJs   and   the   paralyzed   embryos   die   before  birth  (DeChiara  et  al.,  1996).     There  are  several  interaction  partners  of  MuSK,  which  are  required  for  MuSK   pathway  to  induce  AChR  clustering  in  response  to  N-­‐agrin.  One  such  interaction   16   partner  is  the  adaptor  protein  downstream-­‐of-­‐tyrosine-­‐kinase-­‐7  (Dok7)  that  binds   to  tyrosine  phosphorylated  NPXY553  motif  in  JM  domain  of  MuSK  (Bergamin  et  al.   2010;  Okada  et  al.,  2006).  Another  key  molecule  in  this  pathway  is  the  tumorous   imaginal  disc  protein  (Tid1),  which  binds  constitutively  to  the  cytoplasmic  domain   of  MuSK  (Linnoila  et  al.,  2008).  Dvl  binds  to  the  JM  and  kinase  domains  of  MuSK  and   couples  MuSK  to  p21-­‐activated  kinase  (Luo  et  al.,  2002).       The  signaling  pathways  in  which  MuSK  acts  as  the  master  regulator  for  building  the   postsynaptic  membrane  at  NMJs  involve  various  other  kinases  and  adaptor   molecules.  The  assembly  of  AChRs  into  clusters  requires  the  small  GTPases  Rac  and   Rho  (Weston  et  al.,  2003).  MuSK  activation  by  N-­‐agrin  leads  to  the  activation  of  Rac  I   and  the  formation  of  AChR  micro-­‐aggregates  (Luo  et  al.,  2003).  Rac  activation  is   followed  by  the  activation  of  Rho,  which  is  thought  to  act  through  PAK  I  in  order  to   enlarge  AChR  micro-­‐aggregates  into  clusters  bigger  than  10  µm  (Luo  et  al.,  2002).   Moreover,  cytoplasmic  tyrosine  kinases  Abl  and  Src  are  also  activated  by  MuSK  and   they  increase  AChR  clustering  (Mittaud  et  al.,  2004).  Tyrosine  phoshorylation  of   AchR  β-­‐subunit  recruits  the  adaptor  protein,  rapsyn,  after  which  the  AChR  clusters   are  stabilized  (Borges  et  al.,  2008).         Recently,  Wnt11  has  been  also  shown  to  bind  to  MuSK  and  induce  AChR  clusters  on   cultured  mouse  myotubes.  Interestingly,  Wnt11  clustering  activity  is  not  additive  to   agrin’s,  suggesting  that  agrin  and  Wnt11  may  be  using  similar  pathways  to  induce   17   clustering  (Zhang  et  al.,  2012).  The  significance  of  this  interaction  will  be  explored  in   the  experiments  described  in  Chapter  3.         1.10  Muscle  fiber  types  and  fiber-­type  switch     Mammalian  skeletal  muscles  are  heterogenous  in  their  metabolic,  electrical  and   contractile  properties  (Schiaffino  and  Reggiani,  1996;  Bertchtold  et  al.,  2000).  This   heterogeneity  is  the  basis  for  the  flexibility  that  allows  muscles  to  be  used  for   various  activities  ranging  from  low  or  high  intensity  contractions  to  repeated   motions.  Muscle  fibers  are  divided  into  four  main  categories  based  on  their   expression  of  myosin  heavy  chains  (MyHC)  (Pette  and  Staron,  2000).  The  hindlimb   muscles  of  adult  rats  express  one  slow  myosin  heavy  chain  isoform  (MHC  I  (Myh7))   and  three  fast  myosin  heavy  chain  isoforms  (MHC  IIa  (Myh2),  IId  (Myh1)  and  IIb   (Myh14))  (Schiaffino  and  Reggiani,  1994;  Weiss  et  al.,  1999).  The  nomenclature  for   fiber  types  was  also  determined  according  to  these  myosin  heavy  chain  isoforms:   Type  I,  type  IIa,  type  IId  and  type  IIb.  Although  most  fibers  express  only  one  myosin   isoform,  hence  are  referred  to  as  pure  fibers  (Staron  et  al.,  1993),  some  minor   fraction  of  fibers  expresses  two  myosin  isoforms  (Staron  et  al.,  1993).  The   contractile  properties  of  these  hybrid  fibers  exhibit  an  intermediate  level,  which   increases  the  number  of  fiber  types  and  the  complexity  of  fiber  type  composition  of   different  muscle  tissues  (Pette  and  Staron,  2000).       18   Postural  muscles  are  more  enriched  in  slow  fibers,  which  are  highly  vascularized   and  are  dependent  on  oxidative  phosphorylation  for  energy.  On  the  other  hand,   muscles  that  are  used  for  locomotion  are  more  enriched  in  fast  fibers,  which  are   mostly  dependent  on  glycolytic  metabolism  (Bassel-­‐Duby  and  Olson,  2006).  In   C57BL6J  mice,  soleus  muscle  is  enriched  in  type  I  slow  fibers,  whereas  extensor   digitorum  longus  (EDL)  muscle  is  enriched  in  type  IIb  fast  fibers  (Augusto  et  al.,   2004).     During  embryogenesis  and  early  postnatal  life  muscle  fiber  type  is  regulated  by   several  factors  such  as  the  differences  among  progenitor  cells,  growth  factors  and   neural  activity  (Wigmore  and  Evans,  2002)  (Figure  1.5).  The  fibers  that  are   innervated  by  the  branches  of  the  same  motor  neuron  exhibit  the  same  fiber   phenotype  (Burke  et  al.,  1971,  Burke  et  al.,  1973).  In  the  adult  neural  activity  can   reprogram  muscle  fibers  towards  different  fiber  types  (Gauthier  et  al.,  1983;  Gorza   et  al.,  1988;  Ausoni  et  al.,  1990).  The  roles  of  the  nerve  in  reprogramming  the  fiber   type  was  shown  in  cats  and  rats  by  reinnervating  slow  muscles  with  a  fast  nerve  and   reprogramming  slow  fibers  to  fast  ones  or  vice  versa  (Buller  et  al.,  1960;  Hoh  et  al.,   1975).     Several  signaling  pathways  have  been  demonstrated  to  control  muscle  fiber  type-­‐ specific  gene  programs.  In  mammals,  Six  factors,  Six1-­‐Six4,  as  well  as  the   transcriptional  repressor  Sox6  were  implicated  in  the  control  of  the  fast  fiber   induction  program  (Niro  et  al.,  2010;  Hagiwara  et  al.,  2005;  Hagiwara  et  al.,  2007).   19   Calcineurin-­‐dependent  nuclear  factor  of  activated  T-­‐cells  (NFAT)  transcription   factors  have  been  shown  to  increase  MhHC-­‐β/slow  promoter  activity  in  skeletal   muscle,  whereas  the  expression  of  this  slow  myosin  was  inhibited  in  response  to   thyroid  hormone  in  mouse  heart  (McCullagh  et  al.,  2004;  Haddad  et  al.,  2008).   Moreover,  peroxisome  proliferator-­‐activated  receptors  (PPARs)  and  peroxisome   proliferator-­‐activated  receptor  gamma  coactivator-­‐1α  (PGC-­‐1α)  have  been   associated  with  slow  fiber  phenotype  (Wang  et  al.,  2004;  Baar  et  al.,  2002;  Russell  et   al.,  2003;  Terada  et  al.,  2002).       Conclusion     Signaling  pathways  often  times  do  not  function  individually  but  rather  are  part  of   larger  signaling  networks  in  which  multiple  pathways  crosstalk  with  each  other.  In   this  thesis,  I  explore  the  interactions  between  BMP4  and  MuSK  pathways.  Chapter  2   and  3  comprise  the  main  body  of  this  work.  In  Chapter  2,  I  aimed  to  explore  the   regulation  of  BMP4  pathway  in  myogenic  cells  by  MuSK.  In  Chapter  3,  I  explored   BMP4  regulation  of  MuSK-­‐dependent  AChR  clustering.  Chapter  4  summarizes  the   work  demonstrating  therapeutic  benefits  of  biglycan  in  a  mouse  model  of  Duchenne   Muscular  Dystrophy.    In  the  final  chapter  (Chapter  5),  the  significance  of  these   results  and  future  directions  are  discussed.         20   REFERENCES     Abdul-Ghani, Mohammad, Daniel Dufort, Rebecca Stiles, Yves De Repentigny, Rashmi Kothary, and Lynn A Megeney. “Wnt11 Promotes Cardiomyocyte Development by Caspase-mediated Suppression of Canonical Wnt Signals.” Molecular and Cellular Biology 31, no. 1 (January 2011): 163–178. 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 dimer  and  prevent  it  from  binding  to  the   receptor  complex  (Zimmerman  et  al.,  1996,  Piccolo  et  al.,  1996,  Fainsod  et  al.,  1997).   Dragon  family  co-­‐receptors  associate  with  type-­‐1  BMP  receptors  and  the  BMPs  and   enhance  the  signaling  initiated  by  the  ligand  (Halbrooks  et  al.,  2007).  Other  surface   molecules  such  as  Neogenin  act  as  inhibitory  receptors  for  BMPs  and  their   downstream  signaling  (Hagihara  et  al.,  2011).                                                             42         Figure  1.3                                                         Figure  1.3       Schematic  representation  of  MuSK.  MuSK  has  three  immunoglobulin-­‐like  (Ig)   domains  and  a  cycteine-­‐rich  domain  (CRD)  in  its  ectodomain.  Its  intracellular  part   comprises  a  juxtamembrane  and  a  catalytic  tyrosine  kinase  domain.                   43       Figure  1.4                     44       Figure  1.4.       NMJ  and  AChR  clusters  (Adapted  from  Ferraro  et  al.,  2012).  Neural-­‐derived  agrin   released  from  presynaptic  terminal  binds  to  Lrp4  and  induces  the  phosphorylation   and  activation  of  MuSK  (Herbst  et  al.,  2000,  Zhou  et  al.  1999,  Zhu  et  al.  2008,  Zong  et   al.,  2012,  Zang  et  al.,  2011).  Phosphorylated  MuSK  recruits  Dok-­‐7,  which  further   fosters  MuSK  phosphorylation  (Bergamin  et  al.  2010;  Okada  et  al.,  2006).  This  leads   to  the  formation  of  AChR  clusters  and  their  stabilization.  The  adaptor  protein  rapsyn   anchors  AChRs  and  helps  the  stability  of  the  clusters  (Borges  et  al.,  2008).                                                           45   Figure  1.5                                                     46     Figure  1.5     Skeletal  muscle  fiber  types.  Mononuclear  myoblasts  undergo  fusion  and   differentiate  into  multi-­‐nucleated  myofibers,  which  can  express  different  contractile   properties.  Two  main  categories  of  fibers  and  their  properties  are  shown.  Slow   fibers  have  higher  oxidative  capacity  and  endurance  compared  to  the  fast  fibers   (Bassel-­‐Duby  and  Olson,  2006).  Slow  myosin  heavy  chain  isoforms  are  expressed  in   slow  fibers,  while  fast  myosin  heavy  chain  isoforms  are  expressed  in  fast  fibers.   Differences  among  progenitor  cells,  growth  factors  and  changes  neural  activity  can   trigger  fiber  type  switch  programs  (Wigmore  and  Evans,  2002).                                                           47                                   CHAPTER  2                                                           48   The  receptor  tyrosine  kinase  MuSK  binds   BMPs  and  selectively  regulates  their  signaling   Atilgan  Yilmaz1,3,  Chandramohan  Kattamuri2,  Christoph  Schorl3,  Tom  Thompson2,   Justin  Fallon1     1Department  of  Neuroscience,  Brown  University,  Providence,  Rhode  Island  02912,   USA   2  Department  of  Molecular  Genetics,  Biochemistry  and  Microbiology,  University  of   Cincinnati  Medical  Sciences  Building,  Cincinnati,  OH  45267,  USA   3  Department  of  Molecular  Biology,  Cell  Biology  and  Biochemistry,  Brown   University,  Providence,  Rhode  Island  02912,  USA         SPR  experiments  in  Figure  2  and  Table  1  were  conducted  by  Chandramohan   Kattamuri.   Microarray  experiments  in  Figures  5  and  6  were  conducted  by  Christoph  Schorl  and   me.   The  rest  of  the  experiments  were  conducted  by  me.       49   Abstract:   Bone   morphogenic   proteins   (BMPs)   induce   signals   in   various   tissues   with   unique   outcomes.  They  are  tightly  regulated  both  during  development  and  in  adult.  Various   extracellular   interactions   between   BMPs   and   their   regulator   proteins,   both   secreted   and   membrane-­‐bound   ones   have   been   identified   as   means   of   regulation   of   BMP   pathway.  Here  we  propose  a  cell-­‐type  specific  regulation  of  BMPs  by  Muscle  Specific   Kinase   (MuSK),   a   receptor   tyrosine   kinase   that   is   a   master   regulator   in   neuromuscular  junction  formation  with  high  expression  in  myogenic  cells.    We  show   that   MuSK   directly   binds   to   BMP4,   BMP2   and   BMP7   with   low   nM   affinities.   MuSK   binding   to   BMP4   is   partially   mediated   through   the   third   immunoglobulin-­‐like   domain   (Ig3)   of   MuSK   that   had   been   thought   to   be   dispensable   for   MuSK’s   role   at   the  neuromuscular  junction.  BMP4-­‐induced  pSMAD5  levels  and  ID1  transcript  levels   are  reduced  in  the  absence  of  MuSK  in  undifferentiated  myoblasts.  Furthermore,  in   these   cells   MuSK   is   required   for   BMP4-­‐induced   expression   of   a   subset   of   genes,   including   RGS4.   In   cultured   myotubes,   MuSK   positively   regulates   BMP4-­‐induced   expression   of   myosin   heavy   chain   15   (Myh15)   and   carbonic   anhydrase   3   (Car3)   both  of  which  are  expressed  at  higher  levels  in  muscles  with  more  slow-­‐type  fibers   in  mice.  These  results  indicate  a  potential  role  for  MuSK-­‐BMP4  interaction  in  fiber   type   composition   of   muscle   tissue.   To   our   knowledge   this   is   the   first   study   that   shows   MuSK   as   a   BMP   regulator   and   attributes   a   non-­‐synaptogenic   role   to   it   in   myogenic   cells.   We   hypothesize   that   our   results   point   to   a   general   mechanism   for   regulating   BMP   pathway   with   the   use   of   tissue-­‐specific   proteins.   They   also   50   potentially   give   insights   for   the   functions   of   MuSK   in   non-­‐myogenic   tissues   (i.e.   brain)  where  it  is  expressed  at  low  levels.     Introduction   The  interaction  between  the  cells  and  their  environment  dictates  various  decisions   that   the   cells   have   to   make   in   terms   of   their   survival,   differentiation   or   death.   Intercellular   communication   is   one   such   interaction   that   has   intricately   evolved   in   multicellular   organisms   in   order   to   tightly   control   the   behavior   of   individual   cells   in   complex   tissue   structures.   One   of   the   most   commonly   used   communication   systems   involves   a   secreted   polypeptide   that   is   recognized   by   a   cell   surface   receptor.   The   same   polypeptide   signal   can   act   on   different   cell   types   with   different   outcomes,   including  differential  transcriptional  or  post-­‐transcriptional  events.  Maintaining  this   specificity   is   fundamental   to   distinguish   cell   fates   or   cellular   responses   in   various   tissues.   Therefore,   a   very   crucial   question   about   signaling   molecules   is   how   the   cell-­‐   or  tissue-­‐type  specificity  is  achieved  in  their  responses.     BMP4  belongs  to  the  family  of  Bone  Morphogenic  Proteins  (BMPs),  which  is  a  large   group  of  phylogenetically  conserved  signaling  molecules  that  are  in  turn  a  subfamily   of   the   transforming   growth   factor-­‐β   (TGFβ)   superfamily.   Two   different   classes   of   receptors,  type-­‐1  (Alk2,4  or  6)  and  type-­‐2  (BMPRII,  ActRIIa),  bind  to  BMP4  on  the   cell  surface  (ten  Dijke  et  al.,  1994,  de  Sousa  Lopes  et  al.,  2004,  Nohno  et  al.,  1995,  Xia   et   al.,   2007).   The   binding   of   the   ligand   to   the   type-­‐1   receptor   is   followed   by   the   phosphorylation  of  this  receptor  by  the  constitutively  active  type-­‐2  receptor  (Wrana   51   et   al.,   1994).   This   phosphorylation   activates   type   1   receptor   that   in   turn   phosphorylates   the   cytosolic   intermediate   of   the   pathway,   SMAD1/5/8   (Kretzschmar   et   al.,   1997).   Phosphorylated   SMAD1/5/8   forms   a   complex   with   SMAD4   and   translocates   to   and   accumulates   in   the   nucleus   where   it   functions   as   part   of   a   transcriptional   activator   or   repressor   complex   for   a   range   of   genes   at   different   stages   of   development   or   in   adult   (Lagna   et   al.,   1996,   Liu   et   al.,   1996,   Hoodless  et  al.,  1996).  For  example,  although  BMP4  is  an  inducer  of  differentiation   in  osteoblasts  (Miyama  et  al.,  1999),  in  muscle  precursor  cells  it  is  shown  to  inhibit   differentiation  by  inducing  the  transcription  of  Id1  gene,  a  well-­‐characterized  BMP   target   (Ono   et   al.,   2011).   Understanding   how   BMP4   is   regulated   is   key   to   explain   such   major   phenotypic   changes   seen   in   different   cells   as   a   response   to   the   same   BMP4  signal.       Having   pivotal   roles   at   different   stages   of   development,   in   adult   and   in   various   tissues   it   is   not   unexpected   that   BMPs   and   the   pathway   they   initiate   are   tightly   regulated.  One  of  the  most  common  ways  of  regulation  is  to  control  the  availability   of   the   ligand   extracellularly   by   either   secreted   molecules   or   surface   receptors.   Among   the   best-­‐characterized   examples   of   such   secreted   proteins   are   noggin,   chordin  and  follistatin  among  others.  These  inhibitors  bind  to  BMP4  and  prevent  it   from   associating   with   its   signaling   receptors   (Zimmerman   et   al.,   1996,   Piccolo   et   al.,   1996,   Fainsod   et   al.,   1997).   Another   regulatory   mechanism   involves   surface   molecules  that  may  act  as  stimulatory  or  inhibitory  co-­‐receptors.  For  example,  the   GPI-­‐anchored  Dragon  family  of  co-­‐receptors  was  shown  to  positively  regulate  BMP   52   signaling   (Halbrooks   et   al.,   2007).   On   the   other   hand,   the   transmembrane   protein,   BAMBI,  inhibits  BMP  signaling  by  acting  as  a  pseudo-­‐type1  receptor  and  occupying   the   available   type-­‐2   receptors   (Onichtchouk   et   al.,   1999).   Recently,   Neogenin,   a   receptor   for   netrins   and   proteins   of   the   repulsive   guidance   molecule   family,   was   shown  to  bind  BMPs  and  inhibit  Smad  signal  transduction  through  the  activation  of   RhoA   (Hagihara   et   al.,   2011).   Another   interesting   class   of   surface   molecules   regulating  BMPs  is  the  receptor  tyrosine  kinase  family.  Stem  cell  factor  receptor  (c-­‐ kit)  has  been  shown  to  interact  with  type-­‐2  receptor  BMPRII  and  positively  regulate   BMP   signaling   (Hassel   et   al.,   2006).   Although   it   has   not   yet   been   shown   for   BMPs,   the  receptor  tyrosine  kinase  Ror2  binds  to  the  type-­‐1  receptor  Alk6  and  modulates   the  signaling  mediated  by  another  TGF-­‐β  family  member,  GDF5,  through  inhibition   of  the  Smad-­‐dependent  signaling  and  the  activation  of  a  Smad-­‐independent  pathway   (Sammar  et  al.,  2004,  Sammar  et  al.,  2009).    These  observations  raise  the  question   whether  other  tyrosine  kinases  may  also  regulate  BMP  pathway.     MuSK   is   a   receptor   tyrosine   kinase   that   is   predominantly   expressed   at   the   neuromuscular   junctions   (NMJ)   in   muscle   cells   (Valenzuela   et   al.,   1995).   It   was   shown  to  bind  to  LRP4  (Kim  et  al.,  2008)  and  be  activated  by  autophosphorylation   upon   binding   of   neural   derived   proteoglycan,   agrin   to   LRP4   (Herbst   et   al.,   2000,   Zhou  et  al.  1999,  Zhu  et  al.  2008,  Zong  et  al.,  2012).  This  activation  leads  to  a  cascade   of   events   that   is   crucial   for   NMJ   formation,   maturation   and   stability   and   MuSK   has   a   master   regulatory   role   for   these   events   (DeChiara   et   al.,   1996).   Studies   on   MuSK   have  been  mainly  focused  on  its  roles  at  the  NMJs.  However,  MuSK  is  also  expressed   53   at  lower  levels  at  the  extrajunctional  membrane  of  muscle  cells  (Bowen  et  al.,  1998)   and  in  other  tissues  such  as  brain  (Garcia-­‐Osta  et  al.,  2006).  This  expression  profile   indicates  that  MuSK  may  have  other  roles  than  what  has  been  described  so  far.         Here   we   report   that   MuSK   binds   to   BMP4   and   modulates   BMP4   signaling   in   myogenic   cells.   We   show   that   in   the   absence   of   MuSK   BMP4-­‐induced   SMAD5   phoshorylation  and  ID1  transcription  are  decreased.  MuSK  also  selectively  regulates   BMP4-­‐induced   transcriptional   response   for   a   subset   of   genes.   A   transcriptome/microarray   analysis   revealed   over   250   upregulated   genes   in   myoblasts   and   over   150   up-­‐regulated   genes   in   myotubes   only   in   the   presence   of   MuSK.  We  confirmed  that  BMP4-­‐induced  transcription  of  RGS4,  which  is  important   for  G-­‐protein  signaling,  was  dependent  on  the  presence  of  MuSK.  We  also  identified   two   BMP4-­‐induced   transcripts   in   myotubes,   Carbonic   Anhydrase   3   (Car3)   and   Myosin  Heavy  Chain  15  (Myh15)  that  were  regulated  by  MuSK  and  were  enriched  in   muscles   enriched   with   slow-­‐twitch   muscle   fibers   in   mice.   Our   results   suggest   that   MuSK   is   a   muscle-­‐specific   BMP4   regulator   and   it   modulates   BMP4-­‐induced   transcriptional  response  distinctively  in  undifferentiated  and  differentiated  muscle   cells,  with  potential  downstream  regulation  on  G-­‐protein  signaling  and  muscle  fiber-­‐ type  specification.             54   Results       MuSK  binds  to  BMPs     To  test  for  potential  BMP  regulators  in  myogenic  cells  we  used  the  reporter  cell  line,   C2C12BRA,  which  was  generated  by  stably  transfecting  the  immortalized  myogenic   C2C12   cells   with   a   plasmid   consisting   of   BMP-­‐responsive   elements   from   the   Id1   promoter   upstream   of   a   luciferase   reporter   gene   (Zilberberg   et   al.,   2007).   C2C12BRA   cells   were   treated   with   BMP4   along   with   a   purified   recombinant   MuSK   ectodomain   construct   (Figure   2.1a).   After   8   hours   of   treatment   luciferase   activity   was   measured   for   the   indicated   conditions.   At   a   concentration   between   50   and   100nM   the   soluble   MuSK   ectodomain   inhibited   BMP4   activity   by   50%.   The   irrelevant   his-­‐tagged   control   protein   used   at   200nM   did   not   show   any   significant   inhibition   of   BMP4   activity.   These   experiments   showed   that   the   soluble   MuSK   ectodomain  specifically  inhibits  BMP4  activity,  as  judged  by  the  decreased  luciferase   activity  (Figure  2.1b).       The   ability   of   soluble   MuSK   ectodomain   to   inhibit   BMP4   activity   could   reflect   either   a  direct  binding  between  these  molecules  or  the  presence  of  an  indirect  regulatory   loop  mediated  by  the  MuSK  ectodomain.  In  order  to  differentiate  between  those  two   possibilities,   we   performed   a   solution   binding   experiment.   Soluble   BMP4   was   preincubated   with   MuSK   ectodomain   or   the   irrelevant   His-­‐tagged   protein.   MuSK   ectodomain  and  the  control  protein  were  precipitated  via  their  6xHis-­‐tag.  Residual   BMP4  activity  in  solution  was  then  measured  in  the  C2C12BRA  reporter  line  (Figure   55   2.1c).  When  MuSK  was  pulled  down,  BMP4  activity  decreased  about  40%,  indicating   BMP4  co-­‐precipitated  with  MuSK.  There  was  not  any  significant  inhibition  in  BMP4   activity  when  the  irrelevant  His-­‐tagged  control  was  pulled  down.  Specific  pull-­‐down   of  BMP4  by  the  MuSK  ectodomain  was  confirmed  by  ELISA  analysis  of  the  pelleted   ecto-­‐MuSK-­‐BMP4  complex  (Figure  2.1d).  Taken  together,  these  results  indicate  that   the  MuSK  ectodomain  directly  binds  to  BMP4.       To  confirm  and  extend  these  results  we  assessed  the  kinetics  and  the  affinity  of  the   interaction  between  BMP4  and  MuSK  ectodomain  using  Surface  Plasmon  Resonance   (SPR).   BMP4   was   immobilized   as   the   ligand   and   MuSK   ectodomain   construct   was   used  as  the  analyte.  A  kinetic  analysis  with  the  use  of  a  heterogenous  ligand  model   showed  low  nanomolar  binding  affinity  for  BMP4-­‐MuSK  interaction  (Figure  2.2a  and   Table   2.1).   BMP4   is   closely   related   to   BMP2   (von   Bubnoff   et   al.,   2001)   and   has   been   shown   to   form   heterodimers   with   BMP7   (Suzuki   et   al,   1997).   We   investigated   whether   MuSK   ectodomain   could   also   bind   to   other   closely   related   BMP   family   members.  As  shown  in  Fig.  2.2a  and  in  Table  2.1,  SPR  analysis  revealed  that  BMP2   and  BMP7  bound  to  MuSK  ectodomain  with  similar  affinities  to  that  of  BMP4.  Thus,   the  MuSK  ectodomain  binds  to  a  closely  related  set  of  the  BMPs  that  includes  BMP2,   4  and  7.     MuSK  Ig3  domain  is  required  for  BMP4  binding     56   We  next  asked  if  alternative  splicing  of  MuSK  might  regulate  its  interaction  with   BMP4.  One  major  splice  isoform  of  MuSK  lacks  the  Ig3  domain  (Hesser  et  al.,  1999).   We  designed  Fc-­‐fusion  MuSK  ectodomain  constructs  with  (‘full  length’)  or  without   Ig3  domain  (ΔIg3)  (Figure  2.3a).  Equivalent  amounts  of  these  constructs  were   immobilized  on  96-­‐well  plates  and  were  incubated  with  a  range  of  BMP4   concentrations  (Figure  2.3c).  As  shown  in  Figure  2.3b,  BMP4  showed  saturable,  high   affinity  binding  to  FL-­‐ecto-­‐MuSK,  while  a  lower  and  non-­‐saturable  binding  was   observed  to  the  ΔIg3-­‐ecto-­‐MuSK.  We  also  wanted  to  see  if  the  deletion  of  Ig3  domain   interfered  with  the  overall  MuSK  structure,  which  could  be  one  reason  for  its   decreased  binding  to  BMP4.  For  this,  we  tested  the  binding  of  each  MuSK  construct   to  biglycan,  as  we  recently  reported  that  biglycan  binds  to  MuSK  and  potentiates  its   agrin-­‐induced  phosphorylation  (Amenta  et  al.,  2012).  Biglycan  was  incubated  with   immobilized  full-­‐length  and  ΔIg3-­‐MuSK  ectodomain  constructs  and  bound  biglycan   was  detected.  No  difference  was  seen  in  the  binding  of  biglycan  to  either  construct,   indicating  the  decrease  in  BMP4  binding  was  specific  (Figure  2.3d).  We  conclude   from  these  results  that  the  Ig3  domain  of  MuSK  is  necessary  for  its  BMP4  binding.     MuSK  regulates  canonical  BMP4  signaling     We  wanted  to  assess  the  possibility  whether  MuSK  could  have  any  regulatory  effects   on  canonical  BMP4  signaling,  which  is  marked  by  SMAD1/5/8  phosphorylation   upon  ligand  binding  to  the  receptors  and  the  pSMAD1/5/8  dependent  transcription   of  downstream  immediate  gene  targets  (Kretzschmar  et  al.,  1997,  Liu  et  al.,  1996,   57   Hoodless  et  al.,  1996).  To  test  this,  we  first  compared  the  BMP4-­‐induced   pSMAD1/5/8  levels  between  wild  type  and  MuSK  null  myoblasts  by  western   blotting.  In  the  absence  of  MuSK,  a  reduction  was  seen  in  the  dose-­‐response  curve  of   BMP4-­‐induced  SMAD1/5/8  phosphorylation,  indicating  that  MuSK  positively   regulates  this  early  upstream  event  of  BMP4  pathway  (Figure  2.4a,  2.4b).  We  then   wanted  to  test  any  potential  transcriptional  regulation  by  MuSK  and  analyzed   BMP4-­‐induced  Id1  transcript  levels,  since  Id1  is  one  of  the  best  characterized  BMP   downstream  gene  targets  and  its  BMP4-­‐induced  transcription  depends  on   SMAD1/5/8  phosphorylation  (Ogata  et  al.,  1993;  Hollnagel  et  al.,  1999;  Lopez-­‐ Rovira  et  al.,  2002;  Korchynskyi  et  al.,  2002).  We  analyzed  BMP4-­‐induced  Id1   transcripts  in  wild  type  and  MuSK  null  myoblasts  at  an  early  time  point  at  which  the   Id1  transcription  peaks  (data  not  shown).  In  the  absence  of  MuSK  BMP4-­‐induced   Id1  transcript  levels  were  reduced  compared  to  wild  type  levels  (Figure  2.4c).  This   result  was  in  accord  with  the  decrease  in  BMP4-­‐induced  pSMAD1/5/8  in  MuSK  null   myoblasts.       We  also  wanted  to  know  if  the  reduction  seen  in  the  absence  of  MuSK  could  result   from  differences  in  the  compartmentalization  of  pSMAD1/5/8  in  the  cell,  aside  from   the  reduction  in  its  levels.  To  test  this  idea,  we  immunostained  wild  type  and  MuSK   null  myoblasts  for  pSMAD1/5/8.  Although  we  did  not  observe  any  differential  effect   of  BMP4  treatment  in  the  distribution  of  pSMAD1/5/8  between  wild  type  and  MuSK   null  cultures  (data  not  shown),  there  was  a  striking  difference  in  pSMAD1/5/8   distribution  under  resting  conditions  between  these  cells.  Wild  type  myoblasts   58   expressed  distinct  cytosolic  pSMAD1/5/8  granules,  which  were  reduced   dramatically  in  MuSK  null  myoblasts  (Figure  2.4d,  2.4e).  This  pattern  of  distribution   did  not  change  upon  BMP4  treatment  (data  not  shown).     MuSK  selectively  regulates  distinct  sets  of  BMP4-­induced  genes  in  myoblasts   and  myotubes     We  investigated  whether  MuSK  was  required  for  the  transcription  of  any   downstream  BMP4  target  genes  in  myogenic  cells.    In  order  to  test  this  possibility   we  performed  a  microarray  analysis  and  compared  BMP4-­‐induced  transcripts   between  wild  type  and  MuSK  null  cells.  To  test  whether  cell  context  makes  any   difference  for  MuSK  regulation  we  included  both  the  undifferentiated  myoblast  and   the  differentiated  myotube  cultures  in  our  analysis.  Serum-­‐deprived  wild  type  and   MuSK  null  cultures  were  treated  with  BMP4.  RNA  was  harvested  from  the  cultures   and  reverse  transcribed  into  cDNA,  which  was  then  hybridized  to  Affymetrix  array   chips.  Cultures  were  treated  for  8  hours,  a  relatively  long  time  for  transcript   analysis,  in  order  to  detect  the  transcription  of  potentially  regulated  late  response   genes,  as  well.  In  myoblasts,  269  upregulated  genes  were  identified  only  in  wild-­‐ type  cells,  indicating  that  these  were  MuSK-­‐dependent  BMP4  responses  (Figure  2.5a   and  Supp.  Table  1).  107  genes  were  upregulated  both  in  wild  type  and  MuSK  null   myoblasts  and  therefore  were  not  qualitatively  regulated  by  MuSK  (Figure  2.5a  and   Supp.  Table  2).  108  other  genes  were  upregulated  only  in  MuSK  null  myoblasts   (Figure  2.5a  and  Supp.  Table  3).     59     Using  qRT-­‐PCR  we  validated  the  responses  for  a  group  of  genes  focusing  on  some   shared  and  MuSK-­‐dependent  responses.  Id1  and  Id2,  two  well-­‐characterized   canonical  responses  to  BMPs,  had  a  similar  fold  upregulation  between  wild  type  and   MuSK  null  myoblasts  (Figure  2.5b,  2.5c).  We  also  identified  a  novel  gene  response   downstream  of  BMP4,  Fabp7,  which  was  also  not  regulated  by  MuSK  under  these   conditions  (Figure  2.5d).  The  discrepancy  in  Id1  upregulation  between  the  early   (Figure  2.4c)  and  late  time-­‐points  (Figure  2.5b)  most  likely  stems  from  the  fact  that   Id1  expression  peaks  earlier  (data  not  shown)  at  which  point  MuSK  regulation  can   be  seen  robustly  and  then  it  tapers  to  similar  levels  in  both  wild-­‐type  and  MuSK  null   myoblasts.  Next  we  validated  a  group  of  MuSK-­‐dependent  genes.  BMP4-­‐induced   expression  of  Ptgs2,  which  encodes  for  Cox2,  the  key  enzyme  in  prostaglandin   pathway  and  of  Ptger4,  which  encodes  for  a  prostaglandin  receptor  was  regulated   by  MuSK  under  these  conditions  (Figure  2.5e  and  Figure  2.5f).  BMP4-­‐induced   expression  of  Rgs4,  a  regulator  of  G-­‐protein  signaling,  was  strictly  dependent  on  the   presence  of  MuSK  (Figure  2.5g),  as  in  MuSK  null  myoblasts  BMP4  could  not  induce   any  Rgs4  expression,  as  opposed  to  slight  increases  in  Ptgs2  and  Ptger4.  We  also   tested  shorter  treatment  times  for  Rgs4  by  which  MuSK-­‐regulation  on  BMP4-­‐ induced  Id1  expression  could  be  seen  robustly.  At  2  hours  of  BMP4  treatment,  MuSK   null  myoblasts  failed  to  induce  any  Rgs4  expression  by  BMP4,  which  was  in  accord   with  the  results  of  the  study  with  the  longer  treatment  (Supplementary  Figure  2.1).       60   In  order  to  understand  more  about  MuSK’s  regulation  on  Rgs4  expression,  we   wanted  to  see  if  BMP4-­‐induced  Rgs4  expression  was  dependent  on  the  canonical   BMP4  pathway.  In  wild-­‐type  myoblast  cultures  we  used  a  selective  inhibitor  for   type-­‐1  BMP  receptors,  LDN193189  (Vogt  et  al.,  2011)  and  indirectly  inhibited  SMAD   dependent  signaling.  We  then  analyzed  Rgs4  transcript  levels.  When  LDN193189   was  used  with  BMP4,  Rgs4  expression  was  inhibited  (Figure  2.5h),  indicating  that   BMP4-­‐induced  Rgs4  expression  requires  canonical  BMP4  pathway.                 Like  in  myoblasts,  BMP4-­‐induced  upregulation  of  171  genes  were  dependent  on  the   presence  of  MuSK  in  myotubes  (Figure  2.6a,  Supp.  Table  4).  While  there  are  a  few   shared  genes  between  MuSK-­‐dependent  BMP4  responses  in  myoblasts  and   myotubes,  the  majority  of  the  upregulated  genes  were  different  between  these  two   myogenic  cell  types.  This  result  indicates  that  MuSK  regulates  BMP4  pathway  in  a   cell-­‐context  dependent  manner.  Between  wild  type  and  MuSK  null  myotubes  117   gene  responses  were  shared,  whereas  325  genes  were  uniquely  upregulated  in   MuSK  null  myotubes  (Figure  2.6a,  Supp.  Table  5,  Supp.  Table  6).     Among  the  MuSK-­‐regulated  responses  in  myotubes,  Carbonic  Anhydrase  3  (Car3)   and  Myosin  Heavy  Chain  15  (Myh15)  were  two  interesting  novel  transcript   responses  downstream  of  BMP4  since  both  of  these  proteins  were  previously   suggested  as  slow-­‐type  fiber  markers  (Desjardins  et  al.,  2002;  Lyons  et  al.,  1991).   We  validated  the  regulation  of  their  mRNAs  by  MuSK  with  qRT-­‐PCR  in  myotubes.  On   61   the  other  hand,  in  myoblasts  these  two  genes  were  not  upregulated  by  BMP4,   suggesting  a  cell-­‐context  specific  expression  (Figure  2.6b  and  Figure  2.6c).         Fiber  type  composition  of  muscle  tissue  varies  in  different  muscles.  Among  the  four   major  fiber  types,  type-­‐1  slow  muscle  fibers  are  the  most  oxidative,  fatigue  resistant   ones  and  are  more  suitable  for  prolonged  low-­‐intensity  activities  (Bassel-­‐Duby  and   Olson,  2006).  Soleus  is  one  of  the  most  highly  enriched  muscles  for  the  slow  fiber   content  (Augusto  et  al.,  2004).  We  wanted  to  see  if  Myh15  and  Car3  expression  was   higher  in  muscles  that  are  enriched  in  slow  fibers  and  therefore  compared  their   expression  between  Soleus  and  Extensor  digitorum  longus  (EDL)  muscles  of  5.5   weeks  old  adult  mice.  The  expression  of  both  of  these  genes  was  higher  in  Soleus  as   expected  (Figure  2.6e  and  Figure  2.6f).  Interestingly,  MuSK  expression  was  also   shown  to  be  higher  in  Soleus  compared  to  EDL,  recently  (Punga  et  al.,  2011).  Our   expression  study  was  in  agreement  with  this  result  (Figure  2.  6d).       MuSK  kinase  activity  is  not  required  for  MuSK  regulation  of  BMP4  signaling     MuSK  is  a  receptor  tyrosine  kinase  that  can  dimerize  and  undergo  auto-­‐ phosphorylation  (Bergamin  et  al.,  2010).  We  wanted  to  know  if  BMP4  binding  to   MuSK  induced  receptor  auto-­‐phosphorylation.  In  order  to  test  this,  we  used  wild   type  myotubes  in  which  neural-­‐derived  proteoglycan  agrin  is  known  to  induce   MuSK  phosphorylation  (Glass  et  al.,  1996).  Myotube  cultures  were  treated  with   agrin  for  1  hour,  along  with  BMP4  for  various  time-­‐points  ranging  from  10  minutes   62   to  3  hours.  MuSK  was  immunoprecipitated,  run  in  an  SDS-­‐PAGE  and  phospho-­‐MuSK   was  detected  in  western  blots.  While  agrin  induced  robust  phosphorylation  of  MuSK   at  1  hour,  as  expected,  BMP4  failed  to  induce  the  phosphorylation  of  MuSK  under   these  conditions  (Figure  2.7a).  It  is  important  to  note  that  MuSK  regulation  on  BMP4   pathway  was  seen  at  the  same  ligand  concentration  and  within  the  time  window   that  were  chosen  in  this  experiment.  Given  this  result,  we  conclude  that  BMP4  does   not  induce  MuSK  phosphorylation.     Finally,  we  wanted  to  further  confirm  that  MuSK  regulation  on  the  transcripts   downstream  of  BMP4  did  not  require  MuSK’s  kinase  activity.  To  address  this   question,  we  compared  the  BMP4-­‐induced  Rgs4  responses  of  rescue  cell  lines,  which   were  generated  by  expressing  wild  type  or  mutant  MuSK  (kinase-­‐dead  MuSK  and   MuSK  with  a  point  mutation  in  the  juxta-­‐membrane  Y553)  (Mazhar  et  al.,  2012).   Serum-­‐deprived  cultures  were  treated  with  BMP4  and  Rgs4  transcript  levels  were   analyzed  by  qRT-­‐PCR.  As  expected  from  the  previous  results,  Rgs4  expression  was   not  induced  by  BMP4  in  MuSK  null  myoblasts,  however  wild  type  MuSK  could   rescue  Rgs4  expression  (Figure  2.7b  and  2.7c).  Interestingly,  kinase-­‐dead  MuSK  and   MuSK  with  the  point  mutation  in  the  juxta-­‐membrane  Y533  could  also  rescue  Rgs4   expression  (Figure  2.7d  and  2.7e).  Taken  together,  MuSK  kinase  activity  is   dispensable  for  its  regulation  on  BMP4  pathway.     Discussion   63   Our  data  show  that  the  MuSK  ectodomain  binds  to  BMP2,  4  and  7  with  low   nanomolar  affinities.  Furthermore,  we  demonstrate  that  MuSK  regulates  the   magnitude  of  canonical  BMP4  signaling,  as  judged  by  phospho-­‐SMAD1/5/8  and   induced  Id1  levels  and  is  required  for  the  BMP4-­‐induced  expression  of  a  group  of   genes.  We  propose  that  MuSK  modulates  the  BMP  pathway.  There  are  several   potential  mechanisms  by  which  MuSK  could  regulate  BMP  signaling.  MuSK  may   associate  with  BMP  receptors  in  a  complex  where  BMP  is  bound  both  to  MuSK  and   its  canonical  receptors  (Figure  2.9a).  MuSK  in  such  a  complex  could  serve  as  a   chaperone  for  cell  surface  expression  of  BMP  receptors  and  enhance  the  signaling   by  increasing  cell  surface  expression  of  the  receptors.  MuSK  could  also  regulate  the   assembly  of  BMP  receptors  and  increase  the  signaling.  In  the  same  model,  MuSK   association  with  BMP  receptors  could  also  increase  the  avidity  of  BMPs  for  their   receptor.  Similarly,  MuSK  could  act  as  a  cell  surface  presenter  of  BMPs  to  their   receptors  (Figure  2.9b).  MuSK  intracellular  domain  interacts  with  various  signaling   and  scaffolding  molecules  such  as  Dok7,  Tid1,  Dvl,  Abl,  Src  kinase,  ShcD  (Wu  et  al.,   2010).  Therefore,  association  of  MuSK  with  BMP  receptors  may  bring  such   molecules  in  close  proximity  of  cytosolic  domains  of  BMP  receptors  and  integrate   different  downstream  signaling  events  to  BMP  signaling  (Figure  2.9c).    Alternatively,   BMPs  could  bind  to  MuSK  and  BMP  receptors  separately  and  two  distinct  signaling   events  can  converge  downstream  to  regulate  the  transcriptional  outcome  as  a   response  to  BMP.    While  most  of  these  scenarios  are  not  mutually  exclusive,  they   need  to  be  tested  directly  to  understand  which  ones  could  be  working  together.       64   MuSK  binding  to  BMPs       A  recent  SPR  study  has  shown  that  the  binding  affinity  of  BMP2  to  Alk3  and  Alk6   were  1.1  and  1.6nM,  respectively.  Low  affinity  type-­‐2  receptor,  BMPRII,  showed  a   binding  affinity  of  26.7nM  for  BMP2  (Berasi  et  al.,  2011).  DRAGON,  a  positive   regulatory  BMP  co-­‐receptor,  has  been  shown  to  have  a  binding  affinity  of  1.5nM  for   BMP2  (Samad  et  al.,  2005).  On  the  other  hand,  Neogenin,  a  negative  regulatory  co-­‐ receptor  was  predicted  to  bind  to  BMP2  with  an  affinity  of  25nM  (Hagihara  et  al.,   2011).  Our  results  show  that  BMP  binding  to  MuSK  ectodomain  is  in  the  range  of   high  affinity  type-­‐1  receptor  binding  of  BMPs  and  also  has  a  similar  affinity  as  the   positive  regulatory  DRAGON  co-­‐receptor.       We  also  show  that  Ig3  domain  of  MuSK  is  required  for  its  BMP4  binding.  Previously,   several  extracellular  domains  of  MuSK  have  been  attributed  different  roles.  The  Ig1   domain  is  important  for  dimerization  in  agrin-­‐induced  receptor  activation  and  AchR   clustering  and  biglycan  binding  (Stiegler  et  al.,  2006;  Amenta  et  al.,  2012).  Ig2   domain  is  also  critical  for  agrin-­‐induced  AchR  clustering  (Zhou  et  al.,  1999).  CRD   domain  was  shown  to  be  necessary  for  biglycan  binding  and  has  been  implicated  in   Wnt  binding  (Amenta  et  al.,  2012;  Zhang  et  al.,  2012).    However,  the  function  of  the   third  Ig-­‐like  (Ig3)  domain  has  yet  to  be  shown.  In  addition,  one  of  the  several  MuSK   splice  isoforms  lacks  the  Ig3  domain  (Hesser  et  al.,  1999,  Kuehn  et  al.,  2005).  To  our   knowledge  this  is  the  first  study,  which  shows  a  function  for  the  Ig3  domain.   Furthermore,  it  opens  up  the  possibility  that  MuSK  regulation  of  BMP4  signaling  can   65   be  regulated  by  alternative  splicing,  given  that  there  is  an  alternatively  spliced  ΔIg3-­‐ MuSK  isoform.  It  is  possible  that  in  muscle  tissue,  at  different  stages  of  development   and  in  adult  alternative  splicing  of  MuSK  could  change  the  outcomes  of  the  BMP4   signaling.  Targeted  distribution  of  two  isoforms  in  a  muscle  fiber  can  also  modulate   MuSK-­‐dependent  BMP4  signaling  at  different  compartments  of  muscle  fiber   membrane.  On  the  other  hand,  it  brings  up  the  question  if  lower  levels  of  MuSK   expression  in  other  tissue  types  is  regulated  by  alternative  splicing,  which  would   lead  to  differential  regulation  of  BMP4  pathway  in  these  tissues.  Finally,  the   requirement  of  Ig3  domain  for  BMP4  binding  of  MuSK  distinguishes  this  novel   function  from  MuSK’s  well-­‐known  function  in  agrin  pathway,  as  Ig3  domain  was   shown  to  be  dispensable  for  agrin-­‐MuSK  pathway  leading  to  the  maturation  of  NMJs   (Zhou  et  al.,  1999).     The  receptor  tyrosine  kinase  Ror2  has  a  similar  extracellular  domain  to  MuSK   ectodomain.  It  consists  of  a  single  Ig-­‐like  domain,  followed  by  a  frizzled-­‐domain.   Alk6  interaction  with  Ror2  was  observed  before  (Sammar  et  al.,  2004,  Sammar  et  al.,   2009).  This  interaction  raises  the  question  whether  MuSK  would  also  associate  with   Alk3  or  Alk6  in  myogenic  cells  and  act  as  a  BMP4  co-­‐receptor.  Future  studies  will   reveal  if  the  endogenous  MuSK  and  type-­‐1  receptors  (Alk3  or  Alk6)  in  myogenic   cells  are  interacting.       MuSK  regulation  on  canonical  BMP4  pathway     66   We  show  that  MuSK  positively  regulates  canonical  BMP4  pathway  similar  to   DRAGON  co-­‐receptor.  Future  studies  will  determine  if  MuSK  also  serves  as  a  BMP4   co-­‐receptor  and  what  the  exact  mechanism  is  for  MuSK’s  regulation  on  the   pSMAD1/5/8  levels.  Furthermore,  we  observed  cytosolic  pSMAD1/5/8  granules   being  regulated  by  MuSK.  In  the  absence  of  MuSK,  their  numbers  diminish   drastically.  Interestingly,  the  receptor  Lrp6  has  been  shown  to  induce  cytosolic   pSMAD1  puncta  when  overexpressed  in  Cos7  cells  (Fuentealba  et  al.,  2007).  These   puncta  were  predicted  to  be  Lrp6  signalosomes,  based  on  the  observation  that  they   colocalized  with  the  endogenous  GSK3  protein.  Lrp4,  which  was  shown  to  bind  to   MuSK  (Kim  et  al.,  2008),  is  implicated  to  have  genetic  interactions  with  BMP   pathway  during  tooth  morphogenesis  or  in  the  context  of  bone  properties  and   fracture  (Ohazama  et  al.,  2008;  Kumar  et  al.,  2011).  The  cytosolic  granules  that  we   observed  could  well  be  Lrp4-­‐signalosomes  which  function  as  a  MuSK-­‐dependent   signaling  unit  in  the  cell  and  lead  to  MuSK-­‐dependent  transcriptional  output  of   BMP4  pathway.  In  this  regard,  it  would  be  interesting  to  see  if  Lrp4  associated  with   pSMAD1/5/8  cytosolic  granules  in  myoblasts.       MuSK-­dependence  of  a  subset  of  BMP4  responses     Our  results  show  that  there  are  fundamental  differences  in  transcriptional  outputs   of  myoblasts  and  myotubes  as  a  response  to  BMP4.  MuSK  regulates  these  cell-­‐ context  dependent  responses  in  both  of  these  myogenic  cell  types.  Even  though  we   focused  our  analyses  on  the  upregulated  genes,  there  are  several  downregulated   67   genes  shown  in  Appendix  Tables  4-­‐7  and  MuSK  seems  to  regulate  some  of  these   responses,  as  well.  Further  studies  need  to  be  done  in  order  to  understand  the   importance  of  the  downregulated  genes  for  the  downstream  events.       In  our  expression  studies,  we  identified  several  novel  transcripts  downstream  of   BMP4  signal.  One  of  these  is  Fabp7,  which  was  suggested  as  a  stem  cell  marker  in   neurons  (Yun  et  al.,  2012).  In  our  microarray  analysis,  we  compared  Fabp7   expression  between  myoblasts  and  myotubes.  Myoblasts  had  a  few  fold  higher   expression  of  Fabp7,  which  suggests  that  Fabp7  could  be  a  stem-­‐cell  marker  for   myogenic  cells,  as  well.  This  idea  needs  to  be  tested  further.  Strikingly,  Fabp7  was   the  biggest  hit  in  our  microarray  analyses  for  both  BMP4-­‐treated  myoblasts  and   myotubes  and  it  appeared  to  be  a  MuSK-­‐independent  BMP4  response.       In  myoblasts  we  confirmed  that  Ptgs2,  Ptger4  and  Rgs4  were  regulated  by  MuSK.   Rgs4  expression  by  BMP4  is  strictly  dependent  on  the  presence  of  MuSK  whereas   for  Ptgs2  and  Ptger4  the  presence  of  MuSK  significantly  enhances  the  response.   Ptgs2  and  Ptger4  are  components  of  the  prostaglandin  pathway.  Ptgs2  encodes  for   Cox2  enzyme,  which  is  a  critical  enzyme  in  the  prostaglandin  synthesis  pathway.  On   the  other  hand,  Ptger4  is  a  prostaglandin  receptor.  Cox2  pathway  was  shown  to  be   important  in  myoblast  proliferation,  fusion  and  growth  of  muscle  cells  (Otis  et  al.,   2005;  Bondesen  et  al.,  2006;  Horsley  et  al.,  2003).  Further  studies  will  determine  if   the  Cox2  pathway  is  impaired  in  the  absence  of  MuSK  in  myoblasts.       68   Rgs4  is  a  G-­‐protein  regulator,  which  was  shown  to  inhibit  cell  growth  and   myofilament  organization  in  neonatal  cardiac  myocytes  (Tamirisa  et  al.,  1999).  It   was  also  indicated  to  counteract  the  prostaglandin  pathway,  which  uses  G-­‐protein   coupled  receptors  (Song  et  al.,  2009).  Rgs4  expression  downstream  of  BMP4  could   be  a  negative  feedback  mechanism  against  the  BMP4-­‐induced  expression  of  Ptgs2   and  Ptger4.  Since  both  of  these  prostaglandin  pathway  members  are  regulated  by   MuSK,  regulation  of  BMP4-­‐induced  Rgs4  expression  by  MuSK  would  also  be   expected.  The  same  signaling  modules  could  be  controlling  the  expression  of  these   genes.  We  also  showed  that  BMP4-­‐induced  Rgs4  expression  requires  the  activity  by   type-­‐1  BMP  receptors.  This  result  indicates  that  BMP4  induction  of  Rgs4  may  be   using  the  elements  of  canonical  BMP4  pathway.  However,  the  cytosolic   pSMAD1/5/8  granules  that  are  regulated  by  MuSK  could  be  providing  a  specific   intercellular  signaling  compartment,  which  then  regulates  the  expression  of  MuSK-­‐ dependent  transcripts  like  Rgs4.       One  approach  to  understand  more  about  the  signaling  leading  to  BMP4-­‐induced   MuSK-­‐dependent  Rgs4  expression  would  be  to  generate  a  myogenic  reporter  cell   line  using  Rgs4  promoter  elements.  An  RNAi  screen  in  these  cells  could  yield  in   identification  of  various  signaling  molecules  involved  downstream  of  cell  surface   interaction  between  MuSK  and  BMP4.       We  identified  Myh15  and  Car3  as  novel  BMP4  responses.  Both  of  these  transcripts   were  expressed  by  BMP4  in  a  cell-­‐context  dependent  manner  only  in  myotubes  and   69   this  expression  was  regulated  by  MuSK.  The  importance  of  the  cell-­‐context  may   stem  from  additional  regulatory  factors  of  BMP4  pathway  being  expressed  only  in   myotubes.  The  levels  of  MuSK  in  myotubes  and  myoblasts  could  also  contribute  to   this  observation,  as  MuSK  levels  are  significantly  increased  in  myotubes.   (Valenzuela  et  al.,  1995).       Myh15  was  predicted  to  be  a  type-­‐1  slow  skeletal  muscle  myosin  (Desjardins  et  al.,   2002).  Although  Rossi  et  al.  could  not  detect  any  Myh15  expression  in  skeletal   muscles  (Rossi  et  al.,  2010),  our  expression  studies  showed  that  this  myosin  was   expressed  both  in  skeletal  muscle  tissue  in  animals  and  in  the  cultured  myotubes.   Similar  to  Myh15,  Car3  was  also  shown  to  be  enriched  in  slow  muscle  fibers  (Lyons   et  al.,  1991).  In  adult  mice,  we  showed  that  both  of  these  transcripts  were  present  at   much  higher  levels  in  Soleus  muscle  compared  to  EDL  muscle.  Interestingly,  MuSK   also  shows  the  same  expression  pattern.  Soleus  muscle  is  enriched  with  slow  muscle   fibers.  This  correlation  points  the  possibility  that  MuSK  regulated  and  BMP4-­‐ induced  expression  of  Myh15  and  Car3  could  be  part  of  a  fiber-­‐type  switch  program.   BMPs  have  not  been  indicated  among  several  other  factors  shown  to  induce  fiber-­‐ type  switch  in  muscle  to  date.  Further  studies  focusing  on  longer  BMP4  treatments   and  the  identification  of  downstream  fiber-­‐type  specific  transcripts  will  reveal  if   there  is  a  global  fiber-­‐type  reprogramming  towards  slow  fibers  that  is  induced  and   regulated  by  BMP4  and  MuSK.     MuSK  kinase  activity  and  BMP4  pathway   70     Our  experiments  showed  that  tyrosine  phosphorylation  of  MuSK  is  not  induced  by   BMP4.  This  result  indicates  a  novel  MuSK  pathway  that  is  distinct  from  its  role  in   organizing  the  postsynaptic  apparatus  (Herbst  et  al.,  2000).  Tyrosine  553  at  the   juxtamembrane  domain  and  the  tyrosine  residues  in  the  tyrosine  kinase  domain  of   MuSK  are  necessary  residues  for  agrin-­‐mediated  signaling  of  MuSK  (Figure  2.8b).   However,  these  critical  tyrosines  are  not  required  for  MuSK  regulation  of  BMP4   signaling  (Figure  2.7b-­‐e  and  Figure  2.8a).  This  difference  separates  the  two   functionalities  of  MuSK  and  assigns  a  novel  role  to  MuSK.  On  the  other  hand,  Lrp4   association  of  MuSK  in  the  context  of  agrin-­‐mediated  signaling  raises  the  question  of   whether  Lrp4  also  contributes  to  MuSK  regulation  of  BMP4  signaling  and  if  it  is  in   complex  with  MuSK  in  that  context.     Our  results  show  that  the  receptor  tyrosine  kinase  regulates  BMP4  signaling  in   myogenic  cells.  The  evidence  we  show  here  may  point  to  a  general  mechanism  in   which  other  similar  receptor  tyrosine  kinases  or  surface  molecules  regulate  BMP   signaling  in  other  tissues.  Further  studies  may  also  reveal  if  MuSK  expressed  at   much  lower  levels  in  other  tissues  (i.e.  brain,  Garcia-­‐Osta,  2006)  could  have  a  role  in   BMP  signaling.  Similarly,  these  results  can  potentially  attribute  a  role  to  extra-­‐ synaptic  MuSK  that  is  expressed  in  muscle  fibers,  although  this  has  to  be  tested   more  directly.         Materials  and  Methods   71     Antibodies  and  materials     Purified  recombinant  human  BMP4,  purified  agrin,  anti-­‐BMP4  (1:250),  normal  goat   IgG,  biotinylated  anti-­‐BMP4  (1:500),  anti-­‐MuSK  (1:20  for  IPs  and  1:500  for  western   blots),  anti-­‐Alk3  (1:20  for  IPs  and  1:500  for  western  blots),  anti-­‐Alk6  (1:20  for  IPs   and  1:500  for  western  blots)  antibodies  were  obtained  from  R&D  Systems   (Minneapolis,  MN,  USA).  Stretavidin-­‐HRP  antibody  (1:2000)  was  obtained  from   Thermo.  Anti-­‐mouse  HRP  antibody  (1:2000)  was  obtained  from  KPL.  Anti-­‐SMAD5   (1:1000)  and  anti-­‐phosphoSMAD5  (1:1000  for  western  blots,  1:200  for   immunocytochemistry)  antibodies  were  obtained  from  Epitomics  (Burlingame,  CA,   USA).  Anti-­‐phosphotyrosine  (4G10)  antibody  was  obtained  from  EMD  Millipore   Corporation  (Billerica,  MA,  USA).    Alexa-­‐555-­‐conjugated  goat  anti-­‐rabbit  IgG   (1:1000)  was  obtained  from  Invitrogen.  Expression  vector  containing  His-­‐tagged   MuSK  ectodomain  was  a  gift  from  Dr.  Markus  Ruegg.  His-­‐tagged  TEV  protease  was  a   gift  from  Dr.  Rebecca  Page.  Full  length  and  Ig3-­‐lacking  Fc-­‐Fusion  MuSK  ectodomain   constructs  were  obtained  from  GenScript  (Piscataway,  NJ,  USA).  LDN192189  was   obtained  from  STEMGENT  (Cambridge,  MA,  USA).       Mammalian  cell  culture  and  mice                                 Mouse  C2C12  BRE  cells  (Zilberberg  et  al.,  2007)  were  cultured  in  DMEM   supplemented  with  10%  fetal  bovine  serum,  2%  L-­‐Glutamine  and  1%  Penicillin-­‐ 72   Streptomyocin  and  cultured  at  37  0C  in  8%  CO2.  Immortalized  myoblast  cultures  of   wild-­‐type  mouse  H-­‐2Kb-­‐tsA58  (Morgan  JE  et  al.,  1994),  MuSK-­‐/-­‐  and  MuSK  rescue   lines  (wild  type  MuSK  (B1),  kinase-­‐dead  MuSK  (KD)  and  MuSK  Y553A)  were   cultured  were  cultured  on  gelatin-­‐coated  dishes  in  DMEM  supplemented  with  20%   fetal  bovine  serum,  2%  L-­‐Glutamine,  1%  Penicillin-­‐Streptomyocin,  1%  Chicken   Embryo  Extract,  1U  interferon-­‐γ  and  cultured  under  permissive  temperature  at  33   0C  in  8%  CO2.  Myotubes  were  obtained  by  switching  the  confluent  myoblast   cultures  to  a  medium  with  DMEM  supplemented  with  5%  horse  serum,  2%  L-­‐ Glutamine  and  1%  Penicillin-­‐Streptomyocin  at  37  0C  in  8%  CO2.     Luciferase  reporter  assays     C2C12BRA  cells  were  plated  on  96-­‐well  culture  dishes  at  4-­‐5  x  103  cells/well.  The   cells  were  allowed  to  attach  overnight.  The  indicated  recombinant  proteins  were   premixed  in  DMEM  containing  0.1%BSA  for  20  minutes  at  4  0Cand  the  medium  was   replaced  with  this  solution.  The  cells  were  treated  for  8  hours,  washed  twice  with   PBS  and  cell  extracts  were  prepared  with  50μl/well  1X  cell  lysis  buffer  (Roche).    40   μl  of  the  lysate  was  transferred  to  an  opaque  white  96-­‐well  microplate  and  mixed   with  100  μl  of  Luciferase  substrate  (Roche).  The  luciferase  activity  was  read  in  a   luminometer  and  reported  as  relative  luciferase  unit  (RLU),  which  is  the  value  of   each  condition  after  subtraction  of  mock-­‐treatment  (no-­‐BMP4)  value  and   normalization  to  BMP4-­‐only  condition.  All  assays  were  performed  in  8  replicates   and  repeated  at  least  two  times  with  similar  results.   73     For  testing  the  remaining  activity  in  the  solution  binding  experiments,  the  indicated   recombinant  proteins  were  premixed  in  DMEM  containing  0.1%BSA  for  2  hours  at  4   0C.  After  the  addition  of  the  magnetic  Nickel-­‐bound  beads  (Promega)  into  the   solution,  they  were  incubated  in  the  same  solution  for  another  2  hours  at  4  0C.  The   same  protocol  detailed  above  was  followed  after  this  step  to  test  the  BMP4  activity.     Immunoprecipitation  (IP)  and  co-­immunoprecipitation  (co-­IP)     For  MuSK  phosphorylation  IP,  H-­‐2Kb-­‐tsA58  myotubes  were  lysed  in  extraction   buffer  (10mM  Tris-­‐HCl  (pH  7.4),  1%  Triton-­‐X  100,  0.5%  NP40,  150mM  NaCl,  1mM   EGTA,  1mM  EDTA,  1mM  sodium  orthovanadate,  10mM  sodium  fluoride  and  1X   EDTA-­‐free  protease  inhibitor  cocktail  (Roche  Complete))  after  treatments  with   indicated  proteins.  Lysates  were  pre-­‐cleared  with  Protein-­‐G  bound  magnetic  beads   (Invitrogen)  and  bicinchoninic  acid  (BCA)  assay  was  used  to  quantify  total  protein   levels  in  the  lysates  (Pierce).  Equal  amounts  of  lysates  were  mixed  with  equal   amounts  of  the  indicated  IP/co-­‐IP  antibodies  and  tumbled  overnight  at  4  0C.  After   the  addition  of  Protein-­‐G  bound  magnetic  beads  lysates  were  tumbled  for  4-­‐6  hours   at  4  0C.  Beads  were  washed  with  extraction  buffer  for  3  times.  2X  sample  buffer  was   added  for  elution  of  the  immunoprecipitated  proteins  from  the  beads.  Proteins  were   eluted  by  boiling  the  samples  at  98  0C  for  5  minutes.  Western  blots  were  run  for  the   samples  as  indicated  below.     74   The  extraction  and  bead  washing  buffer  used  for  co-­‐IPs  was  as  follows:  10mM  Tris-­‐ HCl  (pH  7.4),  1%  Triton-­‐X  100,  150mM  NaCl,  1mM  EGTA,  1mM  EDTA,  1mM  sodium   orthovanadate,  10mM  sodium  fluoride  and  1X  EDTA-­‐free  protease  inhibitor  cocktail   (Roche  Complete).     Western  blots       For  pSMAD1/5/8  westerns,  cell  lysates  were  prepared  in  extraction  buffer   containing  10mM  Tris-­‐HCl  (pH  7.4),  1%  Triton-­‐X  100,  0.5%  NP40,  150mM  NaCl,   1mM  EGTA,  1mM  EDTA,  1mM  sodium  orthovanadate,  10mM  sodium  fluoride  and  1X   EDTA-­‐free  protease  inhibitor  cocktail  (Roche  Complete).  Cells  were  serum-­‐deprived   in  DMEM  containing  0.1%  BSA  for  5-­‐6  hours  and  then  treated  with  indicated   amounts  of  BMP4  for  15  minutes.  Cells  were  washed  in  PBS  three  times,  incubated   in  the  extraction  buffer  for  30  minutes  at  4  0C  and  the  lysates  were  cleared  by   centrifugation  at  13k  rpm  for  10  minutes  ar  4  0C.  Protein  quantification  of  the   samples  was  assessed  by  BCA  (Pierce).  Equal  amounts  of  protein  were  run  in  5%– 15%  gradient  SDS-­‐PAGE  gels  and  immunoblotted  with  pSMAD1/5/8  antibody   (Antiphosphotyrosine  antibody  for  phospho-­‐MuSK  IPs;  Alk3,  Alk6,  MuSK  antibody   or  normal  goat  IgG  for  co-­‐IPs).  Membranes  were  then  stripped  and  reprobed  with   SMAD1/5/8  antibody  (MuSK  antibody  for  phospho-­‐MuSK  IP).     ELISAs     75   For  MuSK  binding  to  BMP4,  MuSK  constructs  were  immobilized  on  96-­‐well  plates  at   2µg/ml  overnight.  Plates  were  blocked  with  1%  BSA  in  PBS.  BMP4  (0-­‐200nM)  was   incubated  with  immobilized  MuSK.  Bound  BMP4  was  detected  with  biotinylated   anti-­‐BMP4  antibody  (R&D)  followed  by  Streptavidin-­‐conjugated  HRP  (Thermo).   Color  change  in  chromogenic  substrate  3,3′,5,5′-­‐Tetramethylbenzidine  (TMB)  as  a   result  of  HRP  activity  was  measured  with  spectrophotometer  at  450nm  wavelength.   Graphs  are  generated  with  absorbance  values.  Each  data  point  represents  the   average  of  4  replicate  wells.  For  MuSK  binding  to  biglycan,  MuSK  constructs  were   immobilized  and  His-­‐tagged  non-­‐glycanated  biglycan  was  incubated  with   immobilized  MuSK.  Bound  biglycan  was  detected  with  anti-­‐His  antibody  followed  by   HRP-­‐conjugated  anti-­‐mouse  secondary  antibody.     Immunocytochemistry     12  hours  after  myoblast  cultures  were  fed  with  their  growth  medium,  cells  were   washed  with  PBS  three  times  and  fixed  in  4%  paraformaldehyde  in  PBS  for  15   minutes  at  room  temperature.  Cells  were  then  permeabilized  with  0.2%  Triton-­‐X   100  in  PBS  containing  1%  BSA  for  10  minutes,  blocked  in  PBS  containing  1%  BSA   and  0.1%  Triton-­‐X  100  for  10  minutes,  incubated  with  primary  antibody  (anti-­‐ pSMAD1/5/8)  in  PBS  containing  1%  BSA  and  0.1%  Triton-­‐X  100  for  1  hour  at  room   temperature.  After  3  washes  in  PBS,  cell  were  incubated  with  secondary  antibody   Alexa-­‐555-­‐conjugated  goat  anti-­‐rabbit  IgG  for  1  hour  at  room  temperature.  After   three  PBS  washes,  cells  were  fixed  again  in  cold  methanol  at  -­‐20  0C  for  5  minutes.   76   Mounting  medium  with  DAPI    was  used  to  visualize  the  nuclei.  Protein  localization   was  examined  by  laser-­‐scanning  confocal  microscopy.     RNA  extraction,  reverse  transcription  and  quantitative  real  time  polymerase   chain  reaction  (qRT-­PCR)     Total  RNA  was  isolated  from  cells  with  Trizol  (Invitrogen).  Total  RNA  was  cleaned   up  and  DNase-­‐treated  in  Qiagen  RNeasy  columns.  RNA  was  reverse  transcribed  into   first  strand  cDNA  (Invitrogen).  qRT-­‐PCR  reaction  consisted  of  initial  incubation  at   50  0C  for  2  minutes  and  a  denaturation  at  95  0C  for  5  minutes.  The  cycling   parameters  were  as  follows:  95  0C  for  15  seconds,  60  0C  for  30  seconds.  After  40   cycles,  the  reactions  underwent  a  final  dissociation  cycle  as  follows:  95  0C  for  15   seconds,  60  0C  for  1  minute,  95  0C  15  seconds  and  60  0C  for  15  seconds.   Based  on  the  published  sequences,  the  primer  sequences  used  in  qRT-­‐PCR  reactions   were  as  follows:  5'-­‐  GGGATCTCTGGGAAAGACAC  -­‐3'  and  5'-­‐  TCTCTGGAGGCTGAAAGGTG  -­‐3'   for  mouse  Id1;  5'-­‐  GCCTTTTCACAAAGGTGGAG  -­‐3'  and  5'-­‐  CAGCATTCAGTAGGCTCGTG  -­‐3'  for   mouse  Id2;  5'-­‐  GTATTTCCATCGCTCCTTGG  -­‐3'  and  5'-­‐  TGAGGCCTATAAAGCACATGG  -­‐3'  for   mouse  Rgs4;  5'-­‐  TCTTCGGGCAAGAAACTCTG  -­‐3'  and  5'-­‐  TTGCATGTGACTGCTTCTCC  -­‐3'  for   mouse  Car3;  5'-­‐  CAGGCACACTTCTCCTTTCC-­‐3'  and  5'-­‐  CCTTCCTCATCATGGACCAG  -­‐3'  for   mouse  Myh15;  5'-­‐  -­‐3'  and  5'-­‐  -­‐3'  for  mouse  MuSK;  5'-­‐  TCCTCTCTGTTGCGTGTGTC  -­‐3'  and  5'-­‐   CGTTAAGCAACAGGACATGC  -­‐3'  for  mouse  Ptger4;  5'-­‐  CGCTGATTGGGTTTTCGTAG-­‐3'  and  5'-­‐ CCTGAGCTGAGGTTTTCCTG-­‐3'  for  mouse  Ptgs2;  5'-­‐  CTTTGGGGATATCGTTGCTG  -­‐3'  and  5'-­‐ GCTGGCTAACTCTGGACTC  -­‐3'  for  mouse  Fabp7.     77   Microarrays  and  bioinformatics  analysis     RNA  was  harvested  from  myoblast  and  myotube  cultures  of  wild  type  and  MuSK  null   cells  after  8  hours  of  BMP4  treatment.  150-­‐200  ng  total  RNA  of  good  quality  RNA   (bioanalyzer  RIN  scores  of  >9  )  were  used  as  input  material  for  all  arrays.  Total  RNA   was  converted  to  double-­‐stranded  cDNA  and  then  in-­‐vitro  transcribed  overnight   using  the  WT  expression  kit  from  Invitrogen  (cat  #  4411981).  After  cleanup  10  µg   IVT-­‐cRNA  was  converted  to  dUTP  labeled  cDNA  and  5.5  µg  of  the  generated  single   stranded  cDNA  was  enzymatically  fragmented  followed  by  TdT  mediated  biotin  end   labeling  using  Affymterix  WT  terminal  labeling  kit  (cat  #  900670).  The   fragmentation  resulted  in  DNA  fragments  with  a  distribution  peak  at  approximately   75  nucleotides  and  successful  fragmentation  was  demonstrated  on  the  bioanalyzer   with  RIN  scores  of  2.6.  Approximately  2.5  µg  of  cDNA  was  hybridizied  over  night  at   45  0C  and  60  rpm  to  Affymterix  Mouse  1.0  Gene  ST(cat  #  901168).  The  arrays  were   washed  and  stained  following  Affymetrix  standard  protocol  using  GeneChip®   Hybridization  Wash  and  Stain  Kit  (cat  #  900720)  and  subsequently  scanned  on  a   Affymterix  3000  7G  scanner.     Affymetrix  Expression  console  was  used  to  analyze  the  overall  performance  and   quality  of  the  arrays  and  Partek  Genomics  Suite  was  used  to  detect  differentially   expressed  genes.  To  call  genes  differentially  expressed  between  samples  we  used   false  discovery  rate  (FDR)  as  selection  criteria  or  unadjusted  p-­‐values  of  0.05.     Surface  plasmon  resonance  experiments   78     The   binding   affinities   and   kinetic   parameters   between   BMPs   and   MuSK   were   determined   by   SPR   spectroscopy   using   the   BIAcore3000   optical   biosensor   instrument   (GE   Healthcare   Lifescience).     The   carboxymethylated   surface   of   the   sensor   chip   (CM5)   was   activated   with   N-­‐hydroxysuccinimide   (NHS)   and   1-­‐ethyl-­‐3-­‐ (3-­‐dimethylaminopropyl)carbodiimide   hydrochloride   (EDC).     The   CM5   chip   contains  four  flow  cells  and  among  these  four  cells,  three  were  used  for  the  assay.   Flow  cell  1  was  used  as  a  control  surface,  whereas  flow  cells  2,  3  and  4  were  used  as   test  surfaces.    Recombinant  human  BMP2  [2466  response  units  (RU)],  BMP4  (2108   RU)   and   BMP7   (2050   RU)   were   covalently   coupled   in   flow   cells   2,   3   and   4   respectively.   Unreacted   active   ester   groups   were   blocked   with   1M   ethanolamine   hydrochloride,  pH8.5.    The  control  flow  cell  1  was  treated  in  an  identical  manner  but   without  coupling  protein.    The  binding  assays  were  carried  out  at  25  °C  in  20  mM   Hepes   buffer   (pH   7.5)   500   mM   NaCl,   3.4   mM   EDTA,   and   0.005%   surfactant   P-­‐20.     Various   concentrations   of   MuSK   were   applied   over   the   biosensor   chip   at   a   flow   rate   of  20  μl/min  for  360  s  to  measure  the  association  phase  followed  by  buffer  only  for   600  s  to  measure  the  dissociation  phase.  The  sensor  chip  was  regenerated  with  four   short   pulses   of   2M   guanidine   hydrochloride   at   100   μl/min.     Data   were   evaluated   using   the   software   BIAevaluation   4.1.1   (BIAcore   AB).     SPR   sensorgrams   were   globally   analyzed   using   a   distribution   model   for   continuous   affinity   and   rate   constant  analysis  with  the  program  EVILFIT  (Svitel  et  al.,  2003)     Statistical  analysis   79     All  statistical  analyses  used  Student's  t  test  unless  otherwise  noted.     Figures   Figure  2.1             80     FIGURE  2.1       MuSK  ectodomain  binds  to  BMP4.    A.  Schematic  representation  of  MuSK   ectodomain  construct  used  in  the  reporter  and  the  solution-­‐binding  assays.  The   construct  contains  three  Ig-­‐like  domains  (Ig1,  Ig2  and  Ig3)  and  cycteine-­‐rich  domain   (CRD)  of  ecto-­‐MuSK,  followed  by  a  His-­‐tag  at  the  C-­‐terminus.  B.  Soluble  MuSK   ectodomain  inhibits  BMP4  activity.  BMP4  (45pM)  was  incubated  with  the  indicated   concentrations  of  MuSK  ectodomain  or  an  irrelevant  his-­‐tagged  protein  and  then   added  to  cultured  C2C12BRA  cells  for  8  hours.  Cells  were  then  lysed  and  luciferase   activity  was  determined.  The  average  value  of  untreated  cells  was  subtracted  from   each  condition  and  everything  was  normalized  to  BMP4-­‐only  condition  (100%   activity).  Each  bar  represents  the  average  value  from  8  replicate  cultures.  C.  MuSK   ectodomain  depletes  BMP4  from  solution.  Soluble  BMP4  and  MuSK  ectodomain   were  mixed  for  2  hours  at  4  0C  followed  by  addition  of  magnetic  nickel-­‐beads  for  2   hours.  After  removal  of  the  beads  BMP4  activity  in  the  supernatants  was  measured   using  the  C2C12BRA  reporter  cell  line.  D.  BMP4  co-­‐precipitates  with  MuSK   ectodomain.  MuSK  ectodomain  and  irrelevant  his-­‐tagged  protein  were  eluted  from   the  beads  that  were  removed  from  the  supernatant  in  C.  Co-­‐precipitated  BMP4  in   the  elutes  was  detected  with  an  ELISA.  The  graph  shows  the  BMP4  amount  (pmoles)   pulled  down  with  MuSK  ectodomain  or  his-­‐tagged  control.       81   FIGURE  2.2             82   FIGURE  2.2       SPR  binding  analysis  of  MuSK  with  BMPs.    A-­‐C,  MuSK  binding  to  BMP2,  BMP4  and   BMP7  immobilized  on  a  biosensor  chip.  Representative  SPR  profiles  are  shown  for   various  concentrations  of  MuSK  binding  to  BMP2  (A);  BMP4  (B)  and  BMP7  (C).   Sensograms  were  normalized  for  MuSK  binding  to  a  mock-­‐coupled  flow  cell.  The   black  lines  show  the  experimental  measurements  of  a  two-­‐fold  serial  dilution  over   the  concentration  range  [2  mM-­‐1.96  nM]  of  each  sensorgram  and  the  red  lines   correspond  to  global  fits  of  the  data  to  a  1:1  model  using  a  heterogeneous  surface   model  with  the  program  EVILFIT.                             83   FIGURE  2.3                               84   FIGURE  2.3     The  MuSK  Ig3  domain  is  required  for  BMP4  binding.  A.  Schematic  of  Fc-­‐fusion   full  length  (FL)  and  Ig3-­‐lacking  (ΔIg3)  MuSK  ectodomain  constructs  used  in  the   binding  ELISA.  Both  of  the  constructs  have  an  Fc-­‐domain  at  their  C-­‐termini.  B.  The   MuSK  Ig3  domain  is  required  for  BMP4  binding.  FL  and  ΔIg3  MuSK  ectodomain   fusions  were  immobilized  on  96-­‐well  plates  and  then  incubated  with  BMP4  (0-­‐ 200nM).  Bound  BMP4  was  detected  with  biotinylated  anti-­‐BMP4  antibody  and   Streptavidin-­‐conjugated  HRP.  Note  the  saturable  binding  observed  with   immobilized  FL  MuSK  ectodomain,  while  only  non-­‐specific  binding  was  observed   with  ΔIg3  MuSK.  C.  ELISA  detection  of  the  levels  of  immobilized  FL  and  ΔIg3  MuSK   ectodomain  constructs.  Control  experiments  showed  that  equivalent  amounts  of  FL   and  ΔIg3  MuSK  ectodomain  fusions  were  immobilized  on  the  plastic.  D.  MuSK   binding  to  biglycan  was  not  affected  by  the  deletion  of  Ig3  domain  of  MuSK.  FL  and   ΔIg3  MuSK  ectodomain  fusions  were  immobilized  on  96-­‐well  plates  and  then   incubated  with  biglycan.  Bound  His-­‐tagged  biglycan  was  detected  with  anti-­‐His  and   HRP-­‐conjugated  anti-­‐mouse  secondary  antibodies.  FL  and  ΔIg3  MuSK  ectodomain   fusions  bind  to  biglycan  at  comparable  levels.             85   FIGURE  2.4                     86   FIGURE  2.4     MuSK  regulates  the  canonical  BMP4  pathway.  A.  Phospho-­‐SMAD1/5/8   (pSMAD1/5/8)  induction  is  reduced  in  the  absence  of  MuSK.  Wild  type  H-­‐2Kb-­‐tsA58   and  MuSK  null  myoblasts  were  serum  deprived  for  5-­‐6  hours  and  treated  with   BMP4  at  the  indicated  concentrations  for  15  minutes.  Cells  were  lysed  and   pSMAD1/5/8  levels  were  detected  by  Western  blotting.  B.  Quantification  of   pSMAD1/5/8  induction  in  wild  type  and  MuSK  null  myoblasts.  Fold  increase  for   each  BMP4  concentration  is  shown  on  line  graphs.  C.  BMP4-­‐induced  Id1  expression   decreases  in  the  absence  of  MuSK.  Wild  type  H-­‐2Kb-­‐tsA58  and  MuSK  null  myoblasts   were  serum-­‐deprived  for  5-­‐6  hours  and  treated  with  3.25ng/ml  BMP4  for  2  hours.   Id1  transcript  levels  were  measured  by  qRT-­‐PCR.  Each  bar  represents  at  least  n=3.   Student’s  t-­‐test  was  used  for  assessing  the  statistical  significance.  (p<  0.001)  D.   Cytosolic  pSMAD1/5/8  granules  are  reduced  in  the  absence  of  MuSK.    E.   Quantification  of  the  percentage  of  wild  type  H-­‐2Kb-­‐tsA58  and  MuSK  null  myoblasts   expressing  cytosolic  pSMAD1/5/8  granules  at  detectable  levels.  (n=3,  p<  0.05)                 FIGURE  2.5   87     88     FIGURE  2.5     MuSK  selectively  regulates  BMP4-­induced  expression  of  a  subset  of  genes  in   myoblasts.  A.  MuSK  modulates  transcriptional  output  of  BMP4  in  myobasts.  Wild   type  H-­‐2Kb-­‐tsA58  and  MuSK  null  myoblasts  were  serum  deprived  for  4  hours  and   treated  with  25ng/ml  BMP4  for  8  hours.  RNA  was  isolated,  reverse-­‐transcribed  into   double  stranded  cDNA.  cRNAs  were  synthesized  from  cDNA  templates  and   hybridized  to  Affymetrix  chips  after  fragmentation.  Analysis  of  the  array  data  was   done  with  Partek  Genomics  Suite  software  with  an  FDR  filter.  BMP4  responses  for   upregulated  genes  in  wild  type  and  MuSK  null  myoblasts  are  grouped  into  a  Venn   diagram  as  wild  type  only,  shared  and  MuSK  null  only  responses.  (n=3)  B-­‐E.   Validation  of  microarray  results  for  a  group  of  genes.  A  separate  experiment  under   the  same  conditions  of  microarray  samples  was  performed.  RNA  was  harvested  and   reverse  transcribed  into  cDNA.  Transcript  levels  for  the  shared  responses  of  (B)  Id1,   (C)  Id2  and  (D)  Fabp7  and  wild  type  only  responses  of  (E)  Ptgs2,  (F)  Ptger4  and  (G)   Rgs4  were  measured  by  qRT-­‐PCR.  (n=3)  H.  Inhibition  of  BMP  type-­‐1  receptors  also   inhibits  the  BMP4-­‐induced  Rgs4  expression.  Wild  type  H-­‐2Kb-­‐tsA58  myoblasts  were   serum  deprived  for  5-­‐6  hours  and  treated  with  25ng/ml  BMP4  for  2  hours.  For  the   conditions  with  LDN193189,  cells  were  treated  with  the  drug  (50nM)  for  30   minutes  prior  to  BMP4  treatment  and  the  drug  was  kept  in  cultures  during  the   course  of  the  treatment.  RNA  was  isolated,  reverse-­‐transcribed  into  double  stranded   cDNA.  Rgs4  transcript  levels  were  measured  by  qRT-­‐PCR.  (n=3)   89   FIGURE  2.6                   90     FIGURE  2.6     MuSK  selectively  regulates  BMP4-­induced  expression  of  a  subset  of  genes  in   myotubes.  A.  MuSK  modulates  transcriptional  response  of  BMP4  in  myotubes.  Wild   type  H-­‐2Kb-­‐tsA58  and  MuSK  null  myoblasts  were  grown  into  confluence  and   differentiated  into  myotubes  for  3  days.  Myotube  cultures  were  treated  with   25ng/ml  BMP4  for  8  hours.  RNA  was  isolated,  reverse-­‐transcribed  into  double   stranded  cDNA.  cRNAs  were  synthesized  from  cDNA  templates  and  hybridized  to   Affymetrix  chips  after  fragmentation.  Analysis  of  the  array  data  was  done  with   Partek  Genomics  Suite  software  with  an  FDR  filter.  BMP4  responses  for  upregulated   genes  in  wild  type  and  MuSK  null  myotubes  are  grouped  into  a  Venn  diagram  as   wild  type  only,  shared  and  MuSK  null  only  responses.  (n=3)  B-­‐C.  Validation  of   microarray  results.  A  separate  experiment  under  the  same  conditions  of  microarray   samples  was  performed.  RNA  was  harvested  and  reverse  transcribed  into  cDNA.   Transcript  levels  of  (B)  Myh15  and  (C)  Car3  were  analyzed  in  myoblasts  and   myotubes  by  qRT-­‐PCR.  (n=3)  D-­‐F.  MuSK,  Myh15  and  Car3  expression  is  higher  in   Soleus  muscles  compared  to  EDL  muscles.  Soleus  and  EDL  muscles  were  harvested   from  5.5  month  old  C57Bl6  mice.  RNA  was  isolated,  reverse  transcribed  into  cDNA.   Transcript  levels  of  (D)  MuSK,  (E)  Myh15  and  (F)  Car3  were  analyzed  by  qRT-­‐PCR.   (n=5)       91   FIGURE  2.7                               92   FIGURE  2.7     BMP4  does  not  induce  MuSK  phosphorylation.  A.  Wild  type  H-­‐2Kb-­‐tsA58   myoblasts  were  grown  into  confluence  and  differentiated  into  myotubes  for  3  days.   Myotube  cultures  were  treated  with  1ng/ml  agrin  for  1  hour  or  with  25ng/ml  BMP4   for  10  minutes,  1  hour,  2  or  3  hours.  The  myotubes  were  lysed  and  MuSK  was   immunoprecipitated.  Elutes  were  run  in  an  SDS-­‐PAGE  and  immunoblotted  with  an   anti-­‐phosphotyrosine  antibody  (upper  panel).  The  blots  were  stripped  and   reprobed  with  anti-­‐MuSK  antibody  to  assess  the  total  levels  of  MuSK  in  each   condition  (lower  panel).    B-­‐E.  BMP4-­‐induced  Rgs4  expression  is  rescued  by  wild   type,  kinase  dead  and  tyrosine  553  mutant  MuSK.  A)  MuSK  null,  B)  wild  type  MuSK   expressing,  C)  kinase  dead  MuSK  expressing,  D)  tyrosine  553  mutant  (Y553F)  MuSK   expressing  MuSK  null  myoblasts  were  serum-­‐deprived  for  5-­‐6  hours  and  treated   with  3.25ng/ml  BMP4  for  2.5  hours.  Rgs4  transcript  levels  were  measured  by  qRT-­‐ PCR.  Each  bar  represents  at  least  n=3.                   93     FIGURE  2.8             FIGURE  2.8.  Differences 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 no.  47  (November  25,  2011):  40624– 40630.                      Zhang,  Bin,  Chuan  Liang,  Ryan  Bates,  Yiming  Yin,  Wen-­‐Cheng  Xiong,  and  Lin  Mei.   “Wnt  Proteins  Regulate  Acetylcholine  Receptor  Clustering  in  Muscle  Cells.”   Molecular  Brain  5  (2012):  7.                    Zhu,  Dan,  Zhihua  Yang,  Zhenge  Luo,  Shiwen  Luo,  Wen  C  Xiong,  and  Lin  Mei.  “Muscle-­‐ Specific  Receptor  Tyrosine  Kinase  Endocytosis  in  Acetylcholine  Receptor  Clustering   in  Response  to  Agrin.”  The  Journal  of  Neuroscience  28,  no.  7  (February  13,  2008):   1688–1696.                    Zhou,  H,  D  J  Glass,  G  D  Yancopoulos,  and  J  R  Sanes.  “Distinct  Domains  of  MuSK   Mediate  Its  Abilities  to  Induce  and  to  Associate  with  Postsynaptic  Specializations.”   The  Journal  of  Cell  Biology  146,  no.  5  (September  6,  1999):  1133–1146.     Zilberberg,  Lior,  Peter  ten  Dijke,  Lynn  Y  Sakai,  and  Daniel  B  Rifkin.  “A  Rapid  and   Sensitive  Bioassay  to  Measure  Bone  Morphogenetic  Protein  Activity.”  BMC  Cell   Biology  8  (2007):  41.                    Zimmerman,  L  B,  J  M  De  Jesús-­‐Escobar,  and  R  M  Harland.  “The  Spemann  Organizer   Signal  Noggin  Binds  and  Inactivates  Bone  Morphogenetic  Protein  4.”  Cell  86,  no.  4   (August  23,  1996):  599–606.                    Zong,  Yinong,  Bin  Zhang,  Shenyan  Gu,  Kwangkook  Lee,  Jie  Zhou,  Guorui  Yao,  Dwight   Figueiredo,  Kay  Perry,  Lin  Mei,  and  Rongsheng  Jin.  “Structural  Basis  of  agrin-­‐LRP4-­‐ MuSK  Signaling.”  Genes  &  Development  26,  no.  3  (February  1,  2012):  247–258.             103         Supplemental  Material       Supplementary  Figure  2.1           104               Supplementary  Figure  2.1.  BMP4  induced  Rgs4  expression  at  an  earlier  time-­‐ point.  Wild  type  H-­‐2Kb-­‐tsA58  and  MuSK  null  myoblasts  were  serum  deprived  for  5-­‐ 6  hours  and  treated  with  3.25ng/ml  BMP4  for  2  hours.  RNA  was  isolated,  reverse-­‐ transcribed  into  double  stranded  cDNA.  Rgs4  transcript  levels  were  measured  by   qRT-­‐PCR.  (n=3)     Supplementary  Table  2.1     Fold-­‐ Gene  Symbol   Change   p-­‐value   Rgs4   5.70   0.0000143   Msx2   4.13   0.0000505   Avpr1a   4.01   0.0001288   2310002L13Rik   3.78   0.0005764   Kazald1   3.42   0.0000567   Foxc2   2.92   0.0000421   105   Sema6d   2.88   0.0000498   Lgr4   2.66   0.0000446   Ptx3   2.62   0.0000194   Serpinb2   2.62   0.0001340   Ptger4   2.56   0.0000180   Timp3   2.51   0.0007816   Nog   2.51   0.0000139   Pcp4l1   2.44   0.0002664   Orai2   2.43   0.0002012   Slc5a7   2.40   0.0003592   Orai2   2.38   0.0009485   Gprin3   2.38   0.0001323   Plcb1   2.33   0.0000312   Slc25a13   2.32   0.0001249   Snai2   2.24   0.0000299   Inhba   2.23   0.0000116   Prr9   2.22   0.0008294   Lef1   2.21   0.0006123   Insc   2.17   0.0000814   Hoxc13   2.11   0.0008357   Asphd2   2.09   0.0004314   Tiam2   2.08   0.0002241   106   Rasl11a   2.07   0.0002635   Ptch1   2.05   0.0004419   Itgb1bp3   2.02   0.0002875   Dio2   2.02   0.0001115   Tmem56   2.01   0.0000034   Erc1   2.00   0.0000768   Dlx3   2.00   0.0000510   Trps1   1.98   0.0002073   Hoxc11   1.98   0.0000027   Prr5l   1.96   0.0004528   Kcnf1   1.96   0.0002341   Myf5   1.96   0.0007566   Pappa   1.94   0.0010591   Skil   1.93   0.0000764   Cpne4   1.93   0.0000746   A630033H20Rik   1.89   0.0004233   Thsd7a   1.89   0.0000377   Pdk4   1.89   0.0001955   Vasn   1.89   0.0004233   Lpl   1.88   0.0004933   Rarres1   1.87   0.0001771   Ttc39b   1.87   0.0000794   107   Ier3   1.87   0.0001215   Tm4sf1   1.86   0.0000067   Hoxa11   1.86   0.0001793   Fzd1   1.84   0.0001878   Has2   1.84   0.0000313   Car8   1.83   0.0010320   Htr1b   1.82   0.0001701   Erc1   1.82   0.0001232   Pcdh7   1.82   0.0000127   Pcolce2   1.82   0.0004945   Ptpru   1.82   0.0007651   Mab21l1   1.82   0.0001930   Smoc1   1.80   0.0000532   Fzd7   1.80   0.0004557   Papss2   1.78   0.0005405   Col10a1   1.78   0.0004565   Tmeff1   1.76   0.0000932   Ssfa2   1.75   0.0006513   Junb   1.74   0.0000945   Gcnt4   1.74   0.0000814   Prss35   1.73   0.0000027   Itgb8   1.73   0.0000536   108   2900062L11Rik   1.73   0.0010198   Klf10   1.72   0.0004308   Rpgrip1l   1.71   0.0003224   9330159F19Rik   1.71   0.0010531   Ccdc34   1.70   0.0001323   Smpdl3a   1.70   0.0000142   Flrt2   1.67   0.0000579   Jak2   1.67   0.0000748   Pi15   1.66   0.0010803   Cpm   1.65   0.0010810   Il17ra   1.64   0.0000204   Cldn1   1.63   0.0008935   Mpp2   1.62   0.0004449   Ppap2b   1.61   0.0003542   Alpk1   1.61   0.0001941       1.61   0.0007314   Grb14   1.60   0.0002481       1.60   0.0007166   Tmeff2   1.60   0.0000023   Clcf1   1.60   0.0000406   Epha7   1.60   0.0005076       1.60   0.0010815   109   Lrrc8c   1.59   0.0000875   Podnl1   1.59   0.0002083   Rusc2   1.59   0.0010403   Rhou   1.59   0.0005517   Jhdm1d   1.58   0.0001653   Cxcr4   1.58   0.0002609   Irx5   1.57   0.0003214   Mtmr11   1.56   0.0004627   Ephb2   1.56   0.0005772   Fmn2   1.56   0.0004682   Sh3bp4   1.56   0.0009683   Egr2   1.56   0.0004437   Asap1   1.56   0.0000804   Kremen1   1.55   0.0000011   Fam135a   1.55   0.0000553   Twist2   1.55   0.0002147   Syn1   1.55   0.0003857   Dok4   1.54   0.0004804   Mapkapk3   1.54   0.0002218   Zfp462   1.54   0.0001355   Angptl2   1.53   0.0002387   Tsc22d1   1.53   0.0000225   110   Cenpp   1.53   0.0010767   Sstr2   1.53   0.0004486   Col12a1   1.52   0.0008099   Rai14   1.52   0.0004927   Stmn2   1.52   0.0005157   Stra6   1.51   0.0003715   Pdgfra   1.51   0.0009207   Zbtb2   1.50   0.0007063   Tmem45a   1.49   0.0003357   Tnfrsf12a   1.48   0.0010481   Timp1   1.48   0.0001630   Musk   1.47   0.0003490   Retsat   1.47   0.0002800   Prickle1   1.47   0.0006212       1.47   0.0000158   Pgm2l1   1.47   0.0009379       1.47   0.0006998   Isl1   1.46   0.0008981       1.46   0.0007626   Itpripl2   1.45   0.0009280   Stbd1   1.45   0.0009528   Glis1   1.45   0.0004584   111   Wnt5a   1.44   0.0000137   Mmp28   1.44   0.0005021   Pax3   1.44   0.0000540   Rgnef   1.44   0.0001481   Msx1   1.43   0.0007694   Abcc4   1.43   0.0006269   Ephb6   1.43   0.0002071   Phldb2   1.42   0.0000237   Rnf125   1.42   0.0001259   Rab3a   1.42   0.0006564   Btg3   1.42   0.0004979   Aaas   1.41   0.0007230   Ikzf2   1.41   0.0003251   3110006E14Rik   1.41   0.0007078   2810030E01Rik   1.40   0.0010775   Kalrn   1.40   0.0003756   Tmem119   1.40   0.0000023   Rgl1   1.40   0.0003680   Rufy3   1.40   0.0007387   B3gnt2   1.39   0.0005389   Btg3   1.39   0.0006596   Fam161a   1.39   0.0009682   112   Srxn1   1.39   0.0004043   Rab39b   1.39   0.0007812   Zfp365   1.38   0.0006040   Prrx1   1.38   0.0009289   Rab27b   1.38   0.0004361   Cc2d2a   1.38   0.0007264   Slc39a14   1.38   0.0000693   Phtf2   1.38   0.0001752   Ell2   1.37   0.0007072   Arl10   1.37   0.0006908   Dnajb14   1.37   0.0006226       1.36   0.0003009   Golim4   1.36   0.0008862   Manea   1.36   0.0009809   Lrrk2   1.36   0.0000905       1.35   0.0004983   Kif21b   1.35   0.0008825   Rnf41   1.35   0.0006670   Slc44a2   1.35   0.0003451   Dusp8   1.34   0.0001000   Sertad1   1.34   0.0009626   Mupcdh   1.34   0.0007916   113   E2f5   1.34   0.0002054   Prkaa1   1.34   0.0001647   Nr1d1   1.33   0.0000138   Pqlc3   1.33   0.0007139   Epb4.1l3   1.33   0.0001960   Slc1a4   1.33   0.0001838   Slc9a2   1.33   0.0008544       1.32   0.0000151   Wwc2   1.32   0.0009414   Zeb2   1.32   0.0006631   Elk3   1.30   0.0000771   Slc7a1   1.29   0.0001372   Tmem104   1.29   0.0001430   Derl3   1.29   0.0010716   Akap2   1.29   0.0001063   Carf   1.28   0.0005280   Rnf138   1.28   0.0005703   Adamts12   1.28   0.0002444   Eml3   1.28   0.0009465   Sash1   1.27   0.0006048   Cdc2l6   1.27   0.0003786   Ddr1   1.27   0.0002264   114   Bag3   1.27   0.0000268       1.26   0.0008388   Bcor   1.26   0.0010313   Ppt2   1.26   0.0002808   Tmem54   1.26   0.0009634   Fdxr   1.25   0.0006297   9030425E11Rik   1.25   0.0000867   Slc10a7   1.25   0.0000017   Dbc1   1.24   0.0000049   Rnf149   1.24   0.0006702   Chpf2   1.24   0.0004279   Purg   1.24   0.0009966   Svep1   1.23   0.0003406       1.23   0.0010206   0610007P08Rik   1.23   0.0003946   Synj1   1.23   0.0004797   Mras   1.23   0.0005532   Rdx   1.23   0.0010236   4930402H24Rik   1.22   0.0003672   Tmem173   1.22   0.0003495   Mknk2   1.21   0.0006977   Hdac5   1.20   0.0000641   115   Metrnl   1.20   0.0008875   Pcnx   1.20   0.0001538   Blzf1   1.20   0.0000531   Phlda3   1.20   0.0007703   Shroom4   1.20   0.0010561   9430015G10Rik   1.20   0.0002910     Supplementary  Table  2.1.  Transcripts  upregulated  by  BMP4  only  in  wild  type   myoblasts.  Fold-­‐changes  indicate  the  upregulation  of  the  listed  transcript  by  BMP4.   Fold  changes  greater  than  1.2  are  shown  in  the  table.       Supplementary  Table  2.2     Fold-­‐ Gene  Symbol   Change   p-­‐value   Sp7   10.58   0.0000161   Fabp7   9.08   0.0000059   Grem2   8.22   0.0000176   Id1   5.42   0.0000003   Dlx2   5.38   0.0000138   Ptgs2   5.21   0.0000067   116   Id3   5.08   0.0000081   Smad6   4.88   0.0000018   Alpl   4.39   0.0001661   Foxq1   3.77   0.0000168   Unc5b   3.65   0.0005125   Smad7   3.63   0.0000009   Serpinb8   3.38   0.0000107   Bambi   3.16   0.0000401   Lgr6   3.16   0.0000976   Fgfr2   3.09   0.0002492   Atoh8   3.02   0.0000126   Id2   2.99   0.0000118   Smad9   2.91   0.0000144   Lxn   2.90   0.0002553   Adamts9   2.83   0.0000691   Nkd2   2.80   0.0005255   Gcnt2   2.73   0.0000001   1300014I06Rik   2.70   0.0000098   Dlx1   2.69   0.0000282   Rspo3   2.68   0.0003037   Smoc2   2.67   0.0000152   Enc1   2.63   0.0000118   117   Pgf   2.63   0.0000596   Lfng   2.58   0.0000033   Baalc   2.51   0.0000981   Ctgf   2.46   0.0000830   Adamts9   2.44   0.0000339   Foxl1   2.36   0.0000183   Serpine1   2.20   0.0001436   Gnb4   2.19   0.0000485   Wnt2   2.17   0.0003486   Kif26b   2.12   0.0001033   Kctd12   2.11   0.0000301   Myo1d   2.10   0.0000039   Pde8a   2.05   0.0000085   Fam84a   2.01   0.0002542   Ahcyl2   2.01   0.0000317   Adamts9   2.00   0.0005875   Csrp2   1.99   0.0000552   Wnt4   1.97   0.0009629   Optn   1.95   0.0000805   Fam19a5   1.95   0.0002117   Cspg4   1.94   0.0000833   Gas6   1.89   0.0003323   118   Nfkbia   1.86   0.0005386   Hoxa10   1.85   0.0000558   Prrx2   1.83   0.0000337   Slc7a11   1.82   0.0000160   Cxcl14   1.82   0.0000347   Dock5   1.80   0.0000034   Ank   1.80   0.0000172   Samhd1   1.76   0.0003039   Npr3   1.76   0.0004214   Mmp11   1.76   0.0010683   Chmp2b   1.75   0.0001108   Dpysl3   1.70   0.0000034   Pcdh18   1.69   0.0006087   Jag1   1.69   0.0003387   Cux1   1.63   0.0000370   1110032E23Rik   1.62   0.0004655   Relt   1.60   0.0002817   Adamtsl3   1.57   0.0001643   Tpbg   1.56   0.0000216   Fam38b2   1.55   0.0009779   Sox9   1.55   0.0003365   Epha2   1.53   0.0008823   119   Tnfrsf21   1.53   0.0000233   Ptprm   1.52   0.0001927   Crim1   1.52   0.0002062   Atp10d   1.50   0.0002664   Man1a   1.50   0.0006539   Lyst   1.50   0.0000019   Kif26b   1.50   0.0001586   Fzd6   1.44   0.0000429   Ass1   1.43   0.0005171   Ass1   1.43   0.0005998   Tmem2   1.42   0.0001204   Gclc   1.42   0.0001196   Slc44a1   1.42   0.0001084   Slc5a3   1.39   0.0003938   Palld   1.39   0.0004808   Atp2c1   1.39   0.0002947   Slc7a2   1.38   0.0001401   Kirrel   1.38   0.0002013   Capn5   1.37   0.0003373   6330416G13Rik   1.36   0.0004968   Chst11   1.36   0.0000544   Peli2   1.35   0.0009524   120   Ahr   1.35   0.0003480   Lrp6   1.33   0.0000117   Ednra   1.32   0.0000278   Tln2   1.32   0.0008167   Acvr1   1.31   0.0003443   Galnt2   1.27   0.0004890   Dlst   1.26   0.0004482   Fam65a   1.24   0.0001126   Csnk1g3   1.23   0.0007569     Supplementary  Table  2.2.  Transcripts  upregulated  by  BMP4  both  in  wild-­‐type  and   MuSK  null  myoblasts.  Fold-­‐changes  indicate  the  upregulation  of  the  listed  transcript   by  BMP4.  Fold  changes  greater  than  1.2  are  shown  in  the  table.  (For  simplicity,  only   the  fold-­‐changes  from  wild-­‐type  cultures  are  shown  in  the  table)       Supplementary  Table  2.3     Fold-­‐ Gene  Symbol   Change   p-­‐value   Lin7a   4.94   0.0003900   Gm12824   4.22   0.0004426   121   Hey1   3.89   0.0002575   Selp   3.46   0.0000023   Tbx1   2.72   0.0001291   Stc2   2.68   0.0001532   Slc1a3   2.53   0.0000955   Irf5   2.53   0.0002718   Emb   2.44   0.0005802   Klhl29   2.26   0.0004717   Kcnh5   2.24   0.0000496   Gjb3   2.19   0.0000546   Il15   2.18   0.0001363   Adam12   2.16   0.0000011   Fam55c   2.15   0.0000145   Fst   2.06   0.0000663   Ppp1r3c   2.05   0.0000727   Slc2a13   1.97   0.0003914   Tbx20   1.96   0.0000647   Ankrd44   1.94   0.0000566   Depdc6   1.90   0.0001964   Hmgcll1   1.86   0.0002828   Krt19   1.85   0.0003951   Adcy8   1.82   0.0004721   122   Adamtsl1   1.76   0.0000295   Bmp2k   1.75   0.0001046   Grem1   1.75   0.0001096   Pparg   1.74   0.0001149   Fzd4   1.71   0.0000923   Pdgfrl   1.70   0.0000023       1.69   0.0003142   Acss3   1.69   0.0005332   Osbpl6   1.64   0.0001490   Adamtsl1   1.62   0.0004975   A930038C07Rik   1.62   0.0000051   Rims2   1.61   0.0000332   Iqgap2   1.61   0.0000418   Vegfc   1.59   0.0005939   Asb4   1.58   0.0002124   Efna3   1.56   0.0004736   4931406P16Rik   1.56   0.0005929   Prrg1   1.55   0.0007051       1.54   0.0003099   Grhl1   1.53   0.0005838   Esyt3   1.52   0.0004370   Hoxb2   1.48   0.0002266   123   Sostdc1   1.45   0.0002509   Acsl1   1.45   0.0000729   Zbtb25   1.44   0.0004446   Cish   1.44   0.0003414   Cd47   1.44   0.0001359       1.42   0.0004539   Pgam2   1.42   0.0001249   Il13ra1   1.41   0.0005407   Hoxa9   1.40   0.0001565   Chsy3   1.39   0.0004188   Nrp2   1.39   0.0000050   Trib2   1.38   0.0000093   Gramd2   1.38   0.0000013   Glis3   1.37   0.0004675   Freq   1.37   0.0006113       1.35   0.0002059   Pdlim5   1.32   0.0003320   Calhm2   1.32   0.0001093   Mtag2   1.32   0.0005917   Pmepa1   1.32   0.0003797   Mrps6   1.32   0.0002322       1.31   0.0005392   124   Adam10   1.30   0.0000215   Ceacam9   1.30   0.0002529   Thrb   1.30   0.0005348   Npnt   1.30   0.0004299       1.30   0.0002977   Ppme1   1.29   0.0000400   Mdga1   1.27   0.0004805   Magi1   1.27   0.0003866   Xdh   1.26   0.0003931   Cdc14b   1.25   0.0007062   N4bp2l1   1.25   0.0005779   Alms1   1.25   0.0005051   Hspa1a   1.24   0.0003844   Pkp2   1.22   0.0002850   Ficd   1.22   0.0001747   Olfr177   1.21   0.0004665   Tmem50b   1.20   0.0001369   Tlcd1   1.20   0.0001759   Cbx8   1.20   0.0006164     Supplementary  Table  2.3.  Transcripts  upregulated  by  BMP4  only  in  MuSK  null   myoblasts.  Fold-­‐changes  indicate  the  upregulation  of  the  listed  transcript  by  BMP4.   Fold  changes  greater  than  1.2  are  shown  in  the  table.   125       Supplementary  Table  2.4     Fold-­‐ Gene  Symbol   Change   p-­‐value   Sp7   8.69   0.0000045   Dhrs3   3.04   0.0001081   Rgs4   3.01   0.0000770   Adamts9   2.99   0.0001131   Lfng   2.89   0.0000963   Myh15   2.88   0.0004291   Slc10a4   2.72   0.0003353   Pgf   2.60   0.0000181   Adamts9   2.50   0.0000494   Selp   2.36   0.0001861   Hpgd   2.34   0.0001262   Grin3a   2.21   0.0007673   Arc   2.19   0.0001589   Ccdc74a   2.18   0.0004563   Ptgs2   2.18   0.0000096   Fgf2   2.18   0.0001711   126   Alpl   2.16   0.0000146   Kctd12   2.14   0.0000521   Kif26b   2.13   0.0000401   Nebl   2.11   0.0000497   Ptger4   2.10   0.0000079   Etl4   2.10   0.0000101   Wnt11   2.02   0.0007674   1300014I06Rik   2.02   0.0000951   Tbx1   2.00   0.0005592   Serpine1   1.96   0.0001781   Fam19a5   1.94   0.0004988   Slc24a4   1.93   0.0000356   Junb   1.90   0.0000435   Insc   1.86   0.0000349   St3gal6   1.84   0.0005664   Sdc3   1.84   0.0000133   -­‐-­‐   1.84   0.0006638   Epha7   1.84   0.0000574   Kcnc4   1.82   0.0002959   Adcy1   1.81   0.0000157   Fzd5   1.81   0.0000247   Hoxc11   1.81   0.0003600   127   Cspg4   1.80   0.0005948   -­‐-­‐   1.78   0.0006569   Orai2   1.77   0.0004315   Adamts9   1.77   0.0000795   Areg   1.76   0.0005841   Ptx3   1.75   0.0002167   Avpr1a   1.72   0.0002434   Orai2   1.71   0.0002898   Dpysl3   1.71   0.0000126   P4ha3   1.70   0.0000480   Shroom1   1.69   0.0005762   Nfkbia   1.67   0.0007483   Stbd1   1.67   0.0004681   Kremen1   1.66   0.0000332   Fam84a   1.64   0.0004296   Mex3b   1.62   0.0004509   Thsd7a   1.61   0.0000033   Wnt2   1.60   0.0001420   Chst11   1.60   0.0005794   Tnfrsf13c   1.60   0.0006128   Pank1   1.59   0.0000273   Ctgf   1.59   0.0004759   128   Podn   1.57   0.0005206   Kif26b   1.57   0.0004820   Dok7   1.56   0.0006384   Efna2   1.56   0.0001189   Jag1   1.55   0.0000886   Rps6ka5   1.54   0.0001718   Nr4a2   1.54   0.0002394   Hes6   1.53   0.0005466   Plxnd1   1.53   0.0003838   Slitrk4   1.53   0.0005484   Cyr61   1.52   0.0001222   9030425E11Rik   1.52   0.0005533   Cachd1   1.52   0.0005673   Plekha2   1.51   0.0004628   Rgs12   1.51   0.0001235   Pde5a   1.50   0.0000909   Itga1   1.50   0.0005239   9330159F19Rik   1.50   0.0006368   Fgfr2   1.49   0.0004556   Slc7a2   1.49   0.0000048   Mmp11   1.49   0.0004240   Timp3   1.48   0.0006282   129   Tgm2   1.47   0.0003525   E030011O05Rik   1.47   0.0001135   Inhba   1.47   0.0001435   Lgals3   1.47   0.0002452   Ier3   1.46   0.0001405   Fam135a   1.45   0.0005020   Ext1   1.44   0.0003667   Vdr   1.43   0.0007582   Fam46c   1.41   0.0007399   Myf6   1.41   0.0002346   -­‐-­‐   1.41   0.0000537   Gas6   1.41   0.0004738   Rab3b   1.39   0.0005589   Bdnf   1.38   0.0000905   Tle1   1.38   0.0007446   Suv39h1   1.37   0.0003284   Rarres1   1.36   0.0003906   Pde7b   1.36   0.0007270   Ern1   1.35   0.0000349   Ncf1   1.35   0.0005654   Tln2   1.35   0.0003608   -­‐-­‐   1.35   0.0001115   130   Slc43a2   1.34   0.0000659   -­‐-­‐   1.32   0.0007619   Kif1a   1.32   0.0005083   Serpinf1   1.32   0.0006135   Dync1i1   1.31   0.0005698   BY080835   1.30   0.0003441   Vasn   1.30   0.0001598   Kif21b   1.30   0.0006049   Klf10   1.29   0.0000891   Parp8   1.29   0.0000783   Chmp2b   1.29   0.0000763   Chst2   1.28   0.0006049   Rarg   1.28   0.0007196   Fstl1   1.28   0.0000142   Olfr415   1.28   0.0003046   Capn1   1.28   0.0004650   Mbnl1   1.27   0.0001958   Ntan1   1.27   0.0000545   Zeb2   1.27   0.0000344   Zcchc14   1.27   0.0006383   A630091E08Rik   1.26   0.0004318   Elk3   1.26   0.0004549   131   Nbl1   1.26   0.0002941   Bmp2k   1.26   0.0002739   Rdx   1.24   0.0003980   Fkbp10   1.24   0.0003927   Arntl   1.24   0.0002645   Slc29a4   1.23   0.0001504   Gpr157   1.23   0.0002073   Jarid2   1.22   0.0001485   Maz   1.22   0.0000079   Snx15   1.22   0.0004358   Itfg3   1.21   0.0003037   Htra3   1.20   0.0000322   Mtus1   1.20   0.0004251   Lrp6   1.20   0.0003000   Supplementary  table  2.4.  Transcripts  upregulated  by  BMP4  only  in  wild-­‐type   myotubes.  Fold-­‐changes  indicate  the  upregulation  of  the  listed  transcript  by  BMP4.   Fold  changes  greater  than  1.2  are  shown  in  the  table.       Supplementary  table  2.5     Gene  Symbol   Fold-­‐ p-­‐value   132   Change   Fabp7   19.98   0.0000005   Cxcr4   4.35   0.0000090   Id1   4.14   0.0000046   Id3   3.68   0.0001405   Smad6   3.44   0.0000010   Atoh8   3.38   0.0000022   2310002L13Rik   3.15   0.0000115   Smad7   2.97   0.0000491   Prrx2   2.92   0.0000011   Dlx2   2.88   0.0000523   Gnb4   2.88   0.0000005   Greb1   2.74   0.0000804   Baalc   2.73   0.0002523   Grhl1   2.70   0.0002370   Hey1   2.67   0.0000070   St3gal1   2.60   0.0000172   Smad9   2.49   0.0000067   Palmd   2.36   0.0000462   Spry2   2.34   0.0000465   Kcnf1   2.29   0.0001802   Esr1   2.25   0.0000226   133   Gm12824   2.25   0.0000683   Smoc2   2.18   0.0001098   Tmeff1   2.17   0.0000287   Sema6d   2.16   0.0000715   Rasl11a   2.16   0.0000208   Hbegf   2.08   0.0000916   Serpinb8   2.05   0.0001995   Optn   2.03   0.0000065   Inha   2.03   0.0001186   Tiam2   2.00   0.0000049   Angptl2   1.99   0.0000190   Bambi   1.98   0.0002266   Csrp2   1.97   0.0000023   Unc5b   1.97   0.0000165   9130213B05Rik   1.95   0.0000049   Cthrc1   1.93   0.0001716   Hoxc13   1.93   0.0002313   Dlx1   1.91   0.0001567   Car13   1.89   0.0001202   Lxn   1.89   0.0005318   Tle3   1.86   0.0000084   Prg4   1.86   0.0007615   134   Id2   1.84   0.0001083   Runx2   1.82   0.0001272   Tmeff2   1.80   0.0000042   Enc1   1.80   0.0001272   Snai2   1.79   0.0001333   Ddr1   1.77   0.0000060   Foxc2   1.76   0.0000225   Gcnt2   1.75   0.0002086   Net1   1.73   0.0000298   Gse1   1.71   0.0001256   Tgfb1   1.71   0.0000291   S  ept5   1.70   0.0000301   Gm12824   1.69   0.0005883   Sh3bp4   1.69   0.0000496   Tmem119   1.68   0.0000646   Pde8a   1.67   0.0001179   Ilkap   1.66   0.0002247   Dusp1   1.63   0.0002534   Myf5   1.61   0.0000898   Il17ra   1.60   0.0001803   Pdgfc   1.59   0.0000244   Cux1   1.57   0.0000448   135   Grem1   1.57   0.0001576   Lgr6   1.57   0.0007254   Jun   1.56   0.0000517   Tmem47   1.55   0.0004474   Unc5c   1.55   0.0002032   Ptprm   1.54   0.0001361   Golim4   1.51   0.0002435   Slc25a32   1.50   0.0000003   Eya4   1.50   0.0000048   Ankrd28   1.49   0.0003675   Plcd3   1.49   0.0007339   6330442E10Rik   1.47   0.0000921   Creb3l1   1.44   0.0005052   Skil   1.44   0.0006181   Tmem64   1.43   0.0001431   Flrt2   1.43   0.0002298   Fzd1   1.43   0.0000388   Aebp1   1.42   0.0002442   Col12a1   1.41   0.0000003   Ass1   1.41   0.0002622   Prrx1   1.41   0.0000528   Socs2   1.40   0.0001829   136   Ass1   1.40   0.0003002   6330416G13Rik   1.40   0.0003925   Snai1   1.40   0.0007220   Nav3   1.39   0.0001079   Snx25   1.36   0.0001355   3425401B19Rik   1.36   0.0005191   Dock5   1.35   0.0003001   Zbtb38   1.35   0.0005623   Raph1   1.34   0.0000467   Slc44a2   1.34   0.0000742   Myo10   1.32   0.0006481   Meis2   1.31   0.0000803   Asb5   1.31   0.0000008   Rai14   1.31   0.0001686   Prr16   1.31   0.0005664   Ugdh   1.29   0.0002734   Atp6v1g1   1.29   0.0000195   Ids   1.28   0.0001785   Epb4.1l1   1.27   0.0001063   Atp6v1g1   1.26   0.0000453   Cdon   1.26   0.0006243   Lysmd3   1.25   0.0007054   137   Osbpl3   1.24   0.0001454   Slc6a8   1.24   0.0005387   Lpin3   1.23   0.0005065   Xirp1   1.23   0.0007062   Car3   1.22   0.0004624     Supplementary  table  2.5.  Transcripts  upregulated  by  BMP4  both  in  wild-­‐type  and   MuSK  null  myotubes.  Fold-­‐changes  indicate  the  upregulation  of  the  listed  transcript   by  BMP4.  Fold  changes  greater  than  1.2  are  shown  in  the  table.  (For  simplicity,  only   the  fold-­‐changes  from  wild-­‐type  cultures  are  shown  in  the  table)         Supplementary  table  2.6     Fold-­‐ Gene  Symbol   Change   p-­‐value   Prr9   6.80   0.0000111   Pcolce2   4.18   0.0000352   A530098C11Rik   4.11   0.0000177   4930412O13Rik   3.66   0.0000523   Olr1   3.52   0.0001038   138   Samd7   3.52   0.0002913   Itgb1bp3   3.27   0.0003820   Alpk2   2.98   0.0000111   Slc7a11   2.89   0.0000278   Tpbg   2.89   0.0000879   Fam55c   2.67   0.0003726   Slc9a2   2.58   0.0000714   Sfrp2   2.52   0.0000026   Gpr37   2.50   0.0004724   Ak3l1   2.46   0.0000657   -­‐-­‐-­‐   2.46   0.0001367   Gata3   2.45   0.0000952   Lin7a   2.42   0.0000257   4930412O13Rik   2.41   0.0002443   2310045A20Rik   2.39   0.0000527   Rrm2   2.37   0.0002429   Hgf   2.37   0.0003182   Grem2   2.29   0.0002533   BC023105   2.28   0.0012488   Slc2a3   2.28   0.0001941   Prkg2   2.24   0.0000147   Ankrd37   2.20   0.0002882   139   Rorb   2.19   0.0004124   Sfrp4   2.18   0.0005419   Slco2a1   2.17   0.0000705   Dhrs9   2.13   0.0001824   Adm   2.04   0.0003952   Ppp1r3c   2.03   0.0006259   Nxf7   2.03   0.0001944   Atp10d   2.02   0.0012311   Tec   1.99   0.0000423   Timp1   1.98   0.0002008   Kcnq4   1.98   0.0001019   Adcy8   1.97   0.0000116   Tbx20   1.97   0.0008294   Opn3   1.96   0.0000020   -­‐-­‐-­‐   1.94   0.0007543   Has2   1.93   0.0006286   Fosl1   1.93   0.0001022   Ereg   1.91   0.0011558   Itgb3   1.90   0.0003136   -­‐-­‐-­‐   1.88   0.0000037   Ptpn3   1.88   0.0010442   Aldh1a3   1.88   0.0005623   140   Syn1   1.86   0.0002084   Tmem117   1.86   0.0002393   Ano5   1.86   0.0003671   Alcam   1.85   0.0001332   Ptpn3   1.85   0.0002888   Ptpn3   1.85   0.0001371   Uhrf1bp1l   1.85   0.0002515   Rufy3   1.84   0.0003425   Ifi202b   1.84   0.0002364   Ankrd1   1.84   0.0000325   Jak2   1.84   0.0002009   Nog   1.84   0.0006659   Pdk4   1.84   0.0000367   Fam110a   1.83   0.0004013   Rims2   1.83   0.0007488   2010002N04Rik   1.81   0.0002615   Gja5   1.81   0.0000447   Hcn1   1.81   0.0000544   Mylk2   1.80   0.0007058   Ces2   1.80   0.0009719   Csrp3   1.80   0.0001515   Stap1   1.79   0.0011733   141   Tmsb15a   1.78   0.0003148   Pappa2   1.78   0.0006261   Sdf2l1   1.77   0.0001956   Atp1b1   1.76   0.0000575   Crlf1   1.76   0.0007661   Serpinb1a   1.76   0.0000093   Btg3   1.75   0.0003358   Pappa2   1.75   0.0001141   Btg3   1.74   0.0003832   Tgfb2   1.74   0.0001445   Pfkfb3   1.73   0.0001441   Olfm1   1.72   0.0000829   Gm1078   1.72   0.0001417   Sema3a   1.71   0.0004086   Tmem56   1.71   0.0002192   Nol4   1.71   0.0011101   Kremen2   1.71   0.0004522   Fam178a   1.71   0.0005730   Hspa1l   1.69   0.0009244   Frrs1   1.68   0.0002168   Tspan18   1.68   0.0009213   Herc3   1.68   0.0002117   142   Irf6   1.68   0.0010943   Dnajb4   1.67   0.0011273   Rrad   1.66   0.0010142   Dapk2   1.66   0.0001043   Ptpn3   1.65   0.0000242   Erc1   1.65   0.0005432   Bnip3   1.65   0.0002628   Fhdc1   1.64   0.0005928   Hspb6   1.63   0.0000432   Tnfrsf12a   1.63   0.0008864   Dnajb9   1.63   0.0000073   Btg2   1.61   0.0007912   Ptpn3   1.61   0.0007389   Auts2   1.61   0.0000241   Gcnt4   1.61   0.0010965   -­‐-­‐-­‐   1.61   0.0001520   Slc16a1   1.60   0.0000166   Egln3   1.60   0.0000363   Cnnm2   1.60   0.0002476   Pdk1   1.60   0.0006768   Slc2a1   1.60   0.0002767   Calml3   1.59   0.0006882   143   Csf1   1.58   0.0000031   Fst   1.58   0.0003351   Uaca   1.58   0.0000487   Nlrc3   1.58   0.0000295   Slc16a3   1.57   0.0002283   Lpin2   1.57   0.0008676   Lnx2   1.57   0.0000448   Gclc   1.56   0.0001883   Bmper   1.56   0.0007731   Rhou   1.56   0.0001063   Slc5a3   1.56   0.0005752   Fam38b   1.56   0.0005059   Ndst3   1.55   0.0006954   Porcn   1.55   0.0008788   Tmem45a   1.55   0.0006069   Rnf138   1.55   0.0001778   Paqr4   1.55   0.0005520   Ehd3   1.54   0.0001322   Acss2   1.54   0.0007382   Nox4   1.54   0.0000771   Krt80   1.54   0.0011012   Jhdm1d   1.53   0.0007790   144   Adra1b   1.53   0.0001887   Ube2cbp   1.52   0.0007181   Obsl1   1.52   0.0001203   Hspb7   1.52   0.0000132   1700008I05Rik   1.52   0.0003705   Fzd6   1.51   0.0004153   Dlk1   1.51   0.0006232   Cox19   1.51   0.0012160   C1qtnf3   1.51   0.0002855   Leprel1   1.50   0.0005599   Slc1a3   1.50   0.0010638   Parvb   1.50   0.0010983   Ccng2   1.50   0.0006620   Slc44a1   1.49   0.0001075   Pdlim3   1.49   0.0002185   Erc1   1.48   0.0008846   -­‐-­‐-­‐   1.48   0.0001325   Acvr1   1.48   0.0004291   Ccnd1   1.47   0.0009187   Tiparp   1.47   0.0005305   Wbscr27   1.47   0.0001901   Ak5   1.47   0.0000163   145   -­‐-­‐-­‐   1.47   0.0011302   Manf   1.46   0.0002446   Hmga2   1.46   0.0011419   -­‐-­‐-­‐   1.46   0.0001911   Snx18   1.45   0.0007219   Spcs3   1.45   0.0010616   Nrap   1.45   0.0000537   Myo1d   1.44   0.0010639   Hspa1a   1.44   0.0001049   Cilp2   1.44   0.0010480   Prrg4   1.44   0.0012115   Ssfa2   1.43   0.0000731   Inpp4b   1.43   0.0009030   Plcxd2   1.43   0.0001205   Slc5a7   1.43   0.0003299   Ebf1   1.43   0.0009412   Zmynd17   1.42   0.0000140   Frs2   1.42   0.0001723   Nuak1   1.41   0.0001013   Gm129   1.41   0.0008077   Pknox1   1.41   0.0010963   Relt   1.41   0.0006280   146   Arfgap3   1.41   0.0009624   Sp6   1.41   0.0000379   Myh7b   1.41   0.0007000   Mylk4   1.40   0.0000096   Prkar1b   1.40   0.0007132   Arrb1   1.40   0.0000538   Gm9861   1.39   0.0011039   Loxl3   1.39   0.0006197   Ang2   1.39   0.0002183   -­‐-­‐-­‐   1.39   0.0006508   Slc35f5   1.39   0.0006986   Fibin   1.39   0.0003849   Adam17   1.39   0.0006154   Tjap1   1.39   0.0009743   Cast   1.39   0.0004449   Adamtsl3   1.39   0.0009885   Fxyd5   1.39   0.0000197   Ibtk   1.38   0.0001982   Whrn   1.38   0.0001877   -­‐-­‐-­‐   1.37   0.0005115   Spsb2   1.36   0.0012493   Rabgef1   1.36   0.0007778   147   Fance   1.35   0.0005121   Fhl1   1.35   0.0002216   Hk2   1.35   0.0003439   Erf   1.35   0.0005380   Klhl21   1.34   0.0007800   Sertad1   1.34   0.0006213   Abcg2   1.34   0.0010350   Foxp1   1.34   0.0005838   Clic4   1.34   0.0003693   Rel   1.34   0.0002851   Ints9   1.34   0.0000383   Hoxc9   1.34   0.0005140   Creld2   1.33   0.0002281   Plekhh2   1.33   0.0008819   Fat1   1.33   0.0004384   Pgam1   1.33   0.0008862   Pgam1   1.33   0.0008862   Hspa1a   1.33   0.0003968   Etv4   1.33   0.0001280   Mycl1   1.32   0.0008536   Fam72a   1.32   0.0005329   Fam134b   1.31   0.0004316   148   Slc30a4   1.31   0.0007531   Lox   1.31   0.0000602   Zfp451   1.31   0.0010512   Hspa5   1.31   0.0003681   4930503L19Rik   1.31   0.0011781   Myot   1.31   0.0008148   Gpc6   1.31   0.0006981   -­‐-­‐-­‐   1.31   0.0000089   Ltbp2   1.31   0.0003162   Acvr1b   1.30   0.0008132   Fam57a   1.30   0.0003703   Samd8   1.30   0.0008466   Npat   1.30   0.0002675   M  arch8   1.30   0.0008292   Yap1   1.30   0.0001202   Bag2   1.29   0.0004260   Pgam1   1.29   0.0008670   H47   1.29   0.0002790   -­‐-­‐-­‐   1.29   0.0009139   Sorbs1   1.29   0.0002711   Me1   1.29   0.0011375   -­‐-­‐-­‐   1.28   0.0005734   149   Iqgap1   1.28   0.0008691   Wwc2   1.28   0.0003887   Prepl   1.28   0.0006085   Zhx3   1.28   0.0002617   Tacc1   1.28   0.0002677   Ank   1.27   0.0012465   Dnaja4   1.27   0.0004575   Fam65a   1.27   0.0006524   E2f3   1.27   0.0008708   Dlg5   1.27   0.0011113   Myd116   1.27   0.0000237   Ldb3   1.27   0.0006414   Itgb6   1.26   0.0011358   Hipk2   1.26   0.0000079   Ccdc58   1.26   0.0004561   Tmem50b   1.26   0.0000931   Zfp568   1.25   0.0004845   -­‐-­‐-­‐   1.25   0.0004449   Mdfic   1.25   0.0000982   Sec23b   1.24   0.0006352   Fabp3   1.24   0.0010290   Anxa7   1.24   0.0007889   150   Smyd1   1.24   0.0006076   Pfn2   1.24   0.0004366   Il13ra1   1.24   0.0012106   Slc6a6   1.24   0.0002429   Zc3h10   1.24   0.0011189   Atxn1   1.23   0.0000300   -­‐-­‐-­‐   1.23   0.0003211   -­‐-­‐-­‐   1.23   0.0002072   LOC100044416   1.22   0.0007953   Tmem39a   1.22   0.0007496   Dync1li1   1.21   0.0000406   Dnajb14   1.21   0.0005548   Zkscan5   1.21   0.0006842   Tagln2   1.20   0.0001127   Gm249   1.20   0.0000389   Rhoj   1.20   0.0000696     Supplementary  table  2.6.  Transcripts  upregulated  by  BMP4  only  in  MuSK  null   myotubes.  Fold-­‐changes  indicate  the  upregulation  of  the  listed  transcript  by  BMP4.   Fold  changes  greater  than  1.2  are  shown  in  the  table.         151                             CHAPTER  3                                                 152   BMP4 induces acetylcholine receptor clustering in a MuSK- and Wnt11-dependent manner Atilgan  Yilmaz1,2,    Carolyn  Schmiedel1,  Justin  Fallon1     1Department  of  Neuroscience,  Brown  University,  Providence,  Rhode  Island  02912,   USA   2 Department  of  Molecular  Biology,  Cell  Biology  and  Biochemistry,  Brown   University,  Providence,  Rhode  Island  02912,  USA   All experiments were conducted by me. Carolyn Schmiedel performed the AChR cluster counting analysis in Figure 2 and 4. 153   Abstract: Acetylcholine receptor (AChR) clustering on the muscle membrane is a defining step for neuromuscular junction formation. In vertebrate muscle, both the neural-drived extracellular matrix protein agrin as well as Wnt11 have been shown to induce AChR clusters via a mechanism requiring Muscle Specific Kinase (MuSK). Bone morphogenic proteins (BMPs) have been suggested to have roles in retrograde signaling in neuromuscular junctions of invertebrates. However, their roles at the postsynaptic muscle membrane have not been described. Here we show that BMP4 induces AChR clustering on cultured myotubes. This clustering requires MuSK but is agrin-independent. BMP4- induced AChR clusters are morphologically different than the agrin-induced clusters and form only after overnight treatment of cultured myotubes. BMP4 induces Wnt11 expression and Wnt11 activity is required for the clustering activity. Our study suggests that BMP4 acts upstream of Wnt11 to induce MuSK-dependent AChR clustering. 154   Introduction               Acetylcholine  released  from  motor  neuron  terminals  in  vertebrates  binds  to  and   opens  AChRs  in  the  postsynaptic  domains  of  NMJs  initiating  the  endplate  potential   that  in  turn  is  necessary  to  muscle  contraction.  Generation  of  a  sufficiently  large   endplate  potential  requires  a  high  density  of  AChRs  at  the  NMJ.  Thus  AChR   clustering  is  vital  for  the  efficient  neurotransmission,  hence  the  communication   between  neurons  and  the  muscle  tissue.     Muscle  Specific  Kinase  (MuSK)  is  a  receptor  tyrosine  kinase  that  was  originally   purified  from  the  synapse-­‐rich  electric  organ  of  Torpedo  californica  (Jennings  et  al.,   1993).  MuSK  co-­‐localizes  with  AChRs  and  NMJs  (Valenzuela  et  al.,  1995).  It  is   essential  for  the  stability  of  the  AChR  clusters  and  is  concentrated  within  those   (DeChiara  et  al.,  1996;  Kummer  et  al.,  2006).  MuSK-­‐/-­‐  mice  die  prenatally  due  to  the   failure  to  form  NMJs  and  the  MuSK-­‐/-­‐  muscle  fibers  lack  AChR  clusters  (DeChiara  et   al.,  1996;  Lin  et  al.,  2001;  Yang  et  al.,  2001).  Furthermore,  ectopic  MuSK  expression   triggers  NMJ  formation  (Kim  and  Burden,  2008).  These  observations  led  to  the   conclusion  that  MuSK  has  a  master  regulatory  role  in  NMJ  formation,  a  process  that   involves  AChR  clustering.         Neural-­‐derived  proteoglycan  agrin  is  the  best-­‐characterized  inducer  of  AChR   clustering  in  muscle  fibers.  After  being  secreted  from  the  motor  nerve  terminal,   agrin  can  induce  MuSK  autophosphorylation  and  hence  activate  the  receptor   (Mittaud  et  al.,  2004).  MuSK  is  endocytosed  upon  its  activation  and  this   155   internalization  is  required  for  AChR  clustering  (Zhu  et  al.,  2008).  MuSK  activation   eventually  leads  to  diverse  downstream  signaling  events  including  reorganization  of   the  actin  cytoskeleton  and  recruitment  of  AChR-­‐binding  scaffolding  proteins,  both  of   which  are  crucial  for  AChR  clustering  (Bloch  et  al.,  1986;  Dai  et  al.,  2000;  Okada  et   al.,  2006;  Linnoila  et  al.,  2008).       Prior  to  innervation  in  developing  muscle,  aneural,  ‘pre-­‐patterned’  AChR  clusters   are  present.  Upon  innervation  and  the  secretion  of  neural  agrin  these  clusters   mature,  grow  in  size  and  become  stable.  Agrin  is  necessary  for  these  later  stages  in   synapse  differentiation  -­‐  agrin-­‐/-­‐  mice  fail  to  form  AChR  clusters  after  innervation   and  lack  NMJs  (Gautam  et  al.,  1996).  However,  the  pre-­‐patterned  aneural  AChR   clusters  can  still  form  in  agrin-­‐/-­‐  mice,  indicating  that  there  might  be  additional   signals  regulating  the  formation  of  these  early  clusters  (Lin  et  al.,  2001;  Yang  et  al.,   2001).  Indeed,  Wnt11r,  mouse  Wnt11  ortholog  in  zebrafish,  has  been  shown  to   induce  aneural  clusters  and  guide  motor  axons  for  NMJ  formation  in  zebrafish  (Jing   et  al.,  2009).  Notably,  MuSK  and  its  co-­‐receptor  LRP4  are  required  for  the  formation   of  both  aneural  and  neural  AChR  clusters  (Zhang  et  al.,  2004;  Wu  et  al.,  2012).     Wnt11  belongs  to  the  Wnt  family  of  secreted  glycoproteins  that  have  crucial  roles  in   development  (van  Amerongen  et  al.,  2009).  Recently,  Wnt11  has  also  been  shown  to   induce  AChR  clusters  on  cultured  mouse  myotubes  (Zhang  et  al.,  2012).  This  study   showed  that  Wnt11  binds  to  MuSK  and  Wnt11  clustering  activity  is  not  additive  to   156   that  of  agrin,  suggesting  that  agrin  and  Wnt11  may  be  using  similar  pathways  to   induce  clustering.       The  TGFβ  superfamily  of  signaling  molecules  includes  TGFβ,  BMPs  and  activins.   They  are  recognized  by  two  different  types  of  cell  surface  receptors  -­‐  type-­‐1  and   type-­‐2  (ten  Dijke  et  al.,  1994,  de  Sousa  Lopes  et  al.,  2004,  Nohno  et  al.,  1995,  Xia  et   al.,  2007).  Activation  of  the  receptors  leads  to  Smad-­‐dependent  or  Smad-­‐ independent  downstream  signaling,  including  transcription  of  a  variety  of  genes   (Kretzschmar  et  al.,  1997;  Lagna  et  al.,  1996;  Liu  et  al.,  1996;  Hoodless  et  al.,  1996). In  Drosophila,  muscle-­‐derived  BMP  ortholog  Gbb  was  shown  to  be  involved  in   retrograde  signaling  to  coordinate  neuromuscular  synapse  development  and   growth  (McCabe  et  al.,  2003).  In  mice,  on  the  other  hand,  it  is  unknown  if  TGFβ  has   any  function  on  NMJ  development.  Here  we  show  that  BMP4  induces  distinct  AChR   clusters  in  a  MuSK-­‐dependent  manner  by  upregulating  Wnt11  expression.     Results       BMP4  induces  AChR  clusters  in  a  MuSK-­dependent  fashion     Our  recent  work  showed  that  MuSK  binds  BMP4  and  regulates  the  transcriptional   output  of  the  BMP4  signaling  pathway  (Chapter  2,  Yilmaz  et  al.).  Here  we  asked   whether  BMP4  plays  a  role  in  the  canonical  function  of  MuSK  –  AchR  clustering.  In   order  to  test  BMP4’s  potential  involvement  with  AChR  clustering,  we  used  a  well-­‐ 157   established  system  that  is  based  on  labeling  AChR  clusters  formed  in  cultured   mouse  myotubes.  H-2Kb-tsA58 wild type mouse myotubes were treated with agrin or BMP4 overnight. As expected, agrin-treated myotubes formed elongated large clusters after overnight treatment. Interestingly, BMP4 treatment also induced AChR clustering. (Figure  3.1a,  upper  panels).  As  a  negative  control,  we  treated  the  wild  type   myotubes  with  Wnt5a,  which  was  not  implicated  in  the  induction  of  AChR   clustering.  As  expected,  Wnt5a  did  not  form  any  clusters  above  baseline  levels.         We  then  wondered  if  MuSK  was  required  for  BMP4-­‐induced  clusters.  To  test  this   idea,  MuSK  null  myotubes  were  treated  with  agrin  or  BMP4.  Consistent  with   previous  results,  agrin  failed  to  induce  AChR  clusters  in  the  absence  of  MuSK  (Glass   et  al.,  1996).  Notably,  BMP4  induction  of  AChR  clustering  was  also  defective  in  the   absence  of  MuSK.  In  both  cases  AChRs  remained  uniformly  distributed  throughout   the  myotube  membrane  (Figure  3.1a,  lower  panels).  Quantification  of  clusters  per   myotube  segment  showed  BMP4  induced  a  significant  increase  in  the  number  of   AChR  clusters  on  wild  type  myotubes  (Figure  3.1b).     We  then  analyzed  the  time-­‐course  of  BMP4-­‐induced  AChR  clustering  (0-­‐16  hours).   BMP4-­‐induced  clusters  were  only  detected  after  8  hours  of  incubation  (Figure  3.2).   This  time  course  is  distinct  from  that  of  agrin-­‐induced  clusters,  which  are  readily   detected  within  2  hours  of  treatment  (Wallace  et  al.,  1988;  Nastuk  et  al.,  1991)     BMP4  induces  Wnt11  expression   158     The  long  time  course  of  BMP4-­‐induced  AChR  clustering,  together  with  the   observation  that  BMP4  does  not  induce  MuSK  phosphorylation  (Chapter  2,  Yilmaz  et   al.)  suggested  that  there  may  be  an  intermediate  signal  required  for  BMP4-­‐mediated   AChR  clustering.  One  candidate  for  such  a  BMP-­‐dependent  signal  is  Wnt11.  To  test   whether  BMP4  induces  Wnt11  expression,  we  treated  wild  type  myotubes  with   BMP4  and  analyzed  Wnt11  gene  expression  by  qRT-­‐PCR.  BMP4  induced  the   expression  of  Wnt11  by  2-­‐fold  after  8  hours  of  treatment.  Interestingly,  MuSK  was   not  necessary  for  BMP4-­‐induced  Wnt11  upregulation  (Figure  3.3  ).     Wnt11  activity  is  necessary  for  the  formation  of  BMP4-­induced  AchR  clusters     We  next  tested  whether  BMP4-­‐induced  AChR  clustering  requires  Wnt11.  We  used   neutralizing  antibodies  to  inhibit  Wnt11  activity.  BMP4  failed  to  induce  AChR   clusters  in  the  presence  of  anti-­‐Wnt11  antibody,  while  there  was  no  significant   reduction  in  the  presence  of  control  IgG  (Figure  3.4a,  b).  Neutralization  of  Wnt11   worked  best  when  the  antibody  was  added  3-­‐4  hours  after  BMP4  addition.     Discussion       The  Drosophila  BMP  ortholog  Gbb  was  shown  to  regulate  synaptic  growth  at  the   Drosophila  NMJs  via  a  retrograde  signaling  (McCabe  et  al.,  2003).  In  its  absence,   decreased  neurotransmitter  release,  reduced  NMJ  synapse  size  and  aberrant   159   presynaptic  ultrastructure  have  been  observed.  However,  no  function  at  the   postsynaptic  muscle  membrane  organization  has  been  attributed  to  BMPs.  In  this   report  we  show  the  evidence  that  mammalian  BMP4  has  AChR  clustering  activity  in   cultured  mouse  muscle  cells.       AChR  clustering  occurs  at  two  different  stages  during  development.  Prior  to   innervation  of  the  muscle,  a  phenomenon  called  prepatterning  occurs  in  which   Wnt11r  was  suggested  to  be  the  inducer  of  aneural  clusters  and  guide  motor  axons   for  NMJ  formation  in  zebrafish  (Jing  et  al.,  2009;  Nitkin  et  al.,  1987).  On  the  other   hand,  after  innervation  neural-­‐derived  agrin  induces  larger  neural  clusters  (Burden,   2011).  Importantly,  both  aneural  and  neural  clusters  require  MuSK  (Zhang  et  al.,   2004).  Our  results  suggest  that  BMP4  induces  morphologically  different  clusters   compared  to  agrin  induced  ones.  BMP4-­‐induced  clusters  are  smaller  and  rounder  in   shape  and  less  in  numbers.  It  would  be  interesting  to  test  the  idea  if  BMP4  has  any   role  in  prepatterning.  BMP4  signal  from  the  neighboring  tissue  notochord  or   autocrine  BMP  signaling  in  early  muscle  fibers  in  the  embryo  could  regulate   prepatterning.     We  also  show  that  BMP4  upregulates  the  expression  of  Wnt11  message.  To  our   knowledge  this  is  the  first  study  indicating  Wnt11  downstream  of  BMP4  signal.   Given  the  suggested  role  for  Wnt11  in  prepatterning,  BMP4  regulation  of  Wnt11   expression  could  be  a  muscle-­‐specific  mechanism  to  orchestrate  surface  distribution   of  AChRs  and  provide  guidance  for  NMJ  formation.     160     Wnt9a  and  Wnt11  were  shown  to  bind  to  MuSK,  activate  it  and  induce  AChR   clustering  in  cultured  mouse  C2C12  myotubes  (Zhang  et  al.,  2012).  Our  results  show   that  Wnt11  activity  is  required  for  BMP4-­‐induced  AChR  clustering,  as  blocking   Wnt11  with  neutralizing  antibodies  specifically  inhibited  BMP4-­‐induced  clusters.   The  requirement  of  MuSK  and  Wnt11  is  in  accord  with  the  previous  studies  and   attributes  a  regulatory  role  for  BMP4  in  this  process.       In  this  report  we  demonstrate  a  role  for  BMP4  in  the  induction  of  AChR  clusters  in   cultured  muscle  cells.  Our  results  indicate  the  possibilities  that  BMPs  are  a  novel   class  of  regulators  of  prepatterning  or  postsynaptic  muscle  membrane  organization   at  NMJs.  Further  in  vivo  and  in  vitro  studies  will  determine  the  exact  stages  that   BMP4-­‐induced  AChR  clustering  takes  place  in  muscle.       Future  Directions     Some  of  the  future  studies  that  will  help  explain  the  mechanism  of  BMP4  induction   of  AChR  clustering  should  focus  on  the  requirement  of  MuSK  activities.  The  previous   finding  by  Zhang  et  al.  about  Wnt11’s  binding  and  activation  of  MuSK  would  lead  to   prediction  that  MuSK  kinase  activity  would  be  needed  for  BMP4-­‐induced  clustering.   Even  though  BMP4  itself  does  not  induce  phosphorylation  of  MuSK  up  until  3  hours   of  treatment  (Chapter  2,  Yilmaz  et  al.),  MuSK  could  be  phosphorylated  by  Wnt11  at  a   161   later  point  after  Wnt11  is  expressed  and  secreted  from  cells.  The  requirement  of   MuSK  kinase  activity  can  be  tested  by  treating  myotubes  that  express  kinase-­‐dead   MuSK  with  BMP4.       Materials  and  Methods       Antibodies and materials Purified recombinant human BMP4, purified recombinant rat agrin, purified recombinant human/mouse Wnt5a, normal goat IgG, anti-Wnt11 antibody were obtained from R&D Systems (Minneapolis, MN, USA). Rhodamine-α-bungarotoxin was obtained from Invitrogen and was used to label acetylcholine receptors. Mammalian cell culture Wild-type mouse H-2Kb-tsA58 (Morgan JE et al., 1994) and MuSK-/- immortalized myoblasts were cultured on gelatin-coated dishes in DMEM supplemented with 20% fetal bovine serum, 2% L-Glutamine, 1% Penicillin-Streptomyocin, 1% Chicken Embryo Extract, 1U interferon-γ and cultured under permissive temperature at 33 oC in 8% CO2. Myotubes were obtained by switching the confluent myoblast cultures to a medium with DMEM supplemented with 5% horse serum, 2% L-Glutamine and 1% Penicillin- 162   Streptomyocin. Acetylcholine receptor clustering assay Wild-­‐type   and   MuSK   null   myoblasts   were   grown   to   confluence   on   Permanox   chamber  slides  (Nunc  and  Nalgene,  Thermo  Fisher  Scientific)  and  differentiated  to   myotubes   for   2-­‐4   days   as   described.   Myotubes   were   incubated   for   16   hours   with   25ng/ml   BMP4,   1ng/ml   agrin,   400ng/ml   Wnt5a   or   the   indicated   amounts   of   Wnt11   antibody.   AChRs   were   labeled   with   rh-­‐α-­‐bungarotoxin   for   15   minutes   at   33°C.   Myotubes  were  washed  with  PBS  three  times  and  fixed  with  methanol  for  5  minutes   at  −20°C.  AChR  clusters  were  counted  on  a  Nikon  Eclipse  800  microscope.       RNA  extraction,  reverse  transcription  and  quantitative  real  time  polymerase   chain  reaction  (qRT-­PCR)     Total  RNA  was  isolated  from  cells  with  Trizol  (Invitrogen).  Total  RNA  was  cleaned   up  and  DNase-­‐treated  in  Qiagen  RNeasy  columns.  RNA  was  reverse  transcribed  into   first  strand  cDNA  (Invitrogen).  qRT-­‐PCR  reaction  consisted  of  initial  incubation  at   50  0C  for  2  minutes  and  a  denaturation  at  95  0C  for  5  minutes.  The  cycling   parameters  were  as  follows:  95  0C  for  15  seconds,  60  0C  for  30  seconds.  After  40   cycles,  the  reactions  underwent  a  final  dissociation  cycle  as  follows:  95  0C  for  15   seconds,  60  0C  for  1  minute,  95  0C  15  seconds  and  60  0C  for  15  seconds.   Based   on   the   published   sequences,   Wnt11   primer   sequences   used   in   qRT-­‐PCR   163   reactions   were   as   follows: 5'- GCCCATACTATTGCCCTGTC -3' and 5'- GCACATCAGTAGCCACAAGC -3'   Statistical  analysis   All  statistical  analyses  used  Student's  t  test  unless  otherwise  noted.  Quantifications   for  Acetylcholine  clustering  assays  were  performed  by  observers  blinded  to   experimental  conditions.                                       164     FIGURES     FIGURE  3.1                   165         FIGURE  3.1.  BMP4 induces AChR clustering and this activity requires MuSK. A. Agrin and BMP4-induced clusters in wild type and MuSK-/- myotubes. Myotubes of indicated genotype were differentiated from immortalized myoblast lines and treated with 1ng/ml agrin or 25 ng/ml BMP4 for 16 hours. Elongated large clusters formed after agrin treatment, whereas BMP4 treatment led to formation of smaller and rounder clusters in wild type myotubes. Both agrin and BMP4 failed to induced clusters in MuSK-/- myotubes. B. Quantification of AChR clusters. The number of AChR clusters (>4 µm in length and <1 × 103 µm2) per myotube segment on cultures treated with agrin, BMP4 or Wnt5a was scored (n=74 segments/condition). The results were repeated with at least 3 independent experiments. Error bars represent SEM. Wnt5a treatment showed no significant change, all the other conditions were significantly different than each other (One was ANOVA, p<  <0.0001)   166   FIGURE 3.2 167   FIGURE 3.2. BMP4-induced AChR clusters form between 8 and 16 hours after treatment. Time-course of BMP4 induced AChR clustering in wild-type myotubes. Wild type myotubes were treated with 25ng/ml BMP4 for 2, 4, 8 and 16 hours. Short duration BMP4 treatments (2, 4, 8 hours) did not cause any significant changes in the number of AChR clusters. Overnight BMP4 treatment induced AChR clustering. Error bars represent SEM (n=30 segments/condition). 168   FIGURE 3.3 169   FIGURE 3.3. BMP4 induces Wnt11 expression in a MuSK-independent manner. Analysis of mRNA levels of Wnt11 by qRT-PCR after 8 hours of BMP4 treatment. Wild   type   H-2Kb-tsA58 and MuSK null myoblasts were grown into confluence and differentiated into myotubes for 3 days. Myotube cultures were treated with 25ng/ml BMP4  for  8  hours.  RNA was isolated, reverse-transcribed  into  double  stranded  cDNA.   Relative  transcript  level  of  Wnt11  was  analyzed  by  qRT-­‐PCR.  (n=3)  BMP4  induced   Wnt11  expression  both  in  wild  type  and  MuSK-­‐/-­‐  myotubes  by  ~2  fold.       170   FIGURE 3.4. 171   FIGURE 3.4. Wnt11 activity is required for BMP4-induced AchR clusters. A. Quantification of AChR clusters under indicated conditions. Wild  type  H-2Kb-tsA58 myotubes were treated with 1ng/ml agrin or 25ng/ml BMP4 for 16 hours. BMP4 induced AChR clusters (p<0.0001, Student’s t-test). Wnt11 neutralization antibody was added 0.5 and 3.5 hours after BMP4 treatment. When added 3.5 hours after BMP4 treatment, AChR clustering activity of BMP4 was inhibited (p<0.0001, Student’s t-test). (n=28 segments/condition) B. Quantification of AChR clusters under indicated conditions. Wild   type   H-2Kb-tsA58 myotubes were treated with 25ng/ml BMP4 along with Wnt11 antibody or normal goat IgG for 16 hours. Wnt11 antibody inhibited AChR clustering activity of BMP4 significantly (p< 0.002, Student’s t-test), whereas normal goat IgG did not (non-significant, Student’s t-test). 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 bDepartment  of  Molecular  Biology,  Cell  Biology   and  Biochemistry,  Brown  University,  Providence,  RI  02912;  cDepartment  of   Physiology  and  Pennsylvania  Muscle  Institute,  University  of  Pennsylvania  School  of   Medicine,  Philadelphia,  PA  19104;  and  dDivision  of  Hematology  and   Oncology,  University  of  California–Davis  Medical  Center,  Sacramento,  CA  95817   Edited  by  Louis  M.  Kunkel,  Children’s  Hospital  Boston,  Boston,  MA,  and  approved   November  22,  2010  (received  for  review  September  2,  2010) Contributed by Author contributions: A.R.A., A.Y., S.B., B.A.M., T.S.K., and J.R.F. designed research; A.R.A., A.Y., S.B., and B.A.M. performed research; M.A. contributed new reagents/analytic tools; A.R.A., A.Y., S.B., B.A.M., T.S.K., and J.R.F. analyzed data; and A.R.A. and J.R.F. wrote the paper. 178   This work has been adapted from the original article to fit this thesis format. This chapter has been published as: Amenta,  Alison  R,  Atilgan  Yilmaz,  Sasha   Bogdanovich,  Beth  A  McKechnie,  Mehrdad  Abedi,  Tejvir  S  Khurana,  and  Justin  R   Fallon.  “Biglycan  Recruits  Utrophin  to  the  Sarcolemma  and  Counters  Dystrophic   Pathology  in  Mdx  Mice.”  Proceedings  of  the  National  Academy  of  Sciences  of  the   United  States  of  America  108,  no.  2  (January  11,  2011):  762–767. I performed the qRT-PCR analysis in Figures 4.2C and 4.3C the protein analysis in Figures 4.2A and 4.2B 179   ABSTRACT Duchenne  muscular  dystrophy  (DMD)  is  caused  by  mutations  in  dystrophin  and  the   subsequent  disruption  of  the  dystrophin-­‐associated  protein  complex  (DAPC).   Utrophin  is  a  dystrophin  homolog  expressed  at  high  levels  in  developing  muscle  that   is  an  attractive  target  for  DMD  therapy.  Here  we  show  that  the  extracellular  matrix   protein  biglycan  regulates  utrophin  expression  in  immature  muscle  and  that   recombinant  human  biglycan  (rhBGN)  increases  utrophin  expression  in  cultured   myotubes.  Systemically  delivered  rhBGN  up-­‐regulates  utrophin  at  the  sarcolemma   and  reduces  muscle  pathology  in  the  mdx  mouse  model  of  DMD.  RhBGN  treatment   also  improves  muscle  function  as  judged  by  reduced  susceptibility  to  eccentric   contraction-­‐induced  injury.  Utrophin  is  required  for  the  rhBGN  therapeutic  effect.   Several  lines  of  evidence  indicate  that  biglycan  acts  by  recruiting  utrophin  protein  to   the  muscle  membrane.  RhBGN  is  well  tolerated  in  animals  dosed  for  as  long  as  3   months.  We  propose  that  rhBGN  could  be  a  therapy  for  DMD.   180   INTRODUCTION     Duchenne  muscular  dystrophy  (DMD)  is  a  hereditary  disease  that  affects  ~1:3,500   boys,   the   majority   of   whom   will   die   by   their   midtwenties   (Emery   et   al.,   1993).   DMD   is   caused   by   mutations   in   dystrophin   that   result   in   the   faulty   assembly   and   function   of   an   ensemble   of   structural   and   signaling   molecules   at   the   muscle   cell   surface   termed   the   dystrophin-­‐associated   protein   complex   (DAPC)   (Koenig   et   al.,   1987;   Blake   et   al.,   2002;   Muntoni   et   al.,   2003).   There   are   currently   no   treatments   that   target  the  primary  pathology  of  DMD.   One  attractive  therapeutic  approach  for  DMD  is  the  stabilization  of  the  muscle  cell   membrane   through   up-­‐regulation   of   utrophin,   a   dystrophin   homolog.   Transgenic   overexpression   of   utrophin   rescues   dystrophic   pathology   and   restores   function   in   the   dystrophin-­‐deficient   mdx   mouse   (Tinsley   et   al.,   1998;   Khurana   et   al.,   2003;   Miura   et   al.,   2006).   In   mature   muscle,   utrophin   expression   is   restricted   to   the   neuromuscular   and   myotendinous   junctions.   However,   utrophin   is   expressed   over   the   entire   myofiber   in   developing   and   regenerating   muscle   (Khurana   et   al.,   1991;   Clerk   et   al.,   1993;   Pons   et   al.,   1993).   These   observations   raise   the   possibility   that   marshalling   pathways   that   normally   regulate   utrophin   expression   in   developing   muscle  could  be  a  productive  approach  for  developing  DMD  treatments.   The   extracellular   matrix   protein   biglycan   plays   an   important   role   in   developing   muscle.   In   both   humans   and   mice,   biglycan   is   most   highly   expressed   in   immature   and   regenerating   muscle   (Casar   et   al.,   2004,   Lechner   et   al.,   2006).   Biglycan   is   a   181   component  of  the  DAPC,  where  it  binds  to  α-­‐dystroglycan  (Bowe  et  al.,  2000)  and  α-­‐   and   γ-­‐sarcoglycan   (Rafii   et   al.,   2006).   Biglycan   regulates   the   expression   of   the   sarcoglycans   as   well   as   dystrobrevins,   syntrophins,   and   nNOS,   particularly   in   immature   muscle.   Finally,   biglycan   is   important   for   timely   muscle   regeneration   (Casar  et  al.,  2004).   Locally   delivered   recombinant   human   biglycan   (rhBGN)   incorporates   into   the   extracellular  matrix  of  bgn−/o  muscle  where  it  persists  for  at  least  2  wk  and  rescues   the   expression   of   several   DAPC   components   (Mercado   et   al.,   2006).   These   results   raise   the   possibility   that   rhBGN   might   enhance   function   in   muscle   that   lacks   dystrophin.   Here   we   show   that   utrophin   is   down-­‐regulated   in   immature   biglycan   null   (bgn−/o)   mice   and   that   rhBGN   up-­‐regulates   membrane-­‐associated   utrophin   in   cultured   myotubes.   Importantly,   rhBGN   can   be   delivered   systemically   to   dystrophin-­‐deficient   mdx   mice,   where   it   up-­‐regulates   utrophin   and   other   DAPC   components   at   the   sarcolemma,   ameliorates   muscle   pathology,   and   improves   function.  Several  lines  of  evidence  indicate  that  biglycan  acts  by  recruiting  utrophin   to  the  plasma  membrane.  We  propose  rhBGN  as  a  candidate  therapeutic  for  DMD.   RESULTS     Endogenous  Biglycan  Regulates  Utrophin  Expression  in  Immature  Muscle.       At   postnatal   day   14   (P14),   utrophin   is   highly   expressed   in   the   perisynaptic   sarcolemma   (Figure   4.1A)   (Clerk   et   al.,   1993).   To   compare   utrophin   expression   182   levels   in   the   presence   and   absence   of   biglycan,   we   immunostained   sections   of   muscle  from  bgn−/o  mice  and  age-­‐matched  congenic  controls.  In  all  cases,  the  mutant   and   WT   sections   were   mounted   on   the   same   slides,   stained   together   and   imaged   concurrently  (Materials  and  Methods).  Figure  4.1A  shows  that  utrophin  expression   is  decreased  at  the  perisynaptic  sarcolemma  in  bgn−/o  muscle,  whereas  sarcolemmal   dystrophin  expression  was  unchanged.  Quantification  of  50  sarcolemmal  segments   from   each   of   three   animals   from   each   genotype   showed   that   utrophin   levels   were   reduced   by   ~28%   (Figure   4.1B;   Bgn−/o:   0.72   ±   0.03,   WT:   1.0   ±   0.04,   unpaired   Student   t   test,   P   <   0.0001).   In   contrast,   there   was   no   significant   difference   in   the   expression  of  dystrophin  in  the  sarcolemma  (Figure  4.1C;  Bgn−/o:  1.01  ±  0.03,  WT:   1.00   ±   0.03,   unpaired   Student   t   test,   P   =   0.76).   Notably,   the   amount   of   utrophin   transcript   was   indistinguishable   in   WT   as   compared   with   bgn−/o   P14   muscle   (text   below  and  Figure  4.1D).  These  results  indicate  that  utrophin  protein  expression  at   the  sarcolemma  is  selectively  decreased  in  the  absence  of  biglycan.     RhBGN   Treatment   Up-­Regulates   Membrane-­Associated   Utrophin   in   Cultured   Muscle  Cells.       We   next   turned   to   a   cell   culture   system   to   more   precisely   delineate   the   role   of   biglycan   in   regulating   utrophin   association   with   the   sarcolemma.   We   stimulated   bgn−/o   myotubes   with   1   nM   rhBGN   and   assessed   the   levels   of   utrophin   and   γ-­‐ sarcoglycan   in   membrane   fractions   by   Western   blotting.   As   shown   in   Figure   4.2A,   rhBGN   treatment   up-­‐regulates   utrophin   and   γ-­‐sarcoglycan   protein   in   these   183   membrane   fractions.   On   the   other   hand,   there   was   a   reduction   in   utrophin   transcript  levels  following  rhBGN  treatment  (untreated:  1  ±  0.10;  rhBGN  treated:  0.7   ±   0.06;   unpaired   Student   t   test,   P   =   0.02;   n   =   6   separate   experiments   with   three   replicate   flasks   in   each).   Thus,   the   up-­‐regulation   of   utrophin   protein   expression   at   the  membrane  is  not  associated  with  increases  in  the  level  of  its  transcript.     The   results   described   above   suggest   that   biglycan   could   regulate   utrophin   protein   by  mechanisms  involving  elevated  translation,  increased  stability,  and/or  targeting   of   utrophin   to   the   membrane.   To   distinguish   among   these   possibilities,   we   assessed   the  level  of  total  utrophin  protein  in  control  and  biglycan-­‐treated  cultures.  As  shown   in   Figure   4.2,   total   utrophin   protein   levels   are   indistinguishable   in   treated   and   untreated  myotubes.  The  failure  to  detect  changes  in  total  cellular  utrophin  protein   under   conditions   in   which   the   membrane-­‐bound   fraction   is   increased   indicates   that   biglycan  regulates  the  association  of  utrophin  with  the  membrane.   Systemic  Delivery  of  rhBGN.       The   role   for   biglycan   in   recruiting   utrophin   to   the   membrane,   taken   together   with   previous   results,   showing   that   both   endogenous   biglycan   and   intramuscularly   delivered  rhBGN  can  regulate  DAPC  proteins  in  vivo  (Mercado  et  al.,  2006),  raising   the   possibility   that   rhBGN   could   be   a   therapeutic   agent   for   DMD.   As   a   first   step   toward   developing   such   a   therapy,   we   asked   whether   rhBGN   could   be   delivered   systemically.   A   capture   ELISA   showed   that   rhBGN   was   readily   detected   in   the   184   circulation   30   and   60   min   after   i.p.   delivery   (Supp.   Figure   4.1A).   To   detect   the   recombinant  protein  in  tissue,  where  endogenous  biglycan  is  expressed  (Bowe  et  al.,   2000),   we   injected   animals   i.p.   with   rhBGN   conjugated   to   Alexa-­‐555.   As   shown   in   Supp.  Figure  4.1B,  this  rhBGN  is  readily  detected  in  the  muscle  extracellular  matrix   48   h   following   injection.   These   observations   indicate   that   the   circulating   recombinant  protein  partitions  to  muscle  where  it  becomes  stably  associated  with   the  ECM.  This  result  is  in  agreement  with  our  earlier  findings  that  intramuscularly   delivered   rhBGN   is   stable   in   muscle   for   at   least   2   wk   following   a   single   intramuscular   injection   in   bgn−/o   mice   (Mercado   et   al.,   2006).   This   finding   is   also   consistent  with  the  efficacy  of  rhBGN  observed  2  wk  after  a  single  injection  in  mdx   mice  (discussed  below).  Taken  together,  these  findings  indicate  that  rhBGN  can  be   delivered  systemically  and  can  become  localized  to  muscle  for  prolonged  periods.     RhBGN  Up-­Regulates  Utrophin  and  Other  DAPC  Components  in  mdx  Mice.       We   next   asked   whether   rhBGN   can   up-­‐regulate   utrophin   in   mdx   mice.   A   single   i.p.   dose   of   rhBGN   was   delivered   to   ~P18   mdx   mice,   and   utrophin   levels   at   the   sarcolemma   were   assessed   2   wk   later.   Because   utrophin   expression   increases   transiently   in   regenerating   myofibers   (Helliwell   et   al.,   1992)   and   is   known   to   be   enriched   at   synaptic   and   perisynaptic   regions   (Khurana   et   al.,   1991;   Nguyen   et   al.,   1995),   we   restricted   our   analysis   to   extrasynaptic   areas   of   nonregenerated   (peripherally   nucleated)   myofibers.   As   shown   in   Figure   4.3   A   and   B,   rhBGN   treatment   increased   utrophin   expression   at   the   sarcolemma   >2.5-­‐fold   in   quadriceps   185   muscle  mdx  mice  (vehicle:  1.0  ±  0.05,  rhBGN:  2.5  ±  0.08,  unpaired  Student  t  test,  P  <   0.0001,   n   =   200   sarcolemmal   segments   from   two   animals   from   each   group).   Utrophin   levels   at   the   sarcolemma   were   also   significantly   increased   in   the   tibialis   anterior   muscle   (Supp.   Figure   4.2,   vehicle:   1.0   ±   0.1,   rhBGN:   1.7   ±   0.1,   unpaired   Student  t  test;  n  =  300  sarcolemmal  segments  from  three  animals  from  each  group).     The  levels  of  γ-­‐sarcoglycan,  β2-­‐syntrophin,  and  nNOS  are  also  increased  at  the   sarcolemma  following  a  single  dose  of  rhBGN  (Figure  4.4).  We  observed  no  change   in  α-­‐syntrophin  levels.  The  elevation  in  γ-­‐sarcoglycan  and  nNOS  is  in  agreement   with  our  observations  in  cell  culture,  in  which  rhBGN  treatment  increased  the  levels   of  these  proteins  at  the  membrane  (Figure  4.2)  (Mercado  et  al.,  2006).  Furthermore,   these  proteins  as  well  as  β2  syntrophin  are  dysregulated  in  bgn−/o  mice  (Rafii  et  al.,   2006;  Mercado  et  al.,  2006).  Western  blotting  of  membrane  fractions  provided   further  evidence  that  rhBGN  treatment  increased  the  levels  of  both  utrophin  and  γ-­‐ sarcoglycan  mdx  mice  (Figure  4.3  C  and  D).  Taken  together,  these  results  indicate   that  rhBGN  treatment  restores  the  expression  of  utrophin  and  DAPC  proteins  to  the   sarcolemma.     Utrophin   transcript   levels   were   unchanged   in   rhBGN-­‐treated   mdx   (Figure   4.3C).   This   finding   is   in   agreement   with   our   in   vivo   and   cell   culture   results   with   bgn−/o   muscle   (Figure   4.1   and   4.2),   and   indicates   than   rhBGN   regulates   utrophin   in   mdx   mice  at  a  posttranscriptional  level.  Finally,  these  results  show  that  rhBGN  effects  can   be  observed  after  multiple  doses  spanning  6–13  wk  of  treatment  (Figure  4.3D  and   186   E).  Taken  together,  these  immunohistochemical  and  biochemical  results  show  that   systemically   delivered   rhBGN   can   up-­‐regulate   utrophin   and   other   DAPC   protein   in   the  membranes  of  dystrophic  mice.   RhBGN  Reduces  Dystrophic  Pathology  in  mdx  Mice.       To  determine  whether  rhBGN  counters  dystrophic  pathology  in  mdx  mice,  we  first   administered  a  single  i.p.  dose  of  rhBGN  or  vehicle  alone  to  ~P18  mdx  mice  and   assessed  muscle  histologically  2  or  3  wk  later.  Figure  4.5A  (Upper  Panel)  shows  a   section  of  diaphragm  from  vehicle-­‐injected  mice  displaying  characteristic   dystrophic  pathology  including  a  high  proportion  of  centrally  nucleated  fibers   (CNFs)  and  foci  of  necrosis/regeneration  and  areas  of  mononuclear  cell  infiltration   (Coulton  et  al.,  1988).  Strikingly,  rhBGN  treatment  resulted  in  a  ~50%  reduction  in   the  proportion  of  CNFs  observed  in  muscle  from  rhBGN  treated  mice  (17.7%  ±  2.8   and  9.6%  ±  1.7  for  vehicle-­‐  and  rhBGN-­‐injected  animals,  respectively;  unpaired   Student  t  test,  P  =  0.028,  n  =  13  vehicle-­‐  and  11  rhBGN-­‐injected  animals;  Figure   4.5B).  We  also  assessed  serum  creatine  kinase  (CK)  levels,  a  marker  of  muscle   damage,  in  mice  that  had  been  given  1,  2,  or  10  mg/kg  rhBGN.  As  reported  by  others   (Coulton  et  al.,  1988),  there  was  considerable  variation  in  the  baseline  levels  of  CK   among  experiments.  Although  we  observed  a  trend  toward  decreased  CK  levels  in   these  animals,  the  data  did  not  reach  statistical  significance  (Supp.  Figure  4.3).   Taken  together,  these  findings  indicate  that  rhBGN  treatment  reduces  dystrophic   pathology  in  mdx  mice.   187     RhBGN  Efficacy  Is  Utrophin  Dependent.       We  next  asked  whether  the  ability  of  rhBGN  to  counter  dystrophic  pathology  in  mdx   mice  is  dependent  upon  utrophin.  If  utrophin  is  necessary  for  rhBGN  action  in  mdx   mice,   the   pathology   of   mice   mutant   for   both   utrophin   and   dystrophin   would   be   unaffected  by  rhBGN  administration.  Supp.  Figure  4.4  shows  that  the  histology  and   number   of   regenerated   muscle   fibers   in   mdx:utrn−/−   mice   were   indistinguishable   after   a   single   injection   of   rhBGN   or   vehicle.   Thus,   utrophin   is   necessary   for   the   therapeutic  action  of  rhBGN.     RhBGN  Treatment  Improves  Muscle  Function  in  mdx  Mice.       An  effective  treatment  for  DMD  must  improve  muscle  function.  One  of  the  primary   causes  of  myofiber  pathology,  dysfunction,  and  death  in  DMD  is  increased   susceptibility  to  contraction-­‐induced  damage.  Such  muscle  damage  can  be  assessed   ex  vivo  by  measuring  the  force  produced  after  each  of  several  successive  eccentric   (lengthening)  contractions  (ECCs)  (Moens  et  al.,  1993;  Gillis  et  al.,  1999).  In  these  ex   vivo  mdx  muscles,  susceptibility  to  injury  is  evidenced  by  an  increase  in  force  drop   after  a  series  of  ECCs.  We  injected  mdx  mice  at  3-­‐wk  intervals  (starting  at  P14)  with   either  rhBGN  or  vehicle  until  15  wk  of  age,  and  measured  muscle  physiology  as   previously  described  (Bogdanovich  et  al.,  2002;  Bogdanovich  et  al.,  2005).  RhBGN   treatment  improved  performance  on  muscle  function  measurements,  as  shown  by  a   188   reduced  amount  of  force  drop  following  each  consecutive  ECC  (Figure  4.6  C  and  D).   This  improvement  was  robust  and  statistically  significant  from  the  second  ECC   onward  (Figure  4.6C.).  We  observed  no  change  in  other  parameters  of  muscle   function  including  the  amount  of  specific  force  generated  (Supplementary  Table   4.1).  Such  a  profile  of  physiological  improvement—increased  resistance  to  damage   with  no  change  in  specific  force—is  similar  to  that  observed  with  adeno-­‐associated   virus  delivery  of  microdystrophin  (R4–R23)  (Liu  et  al.,  2005)  or  heregulin  treatment   (Krag  et  al.,  2004).  Thus  rhBGN  treatment  improves  muscle  function  in  mdx  mice.     RhBGN  Is  Well  Tolerated  in  mdx  Mice.       We   have   not   observed   deleterious   effects   of   rhBGN   administration   in   mdx   mice,   even  after  3  mo  of  treatment.  Organ  weight  is  a  long-­‐standing  and  widely  accepted   measure   of   pharmacological   toxicity   (Michael   et   al.,   2007;   Peters   et   al.,   1966).   As   shown  in  Supp.  Figure  4.5A,  there  were  no  significant  differences  in  the  weights  of   the   liver,   kidney,   lung,   or   spleen.   There   was   an   8%   decrease   in   the   weight   of   the   heart.   Whole-­‐animal   weights   were   equivalent   in   vehicle-­‐   and   rhBGN-­‐dosed   animals.   Muscle   weights   were   also   unchanged   with   the   exception   of   the   soleus,   which   was   17%  larger  in  rhBGN-­‐treated  animals.  Furthermore,  no  indication  of  kidney  or  liver   dysfunction  was  observed:  there  were  no  significant  changes  in  the  levels  of  serum   creatinine,   blood   urea   nitrogen   (BUN),   aspartate   transaminase   (AST),   or   bilirubin   at   single  doses  ranging  from  1  to  10  mg/kg  (Supp.  Figure  4.5B).     189   DISCUSSION     In   this   report,   we   introduce   a   unique   therapeutic   approach   for   DMD   based   upon   the   systemic   delivery   rhBGN,   a   recombinant   form   of   the   extracellular   matrix   protein   biglycan.   Several   characteristics   of   rhBGN   suggest   that   it   could   be   an   effective   therapy   for   DMD.   (i)   RhBGN   counters   dystrophic   pathology   and   improves   muscle   function.  (ii)  Systemically  delivered  rhBGN  localizes  to  muscle  and  a  single  dose  is   effective  for  up  to  3  wk.  Multiple  doses  at  3-­‐wk  intervals  can  sustain  the  response   for  at  least  3  mo.  (iii)  RhBGN  acts  at  least  in  part  through  utrophin,  a  pathway  that   has  been  extensively  validated  in  animal  studies  (Tinsley  et  al.,  1998,  Ebihara  et  al.,   2000;  Cerletti  et  al.,  2003).  (iv)  RhBGN  restores  the  expression  of  DAPC  components   that   are   important   for   muscle   integrity   and   function.   (v)   RhBGN   could   selectively   target   the   tissues   affected   in   DMD,   as   it   binds   to   α-­‐   and   γ-­‐sarcoglycan   (Rafii   et   al.,   2006),  which  are  components  of  the  dystrophin/utrophin  protein  complex  and  are   expressed  selectively  in  heart  and  skeletal  muscle  (Hack  et  al.,  2000;  Barresi  et  al.,   2000;   Wheeler   et   al.,   2003;   Anastasi   et   al.,   2007).   (vi)   RhBGN   is   well   tolerated   in   mdx   mice.   (vii)   Endogenous   biglycan   is   expressed   in   normal   and   DMD   muscle   (Haslett  et  al.,  2002;  Zanotti  et  al.,  2005)  and  is  a  highly  conserved  protein.  RhBGN   could   thus   be   expected   to   elicit   a   minimal   immune   response.   (viii)   RhBGN   is   nonglycanated   (i.e.,   lacking   glycosaminoglycan   side   chains).   This   relatively   uncomplicated  structure  simplifies  its  manufacture  in  a  homogeneous  form.   Several   lines   of   evidence   suggest   that   rhBGN   counters   pathology   and   improves   190   muscle   function   through   the   up-­‐regulation   of   utrophin   and   other   DAPC   components   at   the   sarcolemma.   First,   rhBGN   treatment   up-­‐regulates   sarcolemmal   utrophin   in   both   acute   (single   dose)   and   prolonged,   multidose   paradigms   (Figure   4.3).   Utrophin   is  necessary  for  rhBGN  action,  as  we  observed  no  improvement  in  muscle  pathology   in  mdx:utrophin  double  null  mice  (Supp.  Figure  4.3).  Importantly,  up-­‐regulation  of   utrophin   by   increasing   gene   expression   (Tinsley   et   al.,   1998)   or   rhBGN   treatment,   which  recruits  utrophin  protein  to  the  sarcolemma,  both  result  in  assembly  of  DAPC   components  and  improvement  in  muscle  function  as  measured  by  resistance  to  ECC.   Furthermore,   as   discussed   below,   the   recruitment   of   utrophin   and   other   DAPC   components   to   the   sarcolemma,   rather   than   the   global   up-­‐regulation   of   utrophin   mRNA  or  protein,  is  likely  to  be  the  therapeutically  salient  feature  of  rhBGN  action.   Our  results  show  that  a  single  systemic  injection  of  rhBGN  is  active  for  a  strikingly   long  period  (Figure  4.3  and  4.5).  This  prolonged  action  of  rhBGN  is  consistent  with   our  previous  studies  in  bgn−/o  mice  showing  that  intramuscularly  delivered  rhBGN   is   stable   and   biologically   active   for   3   wk   (Mercado   et   al.,   2006).   This   protracted   action   seems   likely   to   result   from   the   binding   of   rhBGN   to   the   ECM.   Circulating   levels  of  rhBGN  fall  rapidly  and  are  undetectable  by  24  h  after  i.p.  injection  (Supp.   Figure   4.1A).   However,   rhBGN   is   readily   detected   in   the   muscle   ECM   2   d   after   i.p.   injection.  This  stable  association  could  be  due  in  part  to  binding  to  collagen  VI  in  the   ECM  (Wilberg  et  al.,  2001)  and  to  sarcoglycans  at  the  sarcolemma  (Rafii  et  al.,  2006).   The  long-­‐acting  properties  of  systemically  delivered  rhBGN  in  mice  suggest  that  this   therapeutic   strategy   could   be   practical   for   use   in   humans,   where   treatment   will   191   likely  be  required  for  years.   The  results  presented  here  indicate  that  rhBGN  acts  by  recruiting  utrophin  protein   to  the  sarcolemma.  In  cell  culture,  rhBGN  rapidly  up-­‐regulates  utrophin  content  in   membrane  fractions,  but  there  is  no  increase  in  total  utrophin  protein  levels  (Figure   4.2).   In   vivo,   utrophin   levels   at   the   sarcolemma   of   immature   biglycan−/o   mice   are   decreased,   whereas   transcript   levels   are   unchanged   (Figure   4.1).   Furthermore,   treatment   of   mdx   mice   with   rhBGN   results   in   up-­‐regulation   of   utrophin   at   the   sarcolemma   with   no   increase   in   transcript   levels   (Figure   4.3).   The   posttranscriptional   action   of   rhBGN   is   further   supported   by   its   ability   to   increase   the   levels   of   membrane-­‐associated   utrophin   in   cultured   myotubes   after   8   h   of   treatment;   this   interval   is   far   less   than   the   16   h   required   to   synthesize   a   mature   utrophin  transcript  (Tennyson  et  al.,  1995).  The  data  in  cultured  myotubes  (Figure   4.2)   are   consistent   with   a   model   in   which   increased   levels   of   membrane   (but   not   total)   utrophin   provide   negative   feedback   for   utrophin   transcript   levels.   Taken   together,  our  observation  support  the  proposal  that  the  recruitment  of  utrophin  and   other   DAPC   components   to   the   membrane   is   the   mechanism   by   which   rhBGN   counters   dystrophic   pathology   in   mdx   mice.   It   is   of   particular   note   that   total   utrophin   protein   levels   are   up-­‐regulated   in   DMD   muscle   (Love   et   al.,   1991;   Khurana   et  al.,  1990;  Gramolini  et  al.,  1999).  Therefore  rhBGN  can  be  expected  to  be  effective   in  DMD  patients.   Systemically   delivered   rhBGN   increases   nNOS   at   the   sarcolemma   (Figure   4.4).   We   have  previously  reported  that  biglycan  increases  nNOS  at  the  membrane  in  cultured   192   myotubes   (Mercado   et   al.,   2006).   These   data   are   in   agreement   with   studies   by   Sonnemann   et   al.   (Sonnemann   et   al.,   2009),   in   which   delivery   of   TAT-­‐μutr   protein   restores   sarcolemmal   nNOS   in   mdx   mice.   These   observations   are   of   particular   interest,   as   up-­‐regulation   of   nNOS   could   counter   fatigue   in   dystrophic   muscle   (Kobayashi  et  al.,  2008).  However,  studies  using  viral  delivery  of  utrophin  failed  to   detect   rescue   of   nNOS   expression   at   the   membrane   (Li   et   al.,   2010).   The   basis   for   this   discrepancy   is   unknown.   One   possibility   is   that   are   multiple   mechanisms   of   utrophin-­‐mediated  DAPC  restoration.  For  example,  rhBGN  binds  DAPC  components   at  the  cell  surface,  a  property  that  could  promote  the  assembly  of  a  more  complete   utrophin-­‐associated  complex,  including  nNOS.   The   biglycan-­‐mediated   recruitment   of   utrophin   to   the   sarcolemma   represents   a   novel   pathway   for   DMD   treatment.   Previous   work   has   shown   that   utrophin   expression   is   also   regulated   at   transcriptional   and   translational   levels,   and   efforts   are   underway   to   develop   therapies   that   target   these   mechanisms   (Khurana   et   al.,   2003,  Gramolini  et  al.,  1998;  Gramolini  et  al.,  2001;  Miura  et  al.,  2008).  In  addition  to   having   therapeutic   efficacy   on   its   own,   the   unique   action   of   biglycan   in   recruiting   utrophin   to   the   sarcolemma   could   synergize   with   these   other   utrophin-­‐directed   strategies.   Finally,   rhBGN   could   be   used   in   combination   with   therapies   aimed   at   increasing   muscle   mass   (Bogdanovich   et   al.,   2002;   Bogdanovich   et   al.,   2005),   reducing   inflammation   (Merlini   et   al.,   2003,   Biggar   et   al.,   2004),   or   restoring   dystrophin  by  antisense  oligonucleotide-­‐mediated  exon  skipping  (Mann  et  al.,  2001;   van  Deutekom  et  al.,  2007).   193   Numerous  protein-­‐based  therapies  for  a  range  of  human  disorders  are  currently  in   the  clinic,  and  many  more  are  in  development.  The  methods  for  the  manufacture   and  delivery  of  protein  therapeutics  are  well  understood.  Furthermore,  as  a  class,   protein  therapies  have  proved  to  be  remarkably  safe.  Therefore,  the  path  from  these   laboratory  studies  to  clinical  trials  of  rhBGN-­‐based  DMD  therapies  is  clear.     MATERIALS  AND  METHODS     Biglycan.   Recombinant,   nonglycanated   human   biglycan   (rhBGN)   was   produced   in   mammalian   cells   and   purified   as   previously   described   (Mercado   et   al.,   2006).   This   form   lacks   GAG   side   chains.   The   Alexa   555   protein   labeling   kit   (Invitrogen   Corporation)  was  used  to  conjugate  this  fluor  to  rhBGN.     Animals  and  Injections.  All  protocols  were  conducted  under  accordance  and  with   the   approval   of   Brown   University's   Institutional   Animal   Care   and   Use   Committee.   For  single  injections,  P16-­‐19  mice  received  an  i.p.  injection  of  100  μg  rhBGN  in  25  μL   20   mM   Tris,   0.5M   NaCl,   0.2%   CHAPS,   or   vehicle   (20   mM   Tris,   0.5   M   NaCl,   0.2%   CHAPS).  Multiply  injected  mice  received  additional  i.p.  injections  of  100  μg  rhBGN  or   vehicle  at  3-­‐wk  intervals.  Mice  were  harvested  13–25  d  after  the  final  injection.  For   tracing   studies,   adult   mdx   mice   received   an   i.p.   injection   of   Alexa   555-­‐labeled   rhBGN,  and  diaphragms  were  harvested  48  h  later.     Histology   and   Immunohistochemistry.   Frozen   sections   were   prepared   and   194   stained   as   previously   described   (Mercado   et   al.,   2006).   For   bgn−/o   analysis,   P14   congenic  bgn−/o  and  WT  sections  were  mounted  on  the  same  slide,  immunostained   simultaneously,   and   imaged   with   a   cooled   CCD   camera   in   the   same   session   using   identical   exposures.   All   comparisons   of   sections   from   injected   mice   (vehicle   and   rhBGN)   were   also   mounted,   stained   and   imaged   together.   Sections   were   observed   using   a   Nikon   (Melville,   NY)   Eclipse   E800   microscope   and   images   acquired   with   Scanalytics   IP   Lab   Spectrum   software   or   NIS   Elements   (Nikon).   Utrophin   and   dystrophin   immunoreactivity   intensity   was   quantified   using   Metamorph   image   analysis   software   (Universal   Imaging)   or   ImageJ   software   (National   Institutes   of   Health).   We   also   observed   structures   in   the   interstitial   space,   which   may   be   blood   vessels,   that   showed   increased   utrophin   in   some   experiments   (Fig.   3).   These   structures  were  not  included  in  our  measurements.  The  average  pixel  intensities  of   sarcolemmal   segments   were   measured,   and   the   mean   background   (determined   by   measuring   nonsarcolemmal   regions   from   each   condition)   was   subtracted   from   them.   The   average   background   levels   were   indistinguishable   between   conditions.   Analysis  in  mdx  mice  was  performed  on  quadriceps  from  two  mice  of  each  condition   and  on  TAs  from  three  mice  of  each  condition.  Sources  and  conditions  for  antibodies   are  given  in   SI   Materials   and   Methods.   For   scoring   the   percentage   of   CNFs,   all   cross-­‐ sectioned   myofibers   outside   of   necrosis/regenerative   foci   in   H&E-­‐stained   sections   were  counted  under  a  20×  objective  (270–1,913  fibers/muscle  section).     Quantitative   RT-­PCR   and   Western   Blot   Analysis.   Utrophin   transcript   levels   were   measured  using  SYBR-­‐Green  (Invitrogen)  as  described  in  SI  Materials  and  Methods.   195   Culture   methods,   preparation   of   lysates,   and   membrane   fractions   and   analysis   by   Western  blot  were  by  standard  procedures  detailed  in  SI  Materials  and  Methods.     Muscle  Physiology.  Mdx  mice  were  injected  i.p.  with  rhBGN  (25  μg/animal)  or   vehicle  every  3  wk  starting  at  P14  and  the  physiological  properties  of  the  extensor   digitorum  longus  (EDL)  muscles  were  analyzed  ex  vivo  at  3.5  mo  of  age  as  described   previously  (Bogdanovich  et  al.,  2002;  Bogdanovich  et  al.,  2005).  Muscle  length  was   adjusted  to  achieve  maximal  twitch  response  and  this  length  (Lo)  was  measured.   Eccentric  contraction  force  decrease  was  calculated  for  each  tetanus  of  a  standard   ECC  protocol  of  supramaximal  stimulus  700  ms,  total  lengthening  Lo/10;   lengthening  velocity  0.5  Lo/s.  EDL  sections  were  obtained  and  images  were   acquired  as  above.  Cross-­‐sectional  area  was  measured  using  ImageJ  software   (National  Institutes  of  Health).                                         196     FIGURES     Figure  4.1                   197     Figure  4.1.  Utrophin  is  reduced  at  the  sarcolemma  of  immature  bgn−/o  mice.       (A)  Quadriceps  muscles  from  congenic  P14  WT  (Upper  Panels)  DJS  and  bgn−/o   (Lower  Panels)  mice  were  harvested,  sectioned,  mounted  on  the  same  slides,  and   immunostained  for  dystrophin  and  utrophin.  Utrophin  expression  is  decreased  in   these  developing  biglycan  null  mice  compared  with  WT  mice,  whereas  dystrophin   expression  is  not  detectably  altered.  (Scale  bar  =  25  μm.)  (B)  Quantification  of   sarcolemmal  utrophin  expression.  Images  of  utrophin-­‐stained  muscle  sections  as   prepared  in  A  were  acquired  and  the  levels  of  utrophin  immunostaining  at  the   perijunctional  sarcolemma  were  measured  as  described  in  Materials  and  Methods.  A   total  of  50  sarcolemmal  segments  from  each  of  three  animals  from  each  genotype   were  analyzed.  Utrophin  immunoreactivity  was  decreased  28%  in  sections  from   bgn−/o  muscle  compared  with  WT  (Bgn−/o:  0.72  ±  0.03,  WT:  1.0  ±  0.04,  unpaired   Student  t  test,  P  <  0.0001;  n  =  150  sarcolemmal  segments  from  three  mice  of  each   genotype).  (C)  Quantification  of  perijunctional  sarcolemmal  dystrophin.  Dystrophin-­‐ stained  sections  were  imaged  and  measured  as  in  B.  Dystrophin  immunoreactivity   was  equivalent  in  P14  WT  and  bgn−/o  sections  (Bgn−/o:  1.01  ±  0.03,  WT:  1.00  ±   0.03,  unpaired  Student  t  test,  P  =  0.76).  (D)  Quantitative  real-­‐time  PCR  analysis  of   utrophin  transcripts  in  P14  WT  and  bgn−/o  mice.  Total  RNA  was  extracted  from   quadriceps  muscles  from  WT  and  bgn−/o  mice  and  used  for  cDNA  synthesis.   Expression  of  utrophin  mRNA  was  indistinguishable  in  WT  and  Bgn−/o  muscles   (WT:  1.0  ±  0.26,  Bgn−/o:  0.99  ±  0.09,  n  =  3  animals  from  each  genotype).     198         Figure  4.2.                                                   199             Figure  4.2.  RhBGN  treatment  increases  membrane-­‐associated  utrophin  and  γ-­‐ sarcoglycan  protein  in  cultured  myotubes.       (A)  Cultured  bgn−/o  myotubes  were  incubated  for  8  h  with  either  1  nM  rhBGN  or   vehicle  as  indicated.  Shown  are  Western  blots  of  membrane  fractions  probed  for   utrophin  and  γ-­‐sarcoglycan  (γ-­‐SG).  Note  the  increased  expression  of  both  utrophin   and  γ-­‐sarcoglycan  following  rhBGN  treatment.  (B)  Bgn−/o  myotubes  were  treated   as  in  A  and  whole-­‐cell  extracts  were  prepared.  Proteins  were  separated  by   SDS/PAGE  and  immunoblotted  for  utrophin  and  actin  (loading  control).  Total   utrophin  protein  levels  were  similar  in  untreated  and  rhBGN  treated  cultures.  (C)   Quantitative  RT-­‐PCR  analysis  of  untreated  and  rhBGN  treated  cultured  bgn−/o   myotubes.  RhBGN  treatment  decreased  utrophin  transcript  levels  by  ~30%   (untreated:  1  ±  0.10;  rhBGN  treated:  0.7  ±  0.06;  unpaired  Student  t  test,  P  =  0.02;  n  =   6  separate  experiments  with  three  replicate  flasks  in  each).                             200         Figure  4.3.                                 201         Figure  4.3.  RhBGN  treatment  up-­‐regulates  utrophin  at  the  sarcolemma  of  mdx  mice.       (A)  Utrophin  immunostaining  of  quadriceps  muscles  from  P33  mdx  littermate  mice   that  received  a  single  i.p.  injection  of  either  rhBGN  or  vehicle  at  P19.  (Scale  bar  =  25   μm.)  (B)  Levels  of  immunostaining  at  the  sarcolemma  (e.g.,  arrows  in  A)  of   peripherally  nucleated  fibers.  A  total  of  100  sarcolemmal  segments  from  each  of   four  animals  were  analyzed  (two  littermate  pairs,  one  rhBGN-­‐  and  one  vehicle-­‐ injected  animal  per  pair).  Sarcolemmal  utrophin  immunoreactivity  was  >2.5-­‐fold   higher  in  sections  from  rhBGN-­‐  as  compared  with  vehicle-­‐injected  animals   (unpaired  Student  t  test,  P  <  0.0001).  (C)  qRT  PCR  analysis  of  utrophin  transcripts  in   from  vehicle-­‐  or  rhBGN-­‐injected  mdx  mice.  There  was  no  significant  difference  in   utrophin  transcript  levels  in  rhBGN  treated  mice  compared  with  vehicle-­‐injected   controls  (unpaired  Student  t  test,  P  =  0.057;  n  =  8  vehicle-­‐  and  6  rhBGN-­‐treated   mice).  (D)  RhBGN  treatment  increases  utrophin  expression  in  muscle  membrane   fractions.  Mdx  mice  from  a  single  litter  were  injected  at  P16  and  P38  (Left  Pair)  or   P16,  P38,  and  P63  (Right  Pair)  with  rhBGN  or  vehicle.  Muscles  were  harvested  3  wk   after  the  last  injection.  (E)  RhBGN  treatment  increases  γ-­‐sarcoglycan  expression.   Mdx  mice  were  injected  at  3-­‐wk  intervals  starting  at  P14  with  rhBGN  or  vehicle   alone.  Muscles  were  harvested  at  15  wk  of  age  and  immunoblotted  for  γ-­‐ sarcoglycan.  γ-­‐Sarcoglycan  is  increased  in  the  membrane  fractions  from  rhBGN   treated  mdx  mice  compared  with  vehicle-­‐treated  animals.     202                 Figure  4.4.                                   203                       Figure  4.4.  RhBGN  up-­‐regulates  DAPC  components  at  the  sarcolemma  of  mdx  mice.       Mdx  mice  were  injected  with  rhBGN  or  vehicle  at  P18  and  muscles  were  harvested   at  32P.  Sections  of  TA  from  vehicle-­‐  or  rhBGN-­‐treated  animals  were  immunostained   with  antibodies  to  the  indicated  DAPC  components  as  described  in  Materials  and   Methods.  RhBGN  treatment  increased  the  expression  of  sarcolemmal  γ-­‐sarcoglycan,   β2-­‐syntrophin,  and  nNOS  in  mdx  mice.                         204       Figure  4.5.                                   205       Figure  4.5.  Systemically  administered  rhBGN  counters  dystrophic  pathology  in  mdx   mice.       (A)  H&E-­‐stained  sections  of  diaphragm  from  littermate  mdx  mice  that  were  injected   i.p.  with  vehicle  (Upper  Panels)  or  100  μg  rhBGN  (Lower  Panels)  at  P18  and   harvested  at  P38.  (Right  Panels)  Magnified  view.  Note  the  extensive  areas  of   necrosis/regeneration  and  mononuclear  cell  infiltration  in  muscle  from  vehicle-­‐ injected  as  compared  with  rhBGN-­‐injected  mice.  (Scale  bars  =  50  μm.)  (B)  RhBGN   administration  decreases  proportion  of  CNFs  in  mdx  muscle  compared  with  vehicle-­‐ injected  littermates  (single  injection;  Materials  and  Methods).  Percentages  of  CNFs   were  determined  from  H&E-­‐stained  diaphragm  sections.  RhBGN-­‐treated  mdx  mice   had  ~50%  fewer  centrally  nucleated  myofibers  as  compared  with  vehicle-­‐injected   mdx  mice  (17.7%  ±  2.8  and  9.6%  ±  1.7  for  vehicle-­‐  and  rhBGN-­‐injected  animals,   respectively;  n  =  13  vehicle-­‐injected  and  11  rhBGN-­‐injected  animals;  unpaired   Student  t  test,  P  =  0.028).                           206     Figure  4.6                               207     Figure  4.6.  Physiological  improvement  of  muscle  in  rhBGN-­‐treated  mdx  mice.       Mdx  mice  were  injected  at  3-­‐wk  intervals  starting  at  P14  with  either  rhBGN  (25   μg/injection;  i.p.)  or  vehicle  and  tissue  was  harvested  at  15  wk  of  age.   Representative  first  to  fifth  ECCs  of  extensor  digitorum  longus  (EDL)  muscles  from   mdx  mice  injected  with  (A)  vehicle,  or  (B)  rhBGN.  (C)  Comparisons  of  ECC  force   drop  between  the  first  and  the  second,  third,  fourth,  and  fifth  ECC  of  vehicle-­‐treated   (6.4  ±  1.2%;  12.4  ±  1.9%;  18.4  ±  2.3%;  22.2  ±  7%;  n  =  16)  and  rhBGN-­‐treated  (3.9  ±   0.3%;  7.5  ±  0.5%;  11.6  ±  0.8%;  14.9  ±  1.2%;  n  =  16)  mdx  mice,  respectively.  There  is   significant  difference  in  the  force  drop  between  ECCs  of  vehicle-­‐treated  and  rhBGN-­‐ treated  mdx  mice  on  the  second,  third,  fourth,  and  fifth  contractions  (P  =  0.05,  0.02,   0.01,  0.02,  respectively;  unpaired  Student  t  test).  (D)  Average  force  drop  between   first  and  fifth  ECC  in  vehicle-­‐treated  and  rhBGN-­‐treated  mdx  mice  (22.2  ±  2.7%   vs.14.9  ±  1.2%,  respectively;  P  =  0.02;  n  =  16  muscles  in  each  group;  unpaired   Student  t  test).                               208   REFERENCES     Anastasi  G,  et  al.  (2007)  Sarcoglycan  subcomplex  expression  in  normal  human   smooth  muscle.  J  Histochem  Cytochem  55:831–843.     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oligonucleotide  PRO051.  N  Engl  J  Med  357:2677–2686.     Wheeler  MT,  McNally  EM  (2003)  Sarcoglycans  in  vascular  smooth  and  striated   muscle.Trends  Cardiovasc  Med  13:238–243.     Wiberg  C,  et  al.  (2001)  Biglycan  and  decorin  bind  close  to  the  n-­‐terminal  region  of   the  collagen  VI  triple  helix.  J  Biol  Chem  276:18947–18952.     Zanotti  S,  et  al.  (2005)  Decorin  and  biglycan  expression  is  differentially  altered  in   several  muscular  dystrophies.  Brain  128:2546–2555.                                         212   SUPPLEMENTARY INFORMATION SI  Materials  and  Methods       Western  Blot  Analysis.  For  cell  membrane  preparations,  biglycan  null  myotubes   were  washed  in  PBS,  scraped  from  tissue  culture  flasks  and  homogenized  in   dissection  buffer  (0.3  M  sucrose,  35  mM  Tris,  pH  7.4,  10  mM  EDTA,  10  mM  EGTA   ,and  protease  inhibitor  mixture;  Roche  Applied  Science).  Samples  were  centrifuged   at  7,000  ×  g  at  4  °C  for  5  min.  Membranes  were  then  collected  by  centrifugation  of   the  supernatants  at  38,000  ×  g  for  60  min  at  4  °C.  Protein  concentrations  were   determined  by  the  bicinchoninic  acid  protein  concentration  assay  (Pierce).  For  total   protein  extraction  from  biglycan  null  myotubes,  cells  were  washed  in  PBS  and  sol-­‐   ubilized  in  RIPA  lysis  buffer  (Santa  Cruz  Biotechnology)  for  25  min,  lysates  were   centrifuged  at  10,000  ×  g,  and  supernatants  were  collected.  Membrane  fractions   from  quadriceps  and  biceps  femoris  were  prepared  as  previously  described  (Emery,   1993).     Cell  or  muscle  fractions  were  separated  by  SDS/PAGE  and  proteins  were  transferred   to  nitrocellulose  membranes.  Total  protein  staining  (SYPRO  Ruby;  Invitrogen)  was   visualized  on  a  Storm  Imager  (Amersham  Bioscience).  Blots  were  incubated  with   primary  antibody  followed  by  goat  anti-­‐mouse  IgG  conju-­‐  gated  to  HRP   (Amersham).  Signal  was  detected  with  ECL  plus  (Amersham)  using  a  Storm  Imager.     Quantitative  RT-­PCR.  RNA  extraction  from  the  biglycan  null  immortalized  muscle   213   cell  line  and  quadriceps  femoris  muscles  from  injected  mdx  animals  was  performed   using  the  TRIzol  method  (Invitrogen).  Purified  RNA  was  converted  to  cDNA  using   the  SuperScript  III  First-­‐Strand  Synthesis  System  Kit  (Invitrogen).  qPCR  reactions   were  performed  using  the  SYBR-­‐Green  method  (Invitrogen)  on  the  ABI  PRISM  7300   real-­‐time  thermocycler.  Primers  were  designed  using  DS  Gene  primer  design   software  (Accelrys).  ATP  synthase  was  used  for  normalization.  Data  analysis  was   performed  using  the  standard  curve  method  (Koenig  et  al.,  1987).     The  primers  used  were  as  follows:  ATPSase  forward:  5′-­‐TGG  GAA  AAT  CGG  ACT  CTT   TG-­‐3′;  ATPSase  reverse:  5′-­‐AGT  AAC  CAC  CAT  GGG  CTT  TG;  Utrophin  forward:  5′-­‐   TCC  CAA  GAC  CCA  TTC  AAC  CC;  Utrophin  reverse:  TGG  ATA  GTC  AGT  GTT  TGG  TTC   C  (gi110431377;  3′  UTR  between  bases  10383–12382).     Animals.  Congenic  biglycan  null  mice  on  a  C3H  background  were  generated  as   described  previously  (1)  and  were  compared  with  WT  C3H  from  the  Jackson   Laboratory.  C57BL/10ScSn-­‐mdx/J  mice  were  obtained  from  Jackson  Laboratory;   mdx:utrn−/−  mice  were  bred  as  described  (Blake  et  al.,  2002).     Antibodies.  The  following  primary  antibodies  were  used:  mono-­‐  clonal  anti-­‐ utrophin  (Vector  Labs),  rabbit  anti-­‐utrophin  (a  gen-­‐  erous  gift  of  S.  Froehner,   University  of  Washington,  Seattle,  WA),  rabbit  anti-­‐dystrophin  (Abcam),  monoclonal   anti–γ-­‐sarcoglycan  (Vector),  rabbit  anti-­‐laminin  (Sigma),  rabbit  anti–β2-­‐syntrophin   (Muntoni  et  al.,  2003),  and  rabbit  anti-­‐nNOS  (Invitrogen).  The  specificity  of  the   214   monoclonal  anti-­‐biglycan  (2A5)  (Emery,  1993)  and  rabbit  anti-­‐biglycan  (Tinsley  et   al.,  1998)  was  established  by  Western  blot  (Emery,  1993;  Tinsley  et  al.,  1998;   Khurana  et  al.,  2003)  and  ELISA  (Results);  no  reactivity  was  observed  when  these   reagents  were  tested  on  bi-­‐  glycan  null  samples.  The  following  secondary  antibodies   were  used:  Alexa  488  goat  anti-­‐mouse  IgG  and  Alexa  555  goat  anti-­‐  rabbit  IgG   (Invitrogen),  HRP  goat  anti-­‐mouse  IgG,  and  HRP  goat  anti-­‐rabbit  IgG.     Cell  Culture.  Immortalized  biglycan  null  cells  were  grown  as  previously  described   (Emery,  1993).  Cells  were  differentiated  for  4–5  d  and  then  treated  with  1  nm   rhBGN  in  differentiation  medium  for  8  h.     Serum  Chemistries.  Blood  was  collected  by  cardiac  puncture  from  rhBGN  and   vehicle  injected  mice  and  spun  at  3,300  RPM  for  10  min  to  separate  serum.  Serum   creatine  kinase,  BUN,  creati-­‐  nine,  AST,  and  total  bilirubin  analyses  were  performed   by  the  University  of  Californai–Davis  Comparative  Pathology  Laboratory.   Detection  of  rhBGN  in  Serum.  Adult  C57/B6  mice  were  injected  i.p.  with  10  mg/kg   rhBGN,  and  blood  was  collected  by  cardiac  puncture  30  min,  1  h,  and  24  h   postinjection  (n  =  3–4  mice/  condition).  Control  experiments  showed  that   comparable  levels  of  rhBGN  were  present  in  plasma  (0.12  μg/mL  at  1  h  post-­‐   injection,  n  =  2).  For  two-­‐site  ELISAs,  plates  were  coated  with  mouse  anti-­‐biglycan   antibody,  blocked,  and  incubated  with  se-­‐  rum  samples  or  standard  biglycan   dilutions  followed  by  rabbit  anti-­‐biglycan  and  goat  anti-­‐rabbit  HRP.  Sensitivity  of   the  assays  was  ∼5  ng/mL.   215     References:     Biggar  WD,  et  al.  (2004)  Deflazacort  in  Duchenne  muscular  dystrophy:  A   comparison  of  two  different  protocols.  Neuromuscul  Disord  14:476–482.     Bowe  MA,  Mendis  DB,  Fallon  JR  (2000)  The  small  leucine-­‐rich  repeat  proteoglycan   biglycan  binds  to  alpha-­‐dystroglycan  and  is  upregulated  in  dystrophic  muscle.  J  Cell   Biol  148:801–810.     Bowe  MA,  Mendis  DB,  Fallon  JR  (2000)  The  small  leucine-­‐rich  repeat  proteoglycan   biglycan  binds  to  alpha-­‐dystroglycan  and  is  upregulated  in  dystrophic  muscle.  J  Cell   Biol  148:801–810.     Mann  CJ,  et  al.  (2001)  Antisense-­‐induced  exon  skipping  and  synthesis  of  dystrophin   in  the  mdx  mouse.  Proc  Natl  Acad  Sci  USA  98:42–47.     Mercado  ML,  et  al.  (2006)  Biglycan  regulates  the  expression  and  sarcolemmal  local-­‐   ization  of  dystrobrevin,  syntrophin,  and  nNOS.  FASEB  J  20:1724–1726.     Rafii  MS,  et  al.  (2006)  Biglycan  binds  to  alpha-­‐  and  gamma-­‐sarcoglycan  and   regulates  their  expression  during  development.  J  Cell  Physiol  209:439–447.     van  Deutekom  JC,  et  al.  (2007)  Local  dystrophin  restoration  with  antisense   oligonucleotide  PRO051.  N  Engl  J  Med  357:2677–2686.                                           216     Supplementary  Figures       Supplementary  Figure  4.1                                       217       Supplementary  Figure  4.1.   Systemically  delivered  rhBGN  can  be  detected  in   the  circulation  and  becomes  localized  to  muscle.  (A)  Detection  of  rhBGN  in  serum   following  i.p.  delivery.  Mice  were  injected  i.p.  with  10  mg/kg  rhBGN,  and  serum  was   collected  30  min,  1,  and  24  h  postinjection  (n  =  3–4  animals/group).  Two-­‐site   ELISAs  were  performed  as  described  in  Materials  and  Methods.  Biglycan   (endogenous)  was  not  detected  in  serum  from  uninjected  mice.  However,  rhBGN   was  readily  detected  in  serum  following  a  systemic  injection  of  the  recombinant   protein.  (Scale  bar  =  50  μm.)  (B)  Systemically  delivered  rhBGN  becomes  stably   localized  to  muscle.  Alexa  555-­‐rhBGN  (Materials  and  Methods)  was  injected  i.p.  into   adult  mdx  mice,  and  diaphragms  were  harvested  48  h  later.  Endogenous  laminin   was  detected  by  indirect  immunofluoresence.  Systemically  delivered  Alexa  555-­‐ rhBGN  is  localized  in  the  extracellular  matrix  surrounding  the  myofibers.                       218         Supplementary  Figure  4.2.                         219         Supplementary  Figure  4.2.   RhBGN  treatment  increases  sarcolemmal   utrophin  expression  in  the  tibialis  anterior  of  mdx  mice.       (A)  Utrophin  immunostaining  of  TA  muscles  from  mdx  mice  that  received  one  i.p.   injection  of  rhBGN  or  vehicle.  Systemically  delivered  rhBGN  increased  utrophin   expression  in  TAs  of  mdx  mice  compared  with  vehicle-­‐injected  mice.  (B)   Quantification  of  increased  utrophin  expression  in  TA  muscle  from  rhBGN  treated   mice  (1.74-­‐fold  increase,  *P  <  0.001,  Student  unpaired  t  test;  n  =  300  sarcolemmal   segments  from  three  muscles  for  each  group).  (Scale  bar  =  25  μM.)                         220       Supplementary  Figure  4.3                         221       Supplementary  Figure  4.3.   Creatine  kinase  levels  in  rhBGN-­‐treated  mdx   mice.       Creatine  kinase  levels  in  32P  mdx  mice  that  received  a  single  injection  of  1  mg/kg  (n   =  23),  2  mg/kg  (n  =  12),  or  10  mg/kg  (n  =  11)  rhBGN  or  vehicle  alone  (n  =  24)  at   P18.  RhBGN-­‐treated  mice  showed  trends  of  decreased  CK  levels,  but  the  results  did   not  reach  statistical  significance  (one-­‐way  ANOVA,  P  >  0.05).                               222     Supplementary  Figure  4.4         223       Supplementary  Figure  4.4.  RhBGN  fails  to  counter  dystrophic  pathology  in   mdx:utrn−/−  double  KO  animals.       (A)  Mutant  mice  lacking  both  dystrophin  and  utrophin  (mdx:utrn−/−)  were  injected   at  P19  with  recombinant  rhBGN  or  vehicle.  Diaphragms  were  isolated  3  wk  later,   sectioned,  and  stained  with  H&E.  Characteristic  extensive  muscle  pathology  of  these   double  KO  animals—areas  of  mononuclear  cell  infiltration  and  foci  of   necrosis/regeneration  and  centrally  nucleated  myofibers—  was  comparable  in   rhBGN-­‐  and  vehicle-­‐  injected  animals.  (Scale  bar  =  50  μm.)  (B)  RhBGN   administration  does  not  decrease  CNFs  in  mdx:utrn−/−  mice.  Per-­‐  centages  of   centrally  nucleated  muscle  fibers  were  determined  from  the  H&E-­‐stained   diaphragm  sections  from  rhBGN  and  vehicle  injected  mdx:utrn−/−  (n  =  2  vehicle-­‐ injected  and  3  rhBGN-­‐injected  mice;  unpaired  Student  t  test,  P  =  0.45).                   224       Supplementary  Figure  4.5.                 225         Supplementary  Figure  4.5.   RhBGN  is  well  tolerated  in  mdx  mice.       (A)  P14  mdx  mice  were  injected  at  3-­‐wk  intervals  for  3  mo  with  either  rhBGN  or   vehicle.  Tissues  were  harvested  at  15  wk  and  weighed.  All  organ  and  muscle  weights   are  plotted  relative  to  total  body  weight  in  mg/g  (n  =  8  animals/group;  *P  <  0.05;   unpaired  Student  t  test).  (B)  Liver  and  kidney  function  in  rhBGN  treated  mice.   Serum  was  collected  from  32P  mdx  mice  that  received  an  i.p.  injection  of  1,  2,  or  10   mg/kg  rhBGN  or  vehicle  only.  There  were  no  significant  changes  in  serum  levels  of   BUN,  creatinine,  AST,  or  total  bilirubin.                         226           Supplementary  Table  4.1                             227                     CHAPTER  5   DISCUSSION                         228     BMPs  comprise  one  of  the  largest  growth  factor  families  and  function  during  early   development  throughout  the  embryo  (Ferguson  et  al.,  1992;  Ray  et  al.,  1991;  Urist,   1965).  Furthermore,  this  diverse  group  of  growth  factors  functionally  supports   multiple  processes  such  as  organ  morphogenesis  and  regeneration  in  a  range  of   developing  and  adult  tissues.  Given  their  critical  roles,  it  is  not  surprising  that  tightly   controlled  regulatory  mechanisms  evolved  for  restricting  or  enhancing  the  signaling   that  BMPs  employ.  In  Chapter  2,  I  present  data  demonstrating  MuSK  as  a  novel   regulator  of  BMP  signaling  in  myogenic  cells.  There  are  several  conclusions  to  draw   from  these  results.  First,  the  high  affinity  of  the  binding  between  MuSK  ectodomain   and  BMPs  is  similar  to  what  was  reported  previously  for  type-­‐1  receptor  binding  to   BMPs  using  SPR  (Berasi  et  al.,  2011).  This  puts  MuSK  into  a  high-­‐affinity  receptor   category  along  with  BMPs’  canonical  type-­‐1  receptors,  as  well  as  certain  co-­‐ receptors  such  as  DRAGON  (Samad  et  al.,  2005).  It  is  therefore  expected  that  MuSK   will  interact  with  BMPs  and  its  downstream  pathway  in  the  mature  muscle  fibers   where  it  is  expressed  the  most.  Moreover,  my  results  demonstrate  that  MuSK   regulates  BMP  pathway  also  in  undifferentiated  myoblast  cultures,  where  MuSK  is   expressed  at  lower  levels.  This  observation  raises  the  question  whether  MuSK  acts   as  a  BMP  regulator  in  other  tissues  where  it  is  expressed  at  very  low  levels.  The   kinetics  of  the  binding  suggests  that  MuSK  may  well  be  interacting  with  BMP4   pathway  in  other  tissues,  as  well.  Though  this  binding  would  not  necessarily  mean   that  the  outcomes  of  this  interaction  would  be  the  same  as  in  myogenic  cells.  Indeed,   229   some  of  the  transcripts  regulated  by  MuSK  include  genes  such  as  Car3  and  Myh15   that  are  selectively  enriched  in  muscle.         The  findings  about  the  requirement  of  Ig3  domain  of  MuSK  for  its  BMP4  binding  add   another  level  of  complexity  to  this  regulation.  The  presence  of  a  naturally  occurring   isoform  of  MuSK  lacking  Ig3  domain  suggests  that  MuSK  binding  to  BMP4  can  be   regulated  through  alternative  splicing  of  MuSK.  This  regulation  could  occur  at   multiple  stages  of  development,  as  well  as  under  different  stimuli  in  adult.  This   result  also  attributes  a  function  to  Ig3  domain,  which  has  yet  to  be  shown  as  a   requirement  for  other  known  functions  of  MuSK  in  the  context  of  NMJ.       In  the  absence  of  MuSK,  SMAD-­‐mediated  BMP  signaling  is  downregulated.  This   effect  is  shown  by  decreased  levels  of  BMP4-­‐induced  SMAD1/5/8  phosphorylation,   as  well  as  Id1  transcription.  This  result  suggests  that  MuSK  could  be  acting  as  a   stimulatory  co-­‐receptor  to  BMP4.  The  molecular  mechanisms  of  this  regulation  need   to  be  studied  further.  Future  studies  will  reveal  whether  MuSK  is  indeed  a  co-­‐ receptor  and  binds  to  BMP4  receptors  in  myogenic  cells.  It  is  also  noteworthy  that   MuSK  regulates  the  presence  of  cytosolic  pSMAD1/5/8  granules  in  myoblasts.  We   hypothesize  that  these  granules  may  be  a  specific  signaling  compartment  in  which   MuSK  modulates  BMP4  pathway.  The  interaction  between  MuSK  and  Lrp4  and  the   previously  reported  cytosolic  Lrp  signalosomes  in  other  cellular  contexts  further   support  this  idea  and  raise  the  questions  whether  Lrp4  is  also  present  in  these   230   granules  or  if  the  granules  are  reduced  in  the  absence  of  Lrp4.  These  questions  will   be  addressed  in  future  studies.       Another  key  finding  in  this  study  is  the  modulation  of  the  transcriptional  output  of   BMP4  signaling  by  MuSK.  This  study  identified  a  group  of  BMP4  target  transcripts   that  require  MuSK  in  order  to  be  expressed  in  response  to  BMP4.  This  result  points   to  a  mechanism  in  which  different  surface  interactions  can  potentially  lead  to   specific  transcriptional  outputs  of  BMP  signaling.  It  is  very  tempting  to  speculate   that  the  cytosolic  granules  expressed  at  high  levels  in  the  presence  of  MuSK  are   signaling  compartments  regulated  by  MuSK  and  are  necessary  for  the  transcription   of  a  subset  of  genes  downstream  of  BMP4.  Future  studies  focusing  on  the  roles  and   contents  of  these  granules  will  help  us  understand  if  they  are  such  specific   signalosomes.       BMP4  induces  the  expression  of  distinct  gene  sets  in  undifferentiated  myoblasts  as   compared  to  differentiated  myotubes.  This  result  may  not  be  surprising  given  that   upon  differentiation  the  gene  expression  profile,  and  hence  the  cellular  context   changes  drastically.  However,  it  is  interesting  to  see  that  both  in  myoblasts  and  in   myotubes  there  are  different  sets  of  transcripts  whose  BMP4  responsiveness  is   regulated  by  MuSK.  This  could  be  due  to  differences  in  several  additional  factors   modulating  BMP4  pathway  or  simply  because  of  the  difference  in  MuSK   concentration  between  these  two  cell  types.       231   In  this  thesis,  I  identified  Car3  and  Myh15  as  novel  downstream  targets  of  BMP4  in   myotubes.  Both  Car3  and  Myh15  had  been  suggested  as  slow  muscle  markers.   Interestingly,  their  expression  was  higher  in  slow  fiber  enriched  soleus  muscle   compared  to  fast  fiber  enriched  EDL.  This  correlation  indicates  that  BMP4  may  be  a   novel  regulatory  signal  for  fiber-­‐type  determination  and/or  switching.  Furthermore,   MuSK  regulates  the  transcriptional  response  of  Car3  and  Myh15  to  BMP4.  The   potential  involvement  of  MuSK  and  BMP4  in  fiber  type  determination  or   reprogramming  needs  to  be  tested  more  directly.       In  vivo  studies  will  be  very  useful  to  test  the  functional  outcomes  of  the  MuSK-­‐BMP4   interaction.  Since  MuSK  Ig3  domain  is  dispensable  for  the  roles  of  MuSK  at  the  NMJ,   however  is  necessary  for  MuSK-­‐BMP4  interaction,  the  generation  of  knock-­‐in  mice   expressing  MuSK  that  lacks  Ig3  domain  would  allow  to  have  viable  animals  with   intact  NMJs  and  to  study  the  specific  outcomes  of  BMP4-­‐MuSK  interaction.  The   involvement  of  MuSK-­‐BMP4  interaction  in  muscle  fiber  type  determination  can  be   tested  in  these  mice  by  analyzing  the  distribution  of  fiber  types.  The  role  of  MuSK-­‐ BMP4  interaction  in  fiber  type  switching  can  also  be  addressed  in  these  mice  by   using  re-­‐innervation  models  with  either  slow  or  fast  type  neurons  where  fiber  type   switch  can  be  induced.       Cell-­‐type  specific  effects  of  BMP4  can  vary  tremendously.  This  raises  the  question   how  the  same  signal  can  have  such  diverse  outcomes.  The  data  that  is  presented  in   Chapter  2  supports  the  idea  that  the  cellular  context  and  the  cell  type  specific   232   expression  of  BMP4  regulators  can  modulate  the  signaling  downstream  of  BMP4   and  lead  to  cell  type  specific  outcomes.             In  Chapter  3,  I  demonstrate  a  role  for  BMP4  in  the  induction  of  AChR  clusters  in   cultured  muscle  cells.  High  densities  of  AChRs  expressed  at  the  postsynaptic  muscle   membrane  of  the  vertebrate  NMJ  are  crucial  for  communication  between  neurons   and  muscle.  Several  factors  such  as  laminin,  Wnt11  and  agrin  have  been  previously   shown  to  induce  AChR  clustering.  However,  BMPs  have  not  been  implicated  in  this   process.  The  study  presented  in  Chapter  3  shows  that  BMP4  increases  the   expression  of  Wnt11,  which  is  required  for  BMP4’s  clustering  activity.  Wnt11  binds   to  MuSK  and  induces  AChR  clusters  through  activation  of  MuSK  (Zhang  et  al.,  2012).   Therefore  our  results  suggest  that  BMP4  is  an  upstream  regulator  of  Wnt11   expression  and  indirectly  regulates  clustering,  as  well.       To  our  knowledge,  this  is  the  first  study  showing  Wnt11  downstream  of  BMP4.  As   discussed  above,  Wnt11  could  be  a  muscle  specific  BMP4  response.  Since  Wnt11  has   been  suggested  as  an  inducer  of  prepatterning  before  innervation  of  the  muscle,   BMP4  could  regulate  this  process  upstream  of  Wnt11.  In  the  embryo,  BMP4  released   from  neighboring  embryonic  tissues  before  NMJ  formation  could  upregulate  Wnt11   expression  in  early  myofibers  and  regulate  the  prepatterning.  Further  in  vivo  and  in   vitro  studies  are  needed  to  determine  the  potential  role  of  BMPs  in  synapse   formation  in  muscle.     233   In  Chapter  4,  I  collaborated  with  Dr.  Alison  Amenta  to  examine  biglycan’s  regulation   of  sarcolemmal  utrophin  expression,  the  therapeutic  effect  of  this  regulation  for   Duchenne  Muscular  Dystrophy  (DMD)  and  the  mechanisms  thereof.  We  showed  that   the  non-­‐glycanated  form  of  biglycan  increases  membrane  association  of  utrophin  in   the  absence  of  dystrophin  and  this  increase  counters  the  dystrophic  pathology  in   mdx  mice,  the  mouse  model  of  DMD.  My  contribution  was  to  show  that  in  cultured   biglycan  null  myotubes,  non-­‐glycanated  biglycan  increases  utrophin  levels  in   membrane  fractions,  however  it  does  not  increase  total  protein  or  transcript  levels   of  utrophin.  This  result  points  to  a  post-­‐transcriptional  mechanism  in  which   biglycan  may  regulate  membrane  targeting  of  utrophin.  This  effect  could  either  be   mediated  by  a  structural  stabilization  of  utrophin  at  the  membrane  or  through  a   signaling  mechanism.  Another  alternative  mechanism  for  biglycan  regulation  of   sarcolemmal  utrophin  expression  could  involve  microRNAs.  In  Appendix  Table  3,  I   show  the  list  of  microRNAs  up-­‐  and  downregulated  in  response  to  non-­‐glycanated   form  of  biglycan  in  biglycan  null  myotubes.       In  Appendix  Figure  1,  I  show  that  the  injection  of  the  non-­‐glycanated  form  of   biglycan  into  mdx  mice  increases  the  transcription  of  two  muscle  proteins,  Car3  and   γ-­‐sarcoglycan.  At  two  different  injection  doses,  there  is  also  a  trend  for   downregulation  of  utrophin  transcript.  This  is  in  accord  with  the  published  results   in  Chapter  4.  This  downregulation  could  be  due  to  a  negative  feedback  mechanism   triggered  by  the  stabilization  of  utrophin  protein  at  the  membrane.       234   γ-­‐Sarcoglycan  binds  to  biglycan  and  its  protein  levels  were  shown  to  be  reduced  in   developing  biglycan  null  animals  (Rafii  et  al.,  2006).  In  Chapter  4,  we  also  show  that   biglycan  injection  into  mdx  mice  increases  protein  levels  of  γ-­‐sarcoglycan  at  the   membrane  fractions  of  muscle.  The  result  in  Appendix  Figure  1,  showing  increased   transcript  levels  of  γ-­‐sarcoglycan  in  biglycan  treated  mdx  mice  is  in  accord  with   these  previous  studies  and  further  supports  the  idea  that  γ-­‐sarcoglycan  expression   is  regulated  by  biglycan.         The  increase  in  Car3  transcript  levels  by  non-­‐glycanated  biglycan  is  very  intriguing   as  BMP4  also  increases  Car3  levels  (Chapter  2).  Biglycan  was  also  shown  to  bind  to   BMP4  (Moreno  et  al.,  2005).  In  Appendix  Figure  2,  I  demonstrate  differential   binding  of  non-­‐glycanated  and  proteoglycan  forms  of  biglycan  to  BMP4.  Moreover,   in  Appendix  Table  1  and  2,  differences  in  transcriptional  response  of  biglycan  null   myotubes  in  response  to  both  of  these  forms  of  biglycan  are  shown.  Two  forms  of   biglycan  not  only  bind  to  BMP4  with  different  affinities,  but  they  also  seem  to   regulate  expression  of  different  group  of  genes.  Further  studies  will  determine   whether  non-­‐glycanated  biglycan  increases  Car3  transcript  levels  through  BMP4   and  if  Car3  response  is  specific  to  non-­‐glycanated  form  of  biglycan.       Understanding  the  mechanism  of  action  of  biglycan  in  regulation  of  sarcolemmal   utrophin  expression  is  crucial  for  biglycan’s  future  use  as  a  therapeutic  for  DMD.  It   would  be  interesting  to  see  if  the  mechanism  involves  BMP4  and  it’s  signaling.     235               References:     Berasi,  Stephen  P,  Usha  Varadarajan,  Joanne  Archambault,  Michael  Cain,  Tatyana  A   Souza,  Abe  Abouzeid,  Jian  Li,  et  al.  “Divergent  Activities  of  Osteogenic  BMP2,  and   Tenogenic  BMP12  and  BMP13  Independent  of  Receptor  Binding  Affinities.”  Growth   Factors  (Chur,  Switzerland)  29,  no.  4  (August  2011):  128–139.                      Ferguson,  E  L,  and  K  V  Anderson.  “Localized  Enhancement  and  Repression  of  the   Activity  of  the  TGF-­‐beta  Family  Member,  Decapentaplegic,  Is  Necessary  for  Dorsal-­‐ ventral  Pattern  Formation  in  the  Drosophila  Embryo.”  Development  (Cambridge,   England)  114,  no.  3  (March  1992):  583–597.                     Moreno,  Mauricio,  Rosana  Muñoz,  Francisco  Aroca,  Mariana  Labarca,  Enrique   Brandan,  and  Juan  Larraín.  “Biglycan  Is  a  New  Extracellular  Component  of  the   Chordin-­‐BMP4  Signaling  Pathway.”  The  EMBO  Journal  24,  no.  7  (April  6,  2005):   1397–1405.     Rafii,  Michael  S,  Hiroki  Hagiwara,  Mary  Lynn  Mercado,  Neung  S  Seo,  Tianshun  Xu,   Tracey  Dugan,  Rick  T  Owens,  et  al.  “Biglycan  Binds  to  Alpha-­‐  and  Gamma-­‐ sarcoglycan  and  Regulates  Their  Expression  During  Development.”  Journal  of   Cellular  Physiology  209,  no.  2  (November  2006):  439–447.                    Ray,  R  P,  K  Arora,  C  Nüsslein-­‐Volhard,  and  W  M  Gelbart.  “The  Control  of  Cell  Fate   Along  the  Dorsal-­‐ventral  Axis  of  the  Drosophila  Embryo.”  Development  (Cambridge,   England)  113,  no.  1  (September  1991):  35–54.                     Samad,  Tarek  A.,  Anuradha  Rebbapragada,  Esther  Bell,  Ying  Zhang,  Yisrael  Sidis,   Sung-­‐Jin  Jeong,  Jason  A.  Campagna,  et  al.  “DRAGON,  a  Bone  Morphogenetic  Protein   Co-­‐receptor.”  Journal  of  Biological  Chemistry  280,  no.  14  (April  8,  2005):  14122– 14129.                    Urist,  M  R.  “Bone:  Formation  by  Autoinduction.”  Science  (New  York,  N.Y.)  150,  no.   3698  (November  12,  1965):  893–899.     Zhang,  Bin,  Chuan  Liang,  Ryan  Bates,  Yiming  Yin,  Wen-­‐Cheng  Xiong,  and  Lin  Mei.   “Wnt  Proteins  Regulate  Acetylcholine  Receptor  Clustering  in  Muscle  Cells.”   Molecular  Brain  5  (2012):  7.       236                                                 APPENDIX                               237               Appendix  Figure  1       238       239   Appendix  Figure  1.  Car3  and  γ-­‐sarcoglycan  messages  are  upregulated  by   rhBiglycan  in  mdx  mice.       P18  mdx  mice  were  intraperitoneally  injected  with  vehicle  and  two  concentrations   of  non-­‐glycanated  rhBiglycan  (1  and  10mg/kg).  At  P32  quadriceps  femoris  muscles   were  harvested  from  injected  mice.  RNA  was  harvested  and  reverse  transcribed  into   cDNA.  Transcript  levels  of  (A)  utrophin,  (B)  γ-­‐sarcoglycan  and  (C)  Car3  were   analyzed  by  qRT-­‐PCR.  In  accord  with  the  results  presented  in  Chapter  4,  utrophin   transcripts  were  not  increased  by  non-­‐glycanated  biglycan  injection.  However,  there   was  a  significant  increase  at  the  transcript  levels  of  Car3  and  γ-­‐sarcoglycan  upon   non-­‐glycanated  biglycan  injection.  (n=6)                                         240   Appendix  Figure  2                                                     241   Appendix  Figure  2.  Non-­‐glycanated  biglycan  exhibits  less  binding  to  BMP4  than   proteoglycan  form  of  biglycan.     BMP4  was  immobilized  on  96-­‐well  plates  and  then  incubated  with  His-­‐tagged   proteoglycan  and  non-­‐glycanated  forms  of  biglycan  (0-­‐728nM).  Bound  biglycan  was   detected  with  anti-­‐His  antibody  and  HRP-­‐conjugated  anti-­‐mouse  secondary   antibody.  The  graph  represents  the  absorbance  values  read  for  the  enzymatic   activity  of  HRP.  Each  point  represents  the  average  of  four  replicates.  As  indicated  in   the  shift  in  binding  curves,  proteoglycan  form  of  biglycan  binds  to  BMP4  better  than   the  non-­‐glycanated  form.                                                     242       Appendix  Figure  3                                 243     Appendix  Figure  3.  DAPC  regulation  by  MuSK.       Wild  type  and  MuSK  null  myoblasts  were  grown  into  confluence  and  differentiated   for  3-­‐4  days.  Membrane  fractions  were  isolated  from  myotube  cultures  as  described   in  Chapter  4  Supporting  Information  Materials  and  Methods.  (A)  α-­‐  and  γ-­‐ sarcoglycan  protein  levels  in  membrane  fractions  were  detected  by  Western  blots.   Total  protein  staining  was  used  as  loading  control.  B-­‐C.  Quantifications  of  (B)  α-­‐  and   (C)  γ-­‐sarcoglycan  levels.  In  the  absence  of  MuSK,  α-­‐sarcoglycan  levels  were   decreased  in  myotubes.  Furthermore,  the  migration  pattern  of  α-­‐sarcoglycan  band   was  also  changed  in  the  absence  of  MuSK.  An  additional  band  with  a  slightly  less   molecular  weight  appears  when  MuSK  is  absent,  indicating  that  MuSK  could  also  be   regulating  a  posttranslational  modification  of  α-­‐sarcoglycan.  γ-­‐sarcoglycan  levels,   on  the  other  hand,  were  increased  in  the  absence  of  MuSK,  suggesting  a  negative   regulation  of  γ-­‐sarcoglycan  by  MuSK.                           244     Appendix  Table  1     Transcript   Fold-­‐ ID   Gene  Symbol   p-­‐value   Change   10339808       0.02271   5.25   10343609       0.04192   2.02   10341524       0.00296   2.00   10340228       0.01317   1.98   10338084       0.00590   1.86   10342420       0.02064   1.86   10339273       0.01940   1.80   10340928       0.01707   1.78   10341475       0.03816   1.75   10341200       0.01250   1.70   10343265       0.04457   1.68   10343619       0.02265   1.68   10341953       0.01522   1.64   10343220       0.02409   1.58   10339420       0.00624   1.56   10341887       0.00563   1.55   10340251       0.00891   1.55   10342672       0.04112   1.53   10338131       0.01660   1.53   10339977       0.01585   1.52   10551282   LOC100047728   0.02176   1.52   10339151       0.00556   1.51   10339549       0.01469   1.48   10339060       0.01943   1.46   10466298   Olfr1436   0.00274   1.46   10339724       0.04868   1.45   10341564       0.04673   1.44   10596958       0.02426   1.43   10344298       0.00554   1.43   10342029       0.02100   1.42   10414258       0.00826   1.42   10340815       0.02075   1.42   10338902       0.01725   1.41   10343872       0.00481   1.41   10338100       0.00818   1.39   10339087       0.03898   1.39   10341172       0.03565   1.38   10338533       0.02911   1.37   10339716       0.01309   1.37   245   10340713       0.02260   1.36   10343109       0.03454   1.36   10341584       0.00609   1.36   10340166       0.01041   1.36   10338552       0.00589   1.36   10342342       0.03505   1.35   10559728       0.04882   1.35   10342667       0.04047   1.35   10339946       0.03813   1.34   10344387       0.03732   1.34   10340541       0.04840   1.33   10341139       0.00404   1.33   10599213       0.02632   1.32   10344464       0.02943   1.32   10341386       0.03274   1.32   10340766       0.01097   1.32   10342692       0.02925   1.32   10412646       0.02000   1.31   10339965       0.04016   1.31   10361680   BC013529   0.00048   1.31   10552613   Klk1b4   0.01333   1.31   10342528       0.02707   -­‐1.30   10343888       0.01441   -­‐1.31   10344460       0.03351   -­‐1.31   10340813       0.01756   -­‐1.32   10343930       0.03481   -­‐1.32   10349634       0.03188   -­‐1.32   10342853       0.01926   -­‐1.32   10339179       0.04931   -­‐1.33   10344603       0.03716   -­‐1.33   10484590   Olfr1056   0.02234   -­‐1.33   10591853   Tbx20   0.04557   -­‐1.33   10344088       0.04025   -­‐1.34   10339869       0.00149   -­‐1.34   10339964       0.00010   -­‐1.34   10358785       0.00560   -­‐1.35   10401834   Gm5039   0.01444   -­‐1.36   10545210   Gm1524   0.01009   -­‐1.36   10344401       0.03504   -­‐1.36   10340719       0.01273   -­‐1.36   10341733       0.00218   -­‐1.36   10338584       0.02887   -­‐1.36   10340738       0.01710   -­‐1.37   10344595       0.01353   -­‐1.38   246   10341186       0.04672   -­‐1.38   10490923   Car2   0.02166   -­‐1.39   10340673       0.02694   -­‐1.39   10338470       0.04445   -­‐1.39   10342652       0.02086   -­‐1.39   10340703       0.03562   -­‐1.41   10338539       0.02167   -­‐1.41   10341945       0.04548   -­‐1.41   10343819       0.01720   -­‐1.41   10338692       0.03773   -­‐1.42   10340205       0.01545   -­‐1.42   10341801       0.04163   -­‐1.42   10341559       0.00009   -­‐1.42   10341928       0.01495   -­‐1.43   10343004       0.03890   -­‐1.43   10342840       0.00771   -­‐1.43   10339519       0.02775   -­‐1.43   10338980       0.03170   -­‐1.44   10343700       0.04341   -­‐1.44   10338702       0.00455   -­‐1.45   10341234       0.03702   -­‐1.45   10344257       0.03617   -­‐1.45   10339650       0.02222   -­‐1.46   10341616       0.00680   -­‐1.46   10341886       0.02025   -­‐1.47   10342424       0.01435   -­‐1.47   10342982       0.01087   -­‐1.47   10343755       0.02099   -­‐1.48   10341435       0.04701   -­‐1.49   10344372       0.04955   -­‐1.49   10342061       0.02593   -­‐1.49   10340037       0.00422   -­‐1.49   10344259       0.02984   -­‐1.50   10341074       0.01048   -­‐1.51   10340683       0.03180   -­‐1.51   10343336       0.00197   -­‐1.51   10344551       0.01625   -­‐1.52   10342729       0.02573   -­‐1.52   10339483       0.00187   -­‐1.52   10343392       0.02980   -­‐1.52   10344207       0.00403   -­‐1.53   10341383       0.02160   -­‐1.54   10338846       0.00430   -­‐1.55   10339256       0.03514   -­‐1.55   247   10341148       0.00881   -­‐1.56   10342796       0.00377   -­‐1.56   10342948       0.04466   -­‐1.57   10342469       0.00711   -­‐1.58   10343321       0.00398   -­‐1.60   10547793   Rnu7   0.01068   -­‐1.61   10525914       0.01560   -­‐1.61   10342217       0.01701   -­‐1.62   10340762       0.02333   -­‐1.63   10338350       0.01370   -­‐1.63   10342258       0.03060   -­‐1.64   10343694       0.00498   -­‐1.66   10342648       0.04731   -­‐1.66   10341753       0.04383   -­‐1.67   10339684       0.03328   -­‐1.68   10339838       0.01068   -­‐1.69   10341819       0.02677   -­‐1.70   10344013       0.01706   -­‐1.70   10339268       0.03647   -­‐1.71   10339380       0.00631   -­‐1.71   10338807       0.03441   -­‐1.73   10344267       0.03950   -­‐1.73   10338420       0.04750   -­‐1.74   10341090       0.00371   -­‐1.75   10344021       0.04551   -­‐1.75   10342299       0.01005   -­‐1.76   10338287       0.03949   -­‐1.77   10342874       0.01467   -­‐1.77   10341044       0.03349   -­‐1.78   10339594       0.04275   -­‐1.78   10339278       0.04879   -­‐1.79   10344009       0.03885   -­‐1.79   10339364       0.03825   -­‐1.80   10340222       0.04186   -­‐1.83   10342868       0.00409   -­‐1.84   10342403       0.00232   -­‐1.87   10343326       0.01427   -­‐1.87   10341231       0.04160   -­‐1.89   10341236       0.04004   -­‐1.91   10341177       0.00105   -­‐1.96   10343822       0.00583   -­‐2.00   10341214       0.01906   -­‐2.02   10341229       0.00213   -­‐2.14   10342481       0.01474   -­‐2.18   248   10339113       0.04737   -­‐2.26   10338230       0.04952   -­‐2.27   10344131       0.00721   -­‐2.38   10340970       0.01531   -­‐2.57     Appendix  Table  1.  Genome-­‐wide  gene  expression  analysis  of  non-­‐glycanated   biglycan-­‐treated  biglycan  null  myotubes.     Biglycan  null  myoblasts  were  grown  into  confluence  and  differentiated  for  four   days.  The  myotubes  were  treated  with  non-­‐glycanated  biglycan  for  8  hours.  RNA   was  harvested  from  the  cultures  and  reverse  transcribed  into  cDNA,  which  was  then   hybridized  to  Affymetrix  array  chips.  The  results  of  the  microarray  analysis  are   summarized  in  Table  1.  Fold  changes  greater  than  1.3  are  shown  in  the  table.  The   positive  values  indicate  the  fold  change  in  upregulation  of  the  listed  transcript  by   non-­‐glycanated  biglycan  whereas  the  negative  values  indicate  the  fold  change  in   downregulation  of  the  listed  transcript.  The  majority  of  the  transcript  hits  are   control  probe  sets.  Thus,  overall  there  was  not  a  strong  transcriptional  response  to   non-­‐glycanated  biglycan  treatment  in  biglycan  null  myotubes.         Appendix  Table  2     Transcript   Fold-­‐ ID   Gene  Symbol   p-­‐value   Change   10341131       0.04173   2.71   10338194       0.00034   2.64   10340698       0.03910   2.11   10344256       0.04527   2.10   10339420       0.00049   2.08   10341475       0.01629   2.01   10343669       0.03547   1.98   10413012   Fut11   0.01557   1.93   249   10417068       0.03501   1.91   10340066       0.01631   1.91   10339208       0.00248   1.90   10341484       0.01094   1.89   10338778       0.01937   1.87   10343307       0.00382   1.85   10341448       0.02990   1.84   10340228       0.02052   1.84   10339273       0.01855   1.81   10339121       0.00252   1.81   10338426       0.03860   1.81   10342672       0.01417   1.75   10339254       0.03913   1.74   10342590       0.00359   1.73   10339309       0.02556   1.72   10338756       0.00070   1.71   10344045       0.00112   1.71   10476590   Macrod2   0.00073   1.70   10344366       0.02092   1.70   10341367       0.02315   1.68   10339724       0.01462   1.66   10343084       0.00190   1.66   10596958       0.00592   1.65   10338533       0.00406   1.64   10339418       0.00461   1.63   10566205   Dub2a   0.00322   1.63   10339151       0.00257   1.63   10340482       0.04297   1.62   10343860       0.03282   1.61   10342554       0.02463   1.61   10453688       0.00309   1.61   10343368       0.01933   1.61   10342089       0.02161   1.61   10339240       0.01128   1.59   10338637       0.04188   1.59   10340619       0.02299   1.57   10339894       0.00252   1.56   10343263       0.00356   1.55   10424555       0.04061   1.54   10476393       0.00045   1.54   10339977       0.01414   1.54   10340674       0.04346   1.54   10341868       0.00680   1.54   10344387       0.00870   1.52   250   10524394       0.01029   1.51   10532903       0.01029   1.51   10340199       0.02967   1.51   10341524       0.02931   1.51   10341953       0.03321   1.50   10338092       0.02410   1.50   10338932       0.02614   1.50   10339294       0.03293   1.49   10344298       0.00338   1.49   10339136       0.02799   1.49   10341194       0.00788   1.48   10338309       0.01026   1.48   10458764   LOC100046618   0.03538   1.48   10341456       0.00752   1.47   10343220       0.04489   1.47   10340295       0.03201   1.47   10340713       0.00933   1.47   10341926       0.02174   1.47   10560385   Psg23   0.01917   1.46   10403031   V165-­‐D-­‐J-­‐C   0.01907   1.46   10341172       0.01993   1.46   10339461       0.00005   1.45   10342025       0.02486   1.44   10492165       0.00426   1.44   10338787       0.02749   1.44   10340523       0.00135   1.44   10411506   Gm2524   0.03311   1.43   10491913       0.00018   1.43   10343982       0.02030   1.43   10341068       0.04787   1.42   10340281       0.04639   1.42   10338887       0.01002   1.42   10338553       0.02635   1.41   10340230       0.00354   1.41   10581643       0.01272   1.41   10484720   Olfr1166   0.02012   1.41   10343036       0.00618   1.41   10341633       0.01363   1.41   10340244       0.00122   1.40   10412657       0.02694   1.40   10491474   Gm5708   0.00290   1.40   10340234       0.02044   1.39   10460194       0.01927   1.39   10560164   Obox6   0.00662   1.39   251   10338100       0.00843   1.39   10416897       0.03751   1.39   10339010       0.02633   1.39   10344238       0.03590   1.39   10343904       0.02660   1.39   10340466       0.04657   1.39   10415011       0.01141   1.38   10340946       0.02290   1.38   10344019       0.04762   1.38   10339731       0.01763   1.38   10340749       0.02899   1.38   10339965       0.02160   1.38   10353413       0.04043   1.37   10412663   Gm8429   0.00974   1.37   10536147   Gm16367   0.04767   1.37   10604954       0.00217   1.37   10338441       0.02218   1.36   10360538   Pppde1   0.01726   1.36   10342197       0.00002   1.36   10341058       0.02022   1.36   10341642       0.00218   1.36   10343491       0.03472   1.35   10433197   Olfr161   0.01164   1.35   10362113   Gm10824   0.01347   1.35   10338983       0.00168   1.35   10343872       0.00867   1.35   10421873   Gm10845   0.02435   1.35   10338401       0.00536   1.34   10340251       0.04204   1.34   10600741   Gm5941   0.02938   1.34   10444995   EG547347   0.00111   1.34   10602825       0.01136   1.34   10397538       0.01711   1.34   10550167       0.01132   1.33   10564041       0.03041   1.33   10600720   Gm6027   0.00022   1.33   10461055       0.02779   1.33   10342029       0.04621   1.33   10552613   Klk1b4   0.01061   1.33   10343096       0.03865   1.32   10546432   Adamts9   0.04924   1.32   10340166       0.01494   1.32   10338780       0.03724   1.32   10471569       0.01776   1.32   252   10388284   Olfr389   0.04039   1.32   10607600       0.00800   1.32   10480477   Pax8   0.00259   1.32   10338501       0.04697   1.32   10556573   1110006G14Rik   0.00146   1.32   10464169   1700010L13Rik   0.02225   1.32   10453732   Gm4833   0.04591   1.32   10536363   Tac1   0.00654   1.32   10415013       0.01883   1.31   10430572       0.01626   1.31   10340706       0.00513   1.31   10343216       0.01788   1.31   10447688   4930506C21Rik   0.04823   1.31   10472418   Scn9a   0.04054   1.31   10343765       0.01387   1.31   10368050   Ect2l   0.00227   1.31   10507131   Tal1   0.00066   1.31   10339822       0.03977   1.31   10523903       0.03480   1.31   10343871       0.02852   1.31   10563935       0.03448   1.31   10564045       0.03448   1.31   10564047       0.03448   1.31   10367050   Rdh18   0.00717   1.31   10360398   Ifi202b   0.01953   1.31   10355191   Crygd   0.03009   1.31   10564011   Snord115   0.04429   1.31   10563941       0.01227   1.31   10368175   Pde7b   0.03381   1.31   10607792   Glra2   0.00149   1.30   10503232       0.00780   1.30   10538123   Gimap9   0.00214   1.30   10375838   Col23a1   0.01694   1.30   10438425   Olfr167   0.02131   1.30   10564019       0.02156   1.30   10341053       0.01871   1.30   10344452       0.03920   1.30   10531259   Gm10426   0.01399   1.30   10567380   Umod   0.00771   1.30   10340960       0.01779   1.30   10338723       0.04521   1.30   10563949       0.00638   1.30   10341563       0.04678   1.30   10587000   Dyx1c1   0.03262   1.30   253   10596135       0.01277   1.30   10342389       0.02049   1.30   10340205       0.04734   -­‐1.30   10342878       0.02929   -­‐1.30   10338964       0.04808   -­‐1.30   10344411       0.04787   -­‐1.30   10338977       0.02389   -­‐1.30   10496324   Slc39a8   0.00444   -­‐1.31   10455092   Pcdhb12   0.00904   -­‐1.31   10342652       0.04461   -­‐1.31   10339127       0.00360   -­‐1.31   10472050   Tnfaip6   0.04986   -­‐1.31   10342580       0.04622   -­‐1.32   10338171       0.04293   -­‐1.32   10490923   Car2   0.04102   -­‐1.32   10342889       0.04238   -­‐1.32   10344207       0.02510   -­‐1.32   10340312       0.04477   -­‐1.32   10343006       0.02581   -­‐1.32   10570957   Sfrp1   0.04515   -­‐1.32   10400302       0.03750   -­‐1.33   10342840       0.02087   -­‐1.33   10523156   Cxcl2   0.00797   -­‐1.33   10429564   Ly6a   0.03831   -­‐1.33   10342872       0.02086   -­‐1.33   10341638       0.01615   -­‐1.34   10342290       0.01773   -­‐1.34   10339833       0.00013   -­‐1.34   10438567   2310042E22Rik   0.04918   -­‐1.35   10343151       0.03417   -­‐1.35   10342853       0.01472   -­‐1.35   10343164       0.02380   -­‐1.35   10338702       0.01070   -­‐1.36   10338548       0.00965   -­‐1.36   10341621       0.02767   -­‐1.37   10340053       0.02748   -­‐1.37   10338527       0.00929   -­‐1.38   10338942       0.03479   -­‐1.38   10342159       0.01727   -­‐1.38   10339329       0.00482   -­‐1.38   10584524   1700001J11Rik   0.00633   -­‐1.38   10340741       0.03005   -­‐1.39   10342815       0.00926   -­‐1.39   10340002       0.01977   -­‐1.40   254   10438091   2610318N02Rik   0.04067   -­‐1.41   10344032       0.04545   -­‐1.41   10344539       0.03847   -­‐1.41   10342370       0.03721   -­‐1.41   10338490       0.04275   -­‐1.41   10599680   3830403N18Rik   0.00106   -­‐1.41   10342242       0.04895   -­‐1.42   10473636   Olfr1262   0.01561   -­‐1.42   10339752       0.03869   -­‐1.43   10430679       0.01835   -­‐1.43   10340961       0.04626   -­‐1.43   10367744   LOC629446   0.02226   -­‐1.44   10584496   Olfr960   0.04404   -­‐1.44   10338096       0.04763   -­‐1.45   10338769       0.03071   -­‐1.45   10342217       0.04422   -­‐1.45   10339173       0.01800   -­‐1.46   10436050   Dppa4   0.04943   -­‐1.46   10338896       0.00101   -­‐1.46   10379996       0.03218   -­‐1.47   10341559       0.00005   -­‐1.47   10341865       0.04975   -­‐1.47   10342181       0.02448   -­‐1.49   10338268       0.00865   -­‐1.49   10349634       0.00742   -­‐1.49   10340813       0.00293   -­‐1.50   10591853   Tbx20   0.01127   -­‐1.51   10338700       0.01642   -­‐1.51   10342817       0.03512   -­‐1.51   10343593       0.02034   -­‐1.52   10340703       0.01613   -­‐1.52   10340037       0.00326   -­‐1.53   10339099       0.04763   -­‐1.53   10341543       0.02814   -­‐1.53   10338439       0.04478   -­‐1.54   10342033       0.02664   -­‐1.55   10341850       0.00675   -­‐1.55   10343680       0.04012   -­‐1.58   10338160       0.00726   -­‐1.58   10344149       0.03542   -­‐1.58   10338846       0.00326   -­‐1.59   10339193       0.03765   -­‐1.59   10342657       0.02383   -­‐1.59   10339380       0.01167   -­‐1.60   255   10343321       0.00416   -­‐1.60   10339074       0.03643   -­‐1.60   10344041       0.02589   -­‐1.61   10342874       0.03068   -­‐1.61   10343694       0.00657   -­‐1.61   10338329       0.04629   -­‐1.61   10344372       0.02505   -­‐1.62   10342729       0.01342   -­‐1.64   10343779       0.03391   -­‐1.64   10338820       0.00758   -­‐1.65   10341959       0.02907   -­‐1.65   10339650       0.00655   -­‐1.65   10344200       0.02040   -­‐1.65   10343966       0.04114   -­‐1.66   10344150       0.04214   -­‐1.71   10342575       0.02938   -­‐1.75   10341177       0.00251   -­‐1.76   10339684       0.02096   -­‐1.80   10340179       0.03975   -­‐1.82   10339050       0.00179   -­‐1.82   10339761       0.03695   -­‐1.86   10338807       0.01939   -­‐1.89   10342299       0.00396   -­‐1.99   10341917       0.00566   -­‐2.06   10339242       0.03477   -­‐2.47   10343923       0.01121   -­‐2.66     Appendix  Table  2.  Genome-­‐wide  gene  expression  analysis  of  proteoglycan   biglycan-­‐treated  biglycan  null  myotubes.     Biglycan  null  myoblasts  were  grown  into  confluence  and  differentiated  for  four   days.  The  myotubes  were  treated  with  proteoglycan  biglycan  for  8  hours.  RNA  was   harvested  from  the  cultures  and  reverse  transcribed  into  cDNA,  which  was  then   hybridized  to  Affymetrix  array  chips.  The  results  of  the  microarray  analysis  are   summarized  in  Table  2.  Fold  changes  greater  than  1.3  are  shown  in  the  table.  The   positive  values  indicate  the  fold  change  in  upregulation  of  the  listed  transcript  by   256   proteoglycan  biglycan  whereas  the  negative  values  indicate  the  fold  change  in   downregulation  of  the  listed  transcript.  As  in  the  case  of  non-­‐glycanated  biglycan,   proteoglycan  biglycan  did  not  induce  a  strong  transcriptional  response  in  biglycan   null  myotubes  under  these  conditions.  Among  the  regulated  annotated  transcripts,   there  are  three  common  responses  between  non-­‐glycanated  and  proteoglycan   treated  cultures:  T-­‐box  transcription  factor  TBX20  (Tbx20),  Carbonic  Anhydrase  2   (Car2)  and  kallikrein  1-­‐related  pepidase  b4  (Klk1b4).  Tbx20  and  Car2  transcripts   were  downregulated  by  proteoglycan  and  non-­‐glycanated  forms  of  biglycan,   whereas  Klk1b4  was  upregulated  by  both  forms.  Several  other  transcripts  were   uniquely  regulated  only  by  proteoglycan  or  non-­‐glycanated  form  of  biglycan,   indicating  that  the  two  forms  of  biglycan  can  initiate  different  signaling  events.         Appendix  Table  3     Probeset  ID   p-­‐value   Fold-­‐Change   hsa-­‐miR-­‐640_st   0.0079   5.47   cfa-­‐miR-­‐382_st   0.0119   5.32   cfa-­‐miR-­‐421_st   0.0324   4.56   rno-­‐miR-­‐301a_st   0.0133   3.75   ppy-­‐miR-­‐196_st   0.0430   3.74   mmu-­‐miR-­‐450b-­‐3p_st   0.0105   3.65   cfa-­‐miR-­‐15a_st   0.0055   3.14   mml-­‐miR-­‐196a_st   0.0210   2.96   hsa-­‐miR-­‐1300_st   0.0379   2.76   xtr-­‐miR-­‐199b_st   0.0002   2.61   mdo-­‐miR-­‐212_st   0.0213   2.61   cfa-­‐miR-­‐26b_st   0.0253   2.56   mml-­‐miR-­‐502-­‐5p_st   0.0109   2.55   mmu-­‐miR-­‐26b_st   0.0201   2.52   dre-­‐miR-­‐30a_st   0.0063   2.41   cfa-­‐miR-­‐29c_st   0.0306   2.36   hsa-­‐miR-­‐330-­‐5p_st   0.0076   2.32   257   bta-­‐miR-­‐148b_st   0.0128   2.31   mmu-­‐miR-­‐193_st   0.0181   2.30   mmu-­‐miR-­‐466i_st   0.0279   2.18   dme-­‐miR-­‐34_st   0.0090   2.18   mmu-­‐miR-­‐183_st   0.0070   2.17   xtr-­‐miR-­‐30e_st   0.0414   2.14   ssc-­‐miR-­‐224_st   0.0078   2.13   rno-­‐miR-­‐146b_st   0.0386   2.12   xtr-­‐miR-­‐10a_st   0.0235   2.11   xtr-­‐miR-­‐15c_st   0.0075   2.10   hsa-­‐miR-­‐539_st   0.0134   2.09   cfa-­‐miR-­‐196b_st   0.0103   2.09   mdv2-­‐miR-­‐M17_st   0.0145   2.06   rno-­‐miR-­‐124_st   0.0302   2.04   ppt-­‐miR419_st   0.0263   2.01   rno-­‐miR-­‐126_st   0.0209   2.00   xtr-­‐miR-­‐148a_st   0.0079   2.00   mml-­‐miR-­‐153_st   0.0246   -­‐2.02   hsa-­‐miR-­‐548b-­‐3p_st   0.0393   -­‐2.03   osa-­‐miR408_st   0.0436   -­‐2.05   mml-­‐miR-­‐877_st   0.0112   -­‐2.05   fru-­‐miR-­‐210_st   0.0006   -­‐2.05   cfa-­‐miR-­‐138a_st   0.0003   -­‐2.06   mmu-­‐miR-­‐673-­‐3p_st   0.0071   -­‐2.10   mml-­‐miR-­‐423-­‐5p_st   0.0089   -­‐2.14   hsa-­‐miR-­‐423-­‐5p_st   0.0261   -­‐2.16   mmu-­‐miR-­‐423-­‐5p_st   0.0051   -­‐2.17   hsa-­‐miR-­‐486-­‐3p_st   0.0156   -­‐2.18   rno-­‐miR-­‐23a-­‐star_st   0.0022   -­‐2.18   mmu-­‐miR-­‐483_st   0.0179   -­‐2.18   hsa-­‐miR-­‐23a-­‐star_st   0.0376   -­‐2.20   osa-­‐miR166c_st   0.0172   -­‐2.23   hsa-­‐miR-­‐27a-­‐star_st   0.0090   -­‐2.29   rno-­‐miR-­‐330-­‐star_st   0.0014   -­‐2.30   hsa-­‐miR-­‐25-­‐star_st   0.0041   -­‐2.36   gga-­‐miR-­‐456_st   0.0027   -­‐2.40   cin-­‐let-­‐7e_st   0.0242   -­‐2.41   ath-­‐miR395b_st   0.0061   -­‐2.41   mml-­‐miR-­‐615-­‐5p_st   0.0251   -­‐2.54   mmu-­‐miR-­‐491_st   0.0192   -­‐2.56   hsa-­‐miR-­‐1260_st   0.0086   -­‐2.66   mmu-­‐miR-­‐330-­‐star_st   0.0106   -­‐2.74   rno-­‐miR-­‐27a-­‐star_st   0.0015   -­‐2.80   mml-­‐miR-­‐548a_st   0.0190   -­‐2.80     258     Appendix  Table  3.  Genome-­‐wide  microRNA  expression  analysis  of  non-­‐glycanated   biglycan-­‐treated  (8h)  biglycan  null  myotubes.     Biglycan  null  myoblasts  were  grown  into  confluence  and  differentiated  for  four   days.  The  myotubes  were  treated  with  non-­‐glycanated  biglycan  for  8  hours.  RNA   was  immediately  harvested  with  miRNeasy  purification  columns  (QIAGEN)  and   labeled  with  FlashTag™  Biotin  HSR  RNA Labeling  Kit  (Genisphere).  Samples  were   run  in  Affymetrix  miRNA  arrays.  And  the  results  are  summarized  in  Table  2.  A   number  of  microRNAs  were  up-­‐  and  downregulated  in  biglycan  null  myotubes  by   biglycan  treatment.  A  positive  value  indicates  the  fold  change  for  an  upregulated   microRNA  ,  whereas  a  negative  value  indicates  the  fold  change  for  a  downregulated   microRNA.           Appendix  Tables  4-­7     Further  analysis  of  the  microarray  study  described  in  Chapter  2.  Transcripts  that   are  downregulated  by  BMP4  in  myoblast  and  myotube  cultures  of  wild  type  and   MuSK  null  cells  are  listed  in  Tables  4-­‐7.  The  negative  values  indicate  the  fold  change   in  downregulation  of  the  listed  transcript  by  BMP4.           259   Appendix  Table  4     p-­‐value(N  vs.   Fold-­‐ Gene  Symbol   B)   Change   6330406I15Rik   0.0000214   -­‐4.15   Duxbl   0.0000541   -­‐3.72   Duxbl   0.0000541   -­‐3.72   Duxbl   0.0000639   -­‐3.61   Lrig1   0.0000560   -­‐3.40   Bnc2   0.0005148   -­‐3.34   Mest   0.0000348   -­‐3.31   Adamts5   0.0000029   -­‐3.20   Chst15   0.0000001   -­‐3.16   Sox8   0.0000390   -­‐3.12   Mrgprf   0.0006931   -­‐3.07   Slc40a1   0.0000694   -­‐2.89   Cdkn1c   0.0002003   -­‐2.84   Tgm2   0.0000382   -­‐2.79   E2f2   0.0005093   -­‐2.79   Vipr2   0.0000877   -­‐2.77   Rgs16   0.0000216   -­‐2.64   Lypd6   0.0002860   -­‐2.64   Bdkrb1   0.0006553   -­‐2.63   Sema6a   0.0003070   -­‐2.62   Tm6sf1   0.0001879   -­‐2.60   Lrch1   0.0000196   -­‐2.60   Dtx4   0.0001109   -­‐2.56   Daam2   0.0000656   -­‐2.55   Ramp2   0.0000431   -­‐2.50   Enpp1   0.0000532   -­‐2.48   1810041L15Rik   0.0003347   -­‐2.47   Fgfr4   0.0001609   -­‐2.44       0.0001612   -­‐2.41   Enpp3   0.0002659   -­‐2.41   2610301F02Rik   0.0000533   -­‐2.36   Arhgap28   0.0003329   -­‐2.36   Gm7325   0.0002460   -­‐2.35   Pdlim2   0.0001043   -­‐2.34   Rgma   0.0000702   -­‐2.33   Tmem184a   0.0000144   -­‐2.32   Cpa4   0.0007659   -­‐2.31   2610301F02Rik   0.0000483   -­‐2.29   Pde2a   0.0000306   -­‐2.27   Ccbe1   0.0007084   -­‐2.24   260   Prkag3   0.0003616   -­‐2.23   Ccdc88c   0.0000119   -­‐2.22   2610301F02Rik   0.0010167   -­‐2.21   Cdh15   0.0000005   -­‐2.20   Lrrc32   0.0000532   -­‐2.20   Mamstr   0.0009100   -­‐2.19   Ctnnal1   0.0000288   -­‐2.19   Pip4k2a   0.0001842   -­‐2.17   Zfp238   0.0000058   -­‐2.17   Ralgps2   0.0003000   -­‐2.14   Coro2b   0.0003650   -­‐2.12   Filip1   0.0001155   -­‐2.12   Cdc42ep2   0.0001646   -­‐2.09   Rbm24   0.0000501   -­‐2.07   Gprc5c   0.0001175   -­‐2.06   Ampd3   0.0000043   -­‐2.06   Sema7a   0.0009474   -­‐2.05   Klhl31   0.0000093   -­‐2.02   Pnmal2   0.0005892   -­‐2.01   Frmpd1   0.0004263   -­‐2.00   Aif1l   0.0003115   -­‐1.99   Fam83b   0.0001286   -­‐1.98   Spg21   0.0000623   -­‐1.98   Fam122b   0.0004044   -­‐1.96   Sema5a   0.0000214   -­‐1.95   C330016O10Rik   0.0000032   -­‐1.95   Hdac11   0.0000696   -­‐1.93   Klhl31   0.0000181   -­‐1.92   Ndrg1   0.0000246   -­‐1.92   Fam49a   0.0009917   -­‐1.92   Ston2   0.0002517   -­‐1.91   Ccdc23   0.0002880   -­‐1.90   Il17d   0.0006934   -­‐1.90   Kbtbd5   0.0000863   -­‐1.90   Agap1   0.0002084   -­‐1.89   Tspan15   0.0008423   -­‐1.89   Fam65b   0.0001679   -­‐1.88   Klhl30   0.0004261   -­‐1.88   Egln3   0.0009972   -­‐1.86   Chrnd   0.0000037   -­‐1.86   Rgmb   0.0010311   -­‐1.86   Spry1   0.0003263   -­‐1.86   Dmrt2   0.0001814   -­‐1.85   Frmd4b   0.0009842   -­‐1.84   261   9930013L23Rik   0.0003634   -­‐1.84   Chd7   0.0001923   -­‐1.84   Gprc5c   0.0004738   -­‐1.82   Itga3   0.0010084   -­‐1.82   Notch3   0.0001628   -­‐1.82   Heyl   0.0000305   -­‐1.82   Traf4   0.0000342   -­‐1.82   Clcn5   0.0004309   -­‐1.81   AI464131   0.0009525   -­‐1.81   Maf   0.0002017   -­‐1.80   Chd7   0.0005106   -­‐1.79   Mbnl3   0.0002275   -­‐1.78   Best1   0.0002190   -­‐1.77   Iffo1   0.0000004   -­‐1.77   Gal3st2   0.0004868   -­‐1.77   Gal3st2   0.0006560   -­‐1.77   Pacs2   0.0000134   -­‐1.76   Morc4   0.0002557   -­‐1.76   Chd7   0.0001987   -­‐1.76   Svil   0.0000311   -­‐1.76   Smtnl2   0.0000119   -­‐1.74   Fcgr4   0.0000166   -­‐1.74   Gfra1   0.0001197   -­‐1.74   Zeb1   0.0002503   -­‐1.74   Cap2   0.0000325   -­‐1.74   Plxna1   0.0002050   -­‐1.73   Chd7   0.0002448   -­‐1.73   Fam53b   0.0006727   -­‐1.73   Bicd2   0.0000716   -­‐1.72   Phf17   0.0005239   -­‐1.72   Chd7   0.0000289   -­‐1.71   Olfml2a   0.0000293   -­‐1.71   Ehd4   0.0004606   -­‐1.71   Chd7   0.0006552   -­‐1.71   Nup210   0.0004276   -­‐1.70   Cdk5r1   0.0003189   -­‐1.70   Rassf4   0.0005223   -­‐1.70   Dapk2   0.0003511   -­‐1.70   Chrng   0.0000015   -­‐1.70   Sema3c   0.0000007   -­‐1.70   Sema3e   0.0000277   -­‐1.69   Gdpd1   0.0000414   -­‐1.69   Lrrc30   0.0006275   -­‐1.69   Popdc2   0.0004407   -­‐1.69   262   Asb2   0.0000257   -­‐1.69   Xrcc5   0.0000456   -­‐1.68   Trim55   0.0009039   -­‐1.68   Kif24   0.0000592   -­‐1.68   Dclre1c   0.0006324   -­‐1.68   Cpa1   0.0000918   -­‐1.67   Arhgap24   0.0006311   -­‐1.67   Arhgap29   0.0000061   -­‐1.67   Rps6ka2   0.0000011   -­‐1.66   Ankrd2   0.0000785   -­‐1.66   Lmnb2   0.0004493   -­‐1.66   Dock11   0.0000073   -­‐1.66   2310015B20Rik   0.0004175   -­‐1.65   Syne1   0.0000040   -­‐1.65   Fbxo10   0.0000281   -­‐1.65   Mfsd2   0.0005685   -­‐1.62   Erbb3   0.0000709   -­‐1.61   Arhgap18   0.0007159   -­‐1.61   Plxdc1   0.0004110   -­‐1.60   Chd7   0.0006499   -­‐1.60   Smad3   0.0000005   -­‐1.60       0.0002819   -­‐1.60   Slc22a23   0.0003138   -­‐1.60   Mybph   0.0000782   -­‐1.59   Slc9a3r1   0.0006011   -­‐1.59   Fzd9   0.0006109   -­‐1.59   Chd7   0.0009537   -­‐1.58   Spsb1   0.0007464   -­‐1.57   Kitl   0.0007540   -­‐1.57   Casz1   0.0004358   -­‐1.57   Rasl11b   0.0006053   -­‐1.57   Cd200   0.0003042   -­‐1.56   Bid   0.0004567   -­‐1.56   Tpcn1   0.0001069   -­‐1.56   Ccdc134   0.0000213   -­‐1.56   Gpt2   0.0005790   -­‐1.56   1190002N15Rik   0.0003878   -­‐1.56   Slc12a2   0.0007791   -­‐1.55   Dok3   0.0004473   -­‐1.55   Cd82   0.0000093   -­‐1.54   Chd7   0.0001008   -­‐1.54   Ablim1   0.0010537   -­‐1.54   Dusp3   0.0001850   -­‐1.54   Cyfip2   0.0003695   -­‐1.54   263   Ptprd   0.0001094   -­‐1.53   Il1r1   0.0001670   -­‐1.53   Purb   0.0008843   -­‐1.53   Arhgap22   0.0007595   -­‐1.53   Syne1   0.0000148   -­‐1.53   Amigo1   0.0008859   -­‐1.53   Stard13   0.0000430   -­‐1.53   Btbd17   0.0001645   -­‐1.53   Ankrd23   0.0001154   -­‐1.53   Scn4a   0.0003361   -­‐1.53   Sdpr   0.0005746   -­‐1.52   Fam171a2   0.0000310   -­‐1.52   D14Ertd449e   0.0005901   -­‐1.52   Chd7   0.0001726   -­‐1.52   Ugcg   0.0005628   -­‐1.52   Slc9a9   0.0000562   -­‐1.52   Wisp1   0.0009822   -­‐1.51   2210020M01Rik   0.0006756   -­‐1.51   Ets2   0.0001837   -­‐1.51   D14Ertd449e   0.0006609   -­‐1.51   D14Ertd449e   0.0004112   -­‐1.51   Epb4.1l5   0.0003958   -­‐1.51   Tanc2   0.0007363   -­‐1.51   Chd7   0.0010659   -­‐1.51   Dync1i1   0.0001684   -­‐1.50   Notch1   0.0005415   -­‐1.50   C78339   0.0004487   -­‐1.50   Pxdn   0.0003548   -­‐1.49   Chd7   0.0006375   -­‐1.49   Gm10336   0.0008154   -­‐1.49   Lama5   0.0001344   -­‐1.49   4-­‐Sep   0.0000913   -­‐1.48   D15Wsu169e   0.0003490   -­‐1.48   Fam20a   0.0006558   -­‐1.48   Myom2   0.0007167   -­‐1.48   Olfml2b   0.0007103   -­‐1.47   Adamts7   0.0004595   -­‐1.47   Tmem62   0.0005440   -­‐1.47   Hfe2   0.0001716   -­‐1.47   Fchsd2   0.0002648   -­‐1.47   Met   0.0010149   -­‐1.46   Afap1l2   0.0006751   -­‐1.46   Ppfia4   0.0007139   -­‐1.46   Cpa5   0.0000342   -­‐1.46   264   Tjp1   0.0003720   -­‐1.46   Tns1   0.0001648   -­‐1.46   Dll1   0.0000406   -­‐1.46   Pygm   0.0001745   -­‐1.46   Txnip   0.0003089   -­‐1.45   Efhd2   0.0005053   -­‐1.45   Plau   0.0006234   -­‐1.45   Mtss1   0.0000074   -­‐1.45   Itga7   0.0009093   -­‐1.45   Atp2a1   0.0000915   -­‐1.45   Baiap2l1   0.0004460   -­‐1.45   Stk17b   0.0000621   -­‐1.44   Reep2   0.0001658   -­‐1.44       0.0009833   -­‐1.44   Cnot6l   0.0000034   -­‐1.44   Lta4h   0.0000616   -­‐1.43   Skp2   0.0001334   -­‐1.43   Actn3   0.0007608   -­‐1.43   Tubb2b   0.0002943   -­‐1.43   Ddx51   0.0001568   -­‐1.43   Mef2a   0.0004708   -­‐1.42       0.0002089   -­‐1.42   Dbndd1   0.0005277   -­‐1.42   Atp13a5   0.0007721   -­‐1.42   Bcam   0.0005741   -­‐1.42   Amigo2   0.0010740   -­‐1.42   Ppbp   0.0009399   -­‐1.42   Sorbs3   0.0001509   -­‐1.41   Pnpla2   0.0002227   -­‐1.41   BC018507   0.0000682   -­‐1.41   Bin1   0.0000008   -­‐1.41   Purb   0.0002797   -­‐1.41   Cd97   0.0001901   -­‐1.40   Klhdc6   0.0009893   -­‐1.40   Zfp608   0.0002994   -­‐1.40   Ezh1   0.0001291   -­‐1.40   Exosc9   0.0000082   -­‐1.39   Il34   0.0006272   -­‐1.39   Amotl1   0.0003035   -­‐1.39   Krt80   0.0006019   -­‐1.39   B4galt4   0.0004063   -­‐1.39   Lmf1   0.0000247   -­‐1.38   Lhfpl2   0.0008023   -­‐1.38   Akr1b8   0.0005933   -­‐1.38   265   Chrnb1   0.0001167   -­‐1.38   Lrba   0.0000498   -­‐1.38   St3gal5   0.0007294   -­‐1.37       0.0009068   -­‐1.37   Ctsc   0.0004288   -­‐1.37   Wnt9a   0.0004058   -­‐1.37   Stk25   0.0005884   -­‐1.37   Lpin3   0.0002048   -­‐1.37   Gramd4   0.0000025   -­‐1.37   Tmem20   0.0002588   -­‐1.37   Top1   0.0002888   -­‐1.36   Lamb1-­‐1   0.0007479   -­‐1.36   Spats2l   0.0001604   -­‐1.36   Dbn1   0.0000668   -­‐1.36   Ttyh2   0.0000039   -­‐1.35   Fam110b   0.0001362   -­‐1.35   Atp7a   0.0001619   -­‐1.35   Gca   0.0009740   -­‐1.34   Arhgap10   0.0001320   -­‐1.34   Dtna   0.0008527   -­‐1.34   Cdk2   0.0005810   -­‐1.33   Itgb1bp2   0.0008065   -­‐1.33   Edil3   0.0009066   -­‐1.33   Tubb6   0.0001897   -­‐1.33   Tcea3   0.0000086   -­‐1.33   Ckm   0.0001867   -­‐1.33   Fam13c   0.0000498   -­‐1.33   Unc93b1   0.0001296   -­‐1.33   Rnd2   0.0001313   -­‐1.32   Cugbp2   0.0002356   -­‐1.32   Foxo4   0.0002154   -­‐1.32   Apod   0.0007457   -­‐1.32   Sh2b1   0.0004921   -­‐1.32   Mboat2   0.0008968   -­‐1.31   Kif13b   0.0009384   -­‐1.31   Acsl1   0.0004434   -­‐1.31   Zfp322a   0.0003003   -­‐1.31   Lrrc1   0.0002445   -­‐1.31   Exoc7   0.0000656   -­‐1.31   Dhx32   0.0009763   -­‐1.30   Fbxo16   0.0009223   -­‐1.30   D17H6S56E-­‐5   0.0002998   -­‐1.30   Gabrb2   0.0008291   -­‐1.30   RP23-­‐100C5.8   0.0000397   -­‐1.30   266   Prpf39   0.0006212   -­‐1.30   Hmgn3   0.0008245   -­‐1.30   Nfatc2   0.0009864   -­‐1.29   Psat1   0.0008636   -­‐1.29   Herc1   0.0003193   -­‐1.29   Pja1   0.0004191   -­‐1.29   Slc29a1   0.0005735   -­‐1.29   Afap1   0.0000391   -­‐1.29   Ddhd1   0.0005818   -­‐1.29   Spg20   0.0003120   -­‐1.28   Gpc4   0.0000631   -­‐1.28   Elmod2   0.0003579   -­‐1.28   Unc84a   0.0008693   -­‐1.28   D930014E17Rik   0.0010117   -­‐1.28   Tmem109   0.0002907   -­‐1.28   Ick   0.0000467   -­‐1.28   Hspa12a   0.0010554   -­‐1.28   Ivns1abp   0.0007133   -­‐1.28   Sox6   0.0006008   -­‐1.27   Lsp1   0.0005521   -­‐1.27   Mfsd7a   0.0004216   -­‐1.26   Olfm1   0.0010299   -­‐1.26   Fam117a   0.0009305   -­‐1.26   Col5a3   0.0007260   -­‐1.25   Pak1   0.0005636   -­‐1.25   Gm12888   0.0003339   -­‐1.25   Macf1   0.0000413   -­‐1.25   Ddr2   0.0002458   -­‐1.25   Bcat2   0.0000990   -­‐1.25   Il17rd   0.0008439   -­‐1.25   Sh3d19   0.0007272   -­‐1.25   Ormdl2   0.0007964   -­‐1.25   Fdft1   0.0008487   -­‐1.25   Il17rc   0.0002456   -­‐1.25   Akr1b3   0.0002085   -­‐1.24   Akr1b3   0.0001740   -­‐1.24   Fbxo40   0.0006168   -­‐1.24   Matn2   0.0008516   -­‐1.24   Nat11   0.0006252   -­‐1.24   Akr1b3   0.0003444   -­‐1.23   Iqcb1   0.0000167   -­‐1.23   Tsga14   0.0008146   -­‐1.23   Zfp345   0.0010402   -­‐1.23   Zfp521   0.0000536   -­‐1.23   267   Col4a1   0.0000539   -­‐1.23       0.0007426   -­‐1.22   Fads3   0.0004095   -­‐1.22   Ank2   0.0001693   -­‐1.22   E230016K23Rik   0.0010596   -­‐1.22   Ppm1l   0.0007170   -­‐1.22   Serinc2   0.0004424   -­‐1.21   Tmpo   0.0001389   -­‐1.21   Kat2b   0.0000499   -­‐1.21   Ephb3   0.0006023   -­‐1.21   Atp10a   0.0008674   -­‐1.20   Rsad2   0.0009606   -­‐1.20   Suhw4   0.0006522   -­‐1.20   Jag2   0.0002328   -­‐1.20       0.0002156   -­‐1.20   Tnnt2   0.0001028   -­‐1.20   Tns3   0.0010653   -­‐1.20   Myof   0.0000245   -­‐1.19   Zfp367   0.0001623   -­‐1.19   Fam53a   0.0001783   -­‐1.18   A530098C11Rik   0.0000096   -­‐1.18   Pcx   0.0002013   -­‐1.18   Plat   0.0000242   -­‐1.17   Hdac9   0.0006160   -­‐1.17   AI987944   0.0010865   -­‐1.17   Brp16   0.0007445   -­‐1.17   Usp31   0.0006690   -­‐1.17   Entpd4   0.0006852   -­‐1.17   Reep4   0.0003595   -­‐1.17       0.0009543   -­‐1.16   Plcl2   0.0008897   -­‐1.16   Myod1   0.0000826   -­‐1.16   Mtap1b   0.0010154   -­‐1.16   Mt2   0.0001084   -­‐1.16   Parp3   0.0004961   -­‐1.15   Nbeal2   0.0007471   -­‐1.15   Adam8   0.0000579   -­‐1.15   Evl   0.0006758   -­‐1.14   Msto1   0.0010184   -­‐1.14       0.0010175   -­‐1.14   Mvd   0.0008178   -­‐1.13   Numa1   0.0007722   -­‐1.13   Dtl   0.0000093   -­‐1.12   Fam54b   0.0007400   -­‐1.12   268   Psmd11   0.0007981   -­‐1.12   Arhgap17   0.0002459   -­‐1.11   Xrcc1   0.0005915   -­‐1.11   Phldb3   0.0009385   -­‐1.11   Dmxl1   0.0001392   -­‐1.10   Thoc2   0.0003223   -­‐1.10   Wdr1   0.0003185   -­‐1.10   Cenpq   0.0002256   -­‐1.09   Clcn2   0.0006650   -­‐1.09   Lamc1   0.0000498   -­‐1.09       Appendix  Table  4.  Transcripts  downregulated  by  BMP4-­‐treatment  in  wild  type   myoblasts.             Appendix  Table  5       p-­‐value(N   Fold-­‐ Gene  Symbol   vs.  B)   Change   Adcyap1r1   0.0000028   -­‐5.20   Cpa1   0.0001414   -­‐4.35   Chrnd   0.0000029   -­‐3.77   Stc1   0.0002201   -­‐3.66   Ndst4   0.0003585   -­‐3.64       0.0002743   -­‐3.53   Hpgd   0.0000091   -­‐3.35       0.0006717   -­‐3.34   2610301F02Rik   0.0002716   -­‐3.10   Fam83b   0.0000565   -­‐2.86   Igfbp5   0.0001906   -­‐2.84   2610301F02Rik   0.0000096   -­‐2.83   2610301F02Rik   0.0000103   -­‐2.81   2610301F02Rik   0.0001942   -­‐2.78   Dtx4   0.0000933   -­‐2.75   Tm6sf1   0.0000383   -­‐2.63   Enpp1   0.0000332   -­‐2.58   Cdh15   0.0000219   -­‐2.55   Sprr1a   0.0002499   -­‐2.48   Chrng   0.0000380   -­‐2.48   269   Ramp2   0.0001086   -­‐2.48   Slc40a1   0.0000193   -­‐2.46   Fgfr4   0.0000137   -­‐2.46   Lypd6   0.0007059   -­‐2.42   Tmem184a   0.0002739   -­‐2.42   Arhgap28   0.0002005   -­‐2.37   Rbm24   0.0000004   -­‐2.34   Chst15   0.0000597   -­‐2.29       0.0004783   -­‐2.27   Hfe2   0.0005804   -­‐2.26   Ttc9   0.0000015   -­‐2.25   Slc24a3   0.0001676   -­‐2.25   Chd7   0.0000302   -­‐2.20   Lrig1   0.0000044   -­‐2.20   Ctnnal1   0.0001247   -­‐2.19   Tsga14   0.0001482   -­‐2.17   Cap2   0.0000057   -­‐2.17   Chd7   0.0000239   -­‐2.17   Unc13c   0.0000814   -­‐2.16   Coro2b   0.0004026   -­‐2.15   Car2   0.0000008   -­‐2.14   Chd7   0.0005032   -­‐2.11   Vipr2   0.0001241   -­‐2.10   Ccdc88c   0.0002697   -­‐2.10   Chd7   0.0001239   -­‐2.08   Chd7   0.0000151   -­‐2.07   Chd7   0.0001643   -­‐2.04   Agap1   0.0003860   -­‐2.03   Chd7   0.0004892   -­‐2.03   Chd7   0.0004570   -­‐2.02   Cpa4   0.0001468   -­‐2.01   Chd7   0.0002146   -­‐2.00   Gm7325   0.0000931   -­‐1.99   Hivep2   0.0001623   -­‐1.99   Klhl31   0.0001572   -­‐1.99   Adamts5   0.0000140   -­‐1.98   Chd7   0.0001457   -­‐1.98   Chd7   0.0005202   -­‐1.98   Zfp238   0.0000044   -­‐1.98   Popdc3   0.0000782   -­‐1.97   Dll1   0.0000821   -­‐1.95   Notch3   0.0000128   -­‐1.94   Chd7   0.0004652   -­‐1.94   Fbxo32   0.0001011   -­‐1.93   270   Tdrkh   0.0006300   -­‐1.93   Clcn5   0.0001442   -­‐1.91   Slc9a9   0.0001118   -­‐1.91   Klhl31   0.0001764   -­‐1.89   Chd7   0.0001120   -­‐1.88   Klhdc10   0.0000899   -­‐1.88   Arhgap22   0.0001903   -­‐1.87   Dcn   0.0006473   -­‐1.86   Lrp4   0.0000161   -­‐1.85   Aif1l   0.0002770   -­‐1.84   Pip4k2a   0.0001586   -­‐1.84   Fmo1   0.0001751   -­‐1.83   Jup   0.0001243   -­‐1.83   Rgs16   0.0002544   -­‐1.83   Xrcc5   0.0001207   -­‐1.82   Chd7   0.0005404   -­‐1.82   Stk10   0.0000990   -­‐1.81   Sntb1   0.0001387   -­‐1.81   Bicd2   0.0000025   -­‐1.78   Itga3   0.0001786   -­‐1.77   Unc13c   0.0000694   -­‐1.76   Nptx1   0.0000105   -­‐1.76   Met   0.0001061   -­‐1.76   Sdpr   0.0000687   -­‐1.75   Scx   0.0004083   -­‐1.75   Fam69a   0.0005059   -­‐1.74   Best3   0.0005315   -­‐1.73   P2rx5   0.0000197   -­‐1.72   Shb   0.0005567   -­‐1.72   Slc9a7   0.0005183   -­‐1.71   Myo1e   0.0000003   -­‐1.71   Cd200   0.0000925   -­‐1.71   Chd7   0.0000277   -­‐1.70   Mest   0.0000073   -­‐1.70   Cldn15   0.0005397   -­‐1.70   Zcchc24   0.0002039   -­‐1.68   Kbtbd10   0.0001782   -­‐1.67       0.0004466   -­‐1.66   Hs6st1   0.0000628   -­‐1.66   Gdpd1   0.0003281   -­‐1.65   Lhfpl2   0.0000146   -­‐1.65   Gem   0.0000656   -­‐1.65   Cd97   0.0003106   -­‐1.65   Gfra1   0.0000231   -­‐1.65   271       0.0000572   -­‐1.65   Galntl4   0.0002151   -­‐1.65   AW551984   0.0003040   -­‐1.64   Mycl1   0.0002966   -­‐1.63   Stard13   0.0000628   -­‐1.62   Gpt2   0.0000802   -­‐1.62   Lrch1   0.0000667   -­‐1.61   Actn3   0.0005938   -­‐1.61   Rgmb   0.0002414   -­‐1.61       0.0002790   -­‐1.61   Syt13   0.0000198   -­‐1.60   Fam122b   0.0001533   -­‐1.60   Chd7   0.0002917   -­‐1.60   Dtna   0.0000980   -­‐1.60   Pion   0.0000019   -­‐1.60   Gas2   0.0006283   -­‐1.60   Cd82   0.0002126   -­‐1.60       0.0004899   -­‐1.59   Ndrg1   0.0000563   -­‐1.59   Chrnb1   0.0000251   -­‐1.58   Traf4   0.0002500   -­‐1.58   Gadd45a   0.0000380   -­‐1.58   Ifih1   0.0001069   -­‐1.57   Tjp1   0.0003129   -­‐1.57   Txnip   0.0000137   -­‐1.57   1190002N15Rik   0.0000147   -­‐1.57   Adamts4   0.0007051   -­‐1.56   Plk2   0.0006916   -­‐1.55   Bicd1   0.0001450   -­‐1.55   Apcdd1   0.0000411   -­‐1.55   Klf5   0.0004701   -­‐1.55   Fhl2   0.0000263   -­‐1.53   Sema3d   0.0000804   -­‐1.53   9930013L23Rik   0.0002632   -­‐1.53   Pcbd1   0.0004990   -­‐1.53   Trim55   0.0006800   -­‐1.52   Akr1c14   0.0006291   -­‐1.52   Itgb6   0.0000148   -­‐1.52   Pdgfa   0.0003712   -­‐1.52   Dusp3   0.0003170   -­‐1.51   Synj2   0.0000838   -­‐1.51   Myod1   0.0001626   -­‐1.51   Ankrd35   0.0004248   -­‐1.50   Tpcn1   0.0002357   -­‐1.49   272   Iqsec1   0.0005769   -­‐1.48   Antxr2   0.0000413   -­‐1.48   Dock9   0.0003769   -­‐1.47   Tmem109   0.0002984   -­‐1.47   Skp2   0.0001525   -­‐1.47   Ralgps2   0.0000971   -­‐1.46   Krt80   0.0001474   -­‐1.46   Mtss1   0.0004086   -­‐1.46   Cdh1   0.0001488   -­‐1.45   Ano5   0.0005794   -­‐1.45   Eda2r   0.0000150   -­‐1.44   Syne1   0.0000071   -­‐1.44   Tubb2b   0.0002275   -­‐1.44   Dmpk   0.0001492   -­‐1.44   Slc29a1   0.0003806   -­‐1.43   Pik3c2b   0.0002730   -­‐1.43   C130092O11Rik   0.0000652   -­‐1.43   Chd7   0.0002514   -­‐1.43   Ugcg   0.0000811   -­‐1.43   Olfm1   0.0003338   -­‐1.42   Lphn2   0.0004620   -­‐1.42   Lpin3   0.0005826   -­‐1.42   Pacs2   0.0006733   -­‐1.41   Itga7   0.0001328   -­‐1.41   Sort1   0.0000292   -­‐1.41   Pdgfc   0.0000736   -­‐1.41   Glrb   0.0001839   -­‐1.41   Rerg   0.0004835   -­‐1.41   Cyld   0.0003028   -­‐1.40   Inpp4b   0.0000321   -­‐1.40   Svil   0.0004115   -­‐1.40   Baiap2   0.0005305   -­‐1.40   Bves   0.0000880   -­‐1.39   Ptpla   0.0002489   -­‐1.39   Stam   0.0000360   -­‐1.38   C78339   0.0001219   -­‐1.38   Wisp1   0.0003277   -­‐1.38   Lphn2   0.0006636   -­‐1.37   Acadl   0.0005546   -­‐1.37   Tubb2b   0.0001265   -­‐1.37   Best1   0.0003734   -­‐1.37   Tbx18   0.0003119   -­‐1.36   Hs6st2   0.0006049   -­‐1.36   Syne1   0.0000651   -­‐1.35   273   Ube2e3   0.0006259   -­‐1.35   Mt2   0.0004739   -­‐1.35   Lphn2   0.0000189   -­‐1.35   Fam178a   0.0005696   -­‐1.34   Ephb6   0.0001508   -­‐1.34   Pitx2   0.0001463   -­‐1.34   Cnot6l   0.0003393   -­‐1.34   Ypel1   0.0003653   -­‐1.33   Gpc1   0.0006417   -­‐1.33   Ivns1abp   0.0003064   -­‐1.33   Ezh1   0.0002966   -­‐1.32   Calcoco1   0.0000480   -­‐1.32   Asb5   0.0003891   -­‐1.32   Lamb1-­‐1   0.0004490   -­‐1.31   St6gal1   0.0000148   -­‐1.31   Maob   0.0005573   -­‐1.31   Sh2b1   0.0004378   -­‐1.31   Akr1b3   0.0006544   -­‐1.30   Syngr2   0.0001531   -­‐1.30   Lphn2   0.0000670   -­‐1.30   Arhgef2   0.0001105   -­‐1.30   Rnf128   0.0001125   -­‐1.30   Akr1b3   0.0000255   -­‐1.30   Akr1b3   0.0002428   -­‐1.30   Ahnak2   0.0000128   -­‐1.29   Fbxo10   0.0001086   -­‐1.29   Prrg4   0.0006853   -­‐1.29   Stard10   0.0005295   -­‐1.29   Ick   0.0002723   -­‐1.28   Stk25   0.0000398   -­‐1.28       0.0002023   -­‐1.28   4930455F23Rik   0.0003160   -­‐1.27   Map3k9   0.0001815   -­‐1.27   Pacsin2   0.0001521   -­‐1.27   Vwa5a   0.0000203   -­‐1.27   Lmf1   0.0003618   -­‐1.27   Akr1b3   0.0001573   -­‐1.26   Rhbdf1   0.0002887   -­‐1.26   Serinc2   0.0003209   -­‐1.26   Abca2   0.0004818   -­‐1.26   Xrn2   0.0003918   -­‐1.25   Klhl7   0.0002300   -­‐1.25   Parp9   0.0003623   -­‐1.25   Il17rd   0.0003754   -­‐1.25   274   Tpm2   0.0005828   -­‐1.25   Ccdc134   0.0002501   -­‐1.23       0.0001714   -­‐1.23   Ext1   0.0006306   -­‐1.23   Vasp   0.0002122   -­‐1.23   Tmem209   0.0005555   -­‐1.23   Stom   0.0001346   -­‐1.23   Klhl24   0.0004269   -­‐1.22   Igf2r   0.0003164   -­‐1.22   Gal3st2   0.0002920   -­‐1.22   Fads3   0.0002755   -­‐1.21   BC031353   0.0001315   -­‐1.20   Zc3h12c   0.0002538   -­‐1.20   Rab11fip5   0.0006123   -­‐1.19   Entpd4   0.0004000   -­‐1.19       0.0006959   -­‐1.19   Ctnna1   0.0001451   -­‐1.17   Dcaf5   0.0005100   -­‐1.17   Mtap6   0.0006455   -­‐1.17   Colec12   0.0000988   -­‐1.17   Eif4e2   0.0002206   -­‐1.17   Dst   0.0001110   -­‐1.17   Zfp36l2   0.0001325   -­‐1.17   Ggta1   0.0001781   -­‐1.16   Nbr1   0.0005050   -­‐1.15   Olfr820   0.0003139   -­‐1.15   Slc38a9   0.0007032   -­‐1.14   Frmd4a   0.0006123   -­‐1.14   Cnnm3   0.0001414   -­‐1.14   Sh3d19   0.0003417   -­‐1.14   1700025G04Rik   0.0004922   -­‐1.13   Lmln   0.0001396   -­‐1.13   Pikfyve   0.0001838   -­‐1.12   Car3   0.0004176   -­‐1.11   Sec24d   0.0006966   -­‐1.11   Lclat1   0.0002311   -­‐1.10   Rprd1b   0.0003377   -­‐1.10   Zfyve1   0.0005029   -­‐1.10   Tspan6   0.0004392   -­‐1.10   Ptpn21   0.0004059   -­‐1.10   Klk13   0.0003505   -­‐1.10   Zbtb45   0.0006870   -­‐1.09   Ndufs8   0.0004926   -­‐1.09   Apol7b   0.0000998   -­‐1.08   275   Ube2l3   0.0001781   -­‐1.07       0.0003404   -­‐1.06   Eif4ebp1   0.0003727   -­‐1.06   Hist1h3f   0.0004130   -­‐1.04     Appendix  Table  5.  Transcripts  downregulated  by  BMP4-­‐treatment  in  MuSK  null   myoblasts.       Appendix  Table  6       p-­‐value(N   Fold-­‐ Gene  Symbol   vs.  B)   Change   Fgf9   0.0000046   -­‐3.75       0.0002845   -­‐2.94   E2f2   0.0000097   -­‐2.90   Lrrc26   0.0005241   -­‐2.89   Mest   0.0006330   -­‐2.85   Duxbl   0.0000890   -­‐2.63   Duxbl   0.0000890   -­‐2.63   Duxbl   0.0001782   -­‐2.53   Nptx1   0.0001370   -­‐2.52   Heg1   0.0000703   -­‐2.45   4921525H12Rik   0.0001037   -­‐2.42   Gpr146   0.0000551   -­‐2.36   Egr3   0.0005306   -­‐2.21   Rhobtb2   0.0000866   -­‐2.20   Rgma   0.0000433   -­‐2.20       0.0000941   -­‐2.19   Mxd1   0.0006945   -­‐2.19   Abra   0.0000652   -­‐2.16   Cdkl5   0.0001211   -­‐2.12   Lrch1   0.0000298   -­‐2.11   Gm10387   0.0002122   -­‐2.11   Lrig1   0.0004189   -­‐2.09   Fam53b   0.0000145   -­‐2.01   Grit   0.0000201   -­‐2.00   Kcnk5   0.0000238   -­‐2.00   Chst15   0.0000297   -­‐1.96   Daam2   0.0000014   -­‐1.95   Sema7a   0.0003653   -­‐1.95   276   Klk1b8   0.0003336   -­‐1.94   Fam49a   0.0004006   -­‐1.94   Lrrc1   0.0002910   -­‐1.94   2610301F02Rik   0.0000383   -­‐1.93   Il1r1   0.0005947   -­‐1.91   Erbb3   0.0000517   -­‐1.90   Ppargc1a   0.0000485   -­‐1.88   Sdpr   0.0000502   -­‐1.87   Plekha7   0.0007074   -­‐1.86   2610301F02Rik   0.0005047   -­‐1.84   Mc4r   0.0001750   -­‐1.84   Rgmb   0.0000535   -­‐1.80   Pstpip2   0.0001208   -­‐1.78   Mospd1   0.0003195   -­‐1.78   Slc7a8   0.0002770   -­‐1.77   Grtp1   0.0000262   -­‐1.77   Arhgap28   0.0000184   -­‐1.73   Asb2   0.0006515   -­‐1.72   Ralgps1   0.0002598   -­‐1.72   Tbx15   0.0000274   -­‐1.72   Man1c1   0.0000360   -­‐1.70       0.0004747   -­‐1.68   Filip1   0.0000149   -­‐1.68   Tspan14   0.0006709   -­‐1.68   Osr1   0.0003587   -­‐1.68   Gpt2   0.0002045   -­‐1.68   Bnc2   0.0000572   -­‐1.68   2610301F02Rik   0.0004670   -­‐1.68   Arhgap18   0.0000334   -­‐1.67   2210020M01Rik   0.0000845   -­‐1.67   Gm10009   0.0003104   -­‐1.66   Unc13c   0.0005220   -­‐1.65   Wnt10a   0.0005906   -­‐1.65   Mamstr   0.0002788   -­‐1.65   Mef2d   0.0001481   -­‐1.64   Fam65b   0.0002804   -­‐1.63   Pdlim2   0.0005653   -­‐1.63   2610301F02Rik   0.0005299   -­‐1.61   Sema4c   0.0007481   -­‐1.61   Heyl   0.0004110   -­‐1.61   Pdgfb   0.0004514   -­‐1.61   Rrad   0.0002232   -­‐1.60   Lmo7   0.0005250   -­‐1.60   Atp13a5   0.0001174   -­‐1.58   277   Sox8   0.0000677   -­‐1.58   Mrgprf   0.0000883   -­‐1.58   Adamts5   0.0006270   -­‐1.58   Tnfaip3   0.0000975   -­‐1.57   Klhl31   0.0000407   -­‐1.57   Slc9a9   0.0002163   -­‐1.56   Cbfa2t3   0.0001574   -­‐1.56   Il33   0.0006369   -­‐1.56   Casz1   0.0000916   -­‐1.55   Thpo   0.0000921   -­‐1.54   Prkag3   0.0000558   -­‐1.54   Unc93b1   0.0005276   -­‐1.54   Rnf122   0.0003810   -­‐1.53   Fn3k   0.0001507   -­‐1.53       0.0003518   -­‐1.53   Hjurp   0.0005089   -­‐1.52   Dhcr7   0.0003801   -­‐1.52   Reep1   0.0000054   -­‐1.51   Tfrc   0.0004259   -­‐1.51   Sema6a   0.0000158   -­‐1.51   Snx30   0.0004785   -­‐1.50   Stard13   0.0007689   -­‐1.50   Pnmal2   0.0000757   -­‐1.50   Doc2b   0.0000910   -­‐1.50   Colq   0.0004442   -­‐1.49   Slc16a2   0.0000138   -­‐1.48   Igfbp3   0.0000305   -­‐1.48   Gtdc1   0.0000988   -­‐1.48   Fgfbp1   0.0001426   -­‐1.48   Plau   0.0002335   -­‐1.47   D15Wsu169e   0.0004967   -­‐1.47   Ablim1   0.0000332   -­‐1.47   Ctnnal1   0.0003709   -­‐1.47   Klhl31   0.0000655   -­‐1.47   Gramd1b   0.0002090   -­‐1.47   Uts2r   0.0000370   -­‐1.47   Kbtbd5   0.0000458   -­‐1.46   Ankrd23   0.0006935   -­‐1.46   Slc12a2   0.0007107   -­‐1.46   Mylk   0.0005990   -­‐1.46       0.0003135   -­‐1.44   Plscr2   0.0005201   -­‐1.44   Gramd4   0.0003804   -­‐1.43   Raf1   0.0000879   -­‐1.43   278   Gprc5c   0.0000216   -­‐1.43   Sh2d4b   0.0006165   -­‐1.43       0.0007315   -­‐1.43   Shroom3   0.0001793   -­‐1.42   Gm672   0.0006405   -­‐1.42   Nup210   0.0006938   -­‐1.42   Cpeb2   0.0002830   -­‐1.41   Trpv3   0.0003333   -­‐1.41   Gprc5c   0.0000064   -­‐1.40   Thbd   0.0001630   -­‐1.40   Zfp30   0.0004737   -­‐1.40   Slc24a6   0.0003028   -­‐1.40   AI464131   0.0000975   -­‐1.40   Daam1   0.0005181   -­‐1.40   Zcchc24   0.0002423   -­‐1.39   Traf4   0.0001968   -­‐1.39   Ccdc23   0.0001919   -­‐1.39   Rassf7   0.0006846   -­‐1.39   Dgkz   0.0001531   -­‐1.39   Cd34   0.0004649   -­‐1.38   C1galt1c1   0.0004925   -­‐1.38   Gulp1   0.0000729   -­‐1.38   Pbx3   0.0001241   -­‐1.38   Myo1b   0.0000043   -­‐1.37   Fam13c   0.0002301   -­‐1.36   Lphn3   0.0002646   -­‐1.36   Ch25h   0.0001386   -­‐1.36   Mef2d   0.0000574   -­‐1.36   Rbm38   0.0001233   -­‐1.36   Dlgap4   0.0001454   -­‐1.35   Cd82   0.0000621   -­‐1.35   ORF63   0.0007558   -­‐1.35   Ptar1   0.0001132   -­‐1.35   Lonrf3   0.0002546   -­‐1.35   Phka2   0.0001652   -­‐1.35   Trim7   0.0005729   -­‐1.35   Mpp3   0.0005133   -­‐1.34   Oas2   0.0001851   -­‐1.34   Pcdh1   0.0001339   -­‐1.34   Zfp57   0.0000663   -­‐1.34   2410042D21Rik   0.0001619   -­‐1.34   5031414D18Rik   0.0000142   -­‐1.34   Rapgef1   0.0002170   -­‐1.34   Fbxo10   0.0003976   -­‐1.33   279   Lrrc8d   0.0000454   -­‐1.33   Cyp3a41a   0.0005199   -­‐1.33   Clcn5   0.0001996   -­‐1.33   Adm   0.0005395   -­‐1.32   Prkaa2   0.0003121   -­‐1.32   Ddit4l   0.0002212   -­‐1.32   Eml4   0.0003962   -­‐1.32   Mid1   0.0004414   -­‐1.32   Ube2d1   0.0000259   -­‐1.31       0.0002508   -­‐1.31   Ppap2a   0.0001298   -­‐1.31   Nfib   0.0000220   -­‐1.31   Dgkd   0.0000890   -­‐1.31   Srgap3   0.0002540   -­‐1.30   Brwd1   0.0004427   -­‐1.30   Cdc42ep4   0.0005583   -­‐1.30   Tet1   0.0002087   -­‐1.30   Ppfia4   0.0002337   -­‐1.30   Arrdc1   0.0002436   -­‐1.30   E860004J03Rik   0.0002803   -­‐1.30   Enox1   0.0002534   -­‐1.29   Tmem38b   0.0005888   -­‐1.28   Ddc   0.0004587   -­‐1.28   9-­‐Sep   0.0003336   -­‐1.28   Hn1   0.0002692   -­‐1.28   Gdpd1   0.0006157   -­‐1.27   Sike1   0.0002917   -­‐1.27   St3gal5   0.0000808   -­‐1.27   Pttg1ip   0.0000887   -­‐1.27       0.0000986   -­‐1.27   Clasp1   0.0002760   -­‐1.26   Agl   0.0007200   -­‐1.26   Irak2   0.0001103   -­‐1.26   Frmd4b   0.0007005   -­‐1.26   Rps6kb1   0.0001071   -­‐1.26   Hsf4   0.0003845   -­‐1.26   Zfp275   0.0003678   -­‐1.25   Svil   0.0006959   -­‐1.25   Hells   0.0002387   -­‐1.25   Zeb1   0.0003729   -­‐1.25   Tpcn1   0.0007305   -­‐1.25   Bmyc   0.0002198   -­‐1.25   Lpgat1   0.0005078   -­‐1.24   Baiap2l1   0.0007333   -­‐1.24   280   9030420J04Rik   0.0005035   -­‐1.24   Pcgf3   0.0001061   -­‐1.24       0.0005791   -­‐1.23   Btbd3   0.0007076   -­‐1.22   Cep68   0.0000037   -­‐1.22   Tmem177   0.0004917   -­‐1.22   Gpsm2   0.0003806   -­‐1.22   Ssbp3   0.0003278   -­‐1.22   Mtap1a   0.0005478   -­‐1.22   Wisp1   0.0002093   -­‐1.21   Cmbl   0.0007405   -­‐1.21   Klk10   0.0001836   -­‐1.21   Rad51   0.0004764   -­‐1.20   Tubb6   0.0000712   -­‐1.20       0.0002425   -­‐1.20   Itgb1bp2   0.0003095   -­‐1.20   Ahnak   0.0004622   -­‐1.19   Lmtk2   0.0006029   -­‐1.19   Foxj3   0.0005675   -­‐1.19   Flnc   0.0003287   -­‐1.19   Mapk8ip1   0.0000038   -­‐1.19   Smpdl3b   0.0002784   -­‐1.19   Nfkb1   0.0005372   -­‐1.19   Cugbp2   0.0004842   -­‐1.18   Arhgap10   0.0003785   -­‐1.18   Tubb2a   0.0002721   -­‐1.18   Exosc9   0.0005084   -­‐1.18       0.0001729   -­‐1.18   Cd97   0.0002628   -­‐1.17   Sgsm2   0.0005912   -­‐1.17   Ep400   0.0007493   -­‐1.17   Myof   0.0002287   -­‐1.16   Ttc3   0.0000294   -­‐1.16   Casq2   0.0006576   -­‐1.15   Tmem41a   0.0005291   -­‐1.15   Gpr137b   0.0004426   -­‐1.15   Crybg3   0.0001346   -­‐1.14   Enpp1   0.0005973   -­‐1.14   Mttp   0.0003324   -­‐1.13   Crkrs   0.0007116   -­‐1.12   Akap6   0.0004537   -­‐1.12   Gm2467   0.0002294   -­‐1.12   Crebbp   0.0005433   -­‐1.12   Dyrk1a   0.0007284   -­‐1.12   281   1810055G02Rik   0.0005265   -­‐1.11   2210404J11Rik   0.0005196   -­‐1.11   Fam96b   0.0006195   -­‐1.11   Ddx17   0.0006333   -­‐1.10   Nudt4   0.0005114   -­‐1.10   Lman2   0.0000868   -­‐1.09   Akr1b3   0.0002253   -­‐1.08       0.0002355   -­‐1.07   Dedd   0.0003254   -­‐1.07   BC033915   0.0004698   -­‐1.06   Lamp1   0.0005543   -­‐1.03     Appendix  Table  6.  Transcripts  downregulated  by  BMP4-­‐treatment  in  wild-­‐type   myotubes.       Appendix  Table  7     p-­‐value(N  vs.   Fold-­‐ Gene  Symbol   B)   Change   Lum   0.0007719   -­‐3.52   Lrrc26   0.0000069   -­‐3.16   Mfsd7c   0.0000287   -­‐3.11   Ddit4l   0.0000138   -­‐3.10   Sema7a   0.0011194   -­‐3.02   Nptx1   0.0000099   -­‐2.94   Rassf2   0.0000064   -­‐2.72   Slc2a6   0.0002215   -­‐2.71   Scn7a   0.0009098   -­‐2.67   Grtp1   0.0005902   -­‐2.65   Mc4r   0.0000356   -­‐2.64   Runx1t1   0.0000058   -­‐2.62   Rgs2   0.0002402   -­‐2.60   Gm7325   0.0001291   -­‐2.58   Osr2   0.0005416   -­‐2.54   Trp63   0.0000324   -­‐2.53   Duxbl   0.0000222   -­‐2.52   Duxbl   0.0000222   -­‐2.52   1600029D21Rik   0.0000589   -­‐2.49   4921525H12Rik   0.0000671   -­‐2.49   Sobp   0.0001756   -­‐2.46   F3   0.0000516   -­‐2.42   282   Neu2   0.0002460   -­‐2.42   Duxbl   0.0000145   -­‐2.34   Frmd4b   0.0000167   -­‐2.32   4732465J04Rik   0.0000264   -­‐2.25   Lrig1   0.0000646   -­‐2.24   Gm10001   0.0001652   -­‐2.24   Pdgfb   0.0001275   -­‐2.23   Crabp2   0.0008856   -­‐2.22   Sdpr   0.0002267   -­‐2.22   1810041L15Rik   0.0005212   -­‐2.19   Mamstr   0.0001331   -­‐2.19   Dtx4   0.0000095   -­‐2.16   Maf   0.0000305   -­‐2.12   Il33   0.0000304   -­‐2.11   Tnik   0.0003297   -­‐2.10   Arhgap18   0.0001599   -­‐2.10   Sertad4   0.0001156   -­‐2.07   Fzd9   0.0000667   -­‐2.07   Chst15   0.0000103   -­‐2.06   C330016O10Rik   0.0009277   -­‐2.05   Itga3   0.0012101   -­‐2.04   Slco3a1   0.0000379   -­‐2.01   Fam53b   0.0005303   -­‐2.01   Filip1   0.0000899   -­‐2.01       0.0011301   -­‐2.00   Sema5a   0.0000178   -­‐1.98   Gulp1   0.0005562   -­‐1.97   6330406I15Rik   0.0003047   -­‐1.97   Mest   0.0000121   -­‐1.97   Camk1g   0.0006890   -­‐1.96   Lmcd1   0.0002407   -­‐1.96   Nceh1   0.0003507   -­‐1.95   I830127L07Rik   0.0000515   -­‐1.94   Acsl1   0.0000000   -­‐1.94   Fmo1   0.0001929   -­‐1.94   Lgals12   0.0008628   -­‐1.93       0.0009695   -­‐1.93   Trim7   0.0002339   -­‐1.93   Slc7a8   0.0003327   -­‐1.92   Lrrc30   0.0007290   -­‐1.92   Arhgap28   0.0000094   -­‐1.91   Sema6a   0.0000789   -­‐1.90   Mfsd7a   0.0004824   -­‐1.90   Tnfrsf19   0.0000284   -­‐1.90   283   Asb15   0.0000336   -­‐1.88   Igsf10   0.0007079   -­‐1.87   Cd24a   0.0000509   -­‐1.86   Tet1   0.0004377   -­‐1.86   Tnfaip3   0.0001109   -­‐1.86   Kif24   0.0011197   -­‐1.86   Scn4a   0.0001671   -­‐1.85   Zcchc5   0.0000302   -­‐1.85   Igsf10   0.0001964   -­‐1.83   Rnf122   0.0001437   -­‐1.83   Prkag3   0.0000307   -­‐1.82   Tnfaip8   0.0000404   -­‐1.82   Igsf10   0.0002984   -­‐1.81   Olfml2a   0.0000650   -­‐1.81   Atp1b2   0.0001944   -­‐1.81   Chodl   0.0011311   -­‐1.80   Igfbp3   0.0000361   -­‐1.80   Fam65b   0.0005582   -­‐1.80   Tecrl   0.0000171   -­‐1.79   Slc5a6   0.0010341   -­‐1.79   Bnc2   0.0000067   -­‐1.79   Mtss1   0.0000033   -­‐1.79   Steap3   0.0000003   -­‐1.79   Rgmb   0.0000049   -­‐1.79   Klhl3   0.0001000   -­‐1.79   Rasgrp3   0.0010547   -­‐1.79   Zcchc24   0.0006526   -­‐1.78   Gpr146   0.0008811   -­‐1.77   Fn3k   0.0001053   -­‐1.77   Casz1   0.0003177   -­‐1.76   Mpp3   0.0000129   -­‐1.76   Rgs16   0.0001789   -­‐1.75   Syt13   0.0010344   -­‐1.74   Fam135b   0.0012472   -­‐1.74   Rhobtb2   0.0000652   -­‐1.74   Glul   0.0000047   -­‐1.73   Aspa   0.0006975   -­‐1.73   Tet1   0.0000379   -­‐1.72   Atp6v1c2   0.0003134   -­‐1.72   Il15   0.0001238   -­‐1.71   Klhl31   0.0000149   -­‐1.71   Sox6   0.0001232   -­‐1.70   Trim16   0.0004831   -­‐1.70   Fam13c   0.0001673   -­‐1.70   284   Tbx15   0.0001356   -­‐1.69   Mrgprf   0.0006433   -­‐1.69   Mafa   0.0001022   -­‐1.69   Slc9a9   0.0005388   -­‐1.68   Flrt1   0.0001026   -­‐1.68   5033413D22Rik   0.0000230   -­‐1.68   Mospd1   0.0003675   -­‐1.67   Klk10   0.0010079   -­‐1.66   Erbb3   0.0000755   -­‐1.66   Adamts16   0.0005347   -­‐1.65   Tmem184a   0.0001427   -­‐1.65   Atp2b3   0.0001302   -­‐1.65   Coro2b   0.0000817   -­‐1.64   Lphn3   0.0010652   -­‐1.64   Fsd1l   0.0002759   -­‐1.64   Tspan14   0.0002283   -­‐1.64   D14Ertd449e   0.0002371   -­‐1.64   Gtdc1   0.0010531   -­‐1.64   Tet1   0.0000212   -­‐1.64   Pnkd   0.0001507   -­‐1.63   Lpar1   0.0004387   -­‐1.63   D14Ertd449e   0.0004424   -­‐1.63   Heg1   0.0007083   -­‐1.63   Slc2a5   0.0003320   -­‐1.63   Lrrc15   0.0010595   -­‐1.63   Lama4   0.0000434   -­‐1.63   Dgkd   0.0000035   -­‐1.63   D14Ertd449e   0.0003057   -­‐1.62   Glul   0.0002872   -­‐1.62   Cpne2   0.0000992   -­‐1.62   Galnt5   0.0001757   -­‐1.62   Ralgps1   0.0004341   -­‐1.62   Reep1   0.0002645   -­‐1.62   Kifc3   0.0008480   -­‐1.62   Popdc2   0.0000025   -­‐1.62   Sorcs2   0.0002661   -­‐1.61   2810432L12Rik   0.0006078   -­‐1.61   Daam1   0.0000718   -­‐1.61   Heg1   0.0003233   -­‐1.61   2610301F02Rik   0.0002838   -­‐1.61   Fam135b   0.0002523   -­‐1.60   Antxr2   0.0004733   -­‐1.60   Cgnl1   0.0002057   -­‐1.60   Myh4   0.0005041   -­‐1.60   285   Ccdc88c   0.0002300   -­‐1.60   Dbp   0.0003715   -­‐1.59   Pnmal2   0.0000124   -­‐1.58   Dhcr7   0.0007005   -­‐1.58   5730419I09Rik   0.0003080   -­‐1.58   Lamb1-­‐1   0.0004967   -­‐1.58   Ctnnal1   0.0005493   -­‐1.58   Lrch1   0.0003398   -­‐1.58   Ccbe1   0.0007573   -­‐1.58   Gas2   0.0001945   -­‐1.58   D0H4S114   0.0003344   -­‐1.58   Ednra   0.0001915   -­‐1.58   Fras1   0.0005443   -­‐1.58   Klhl31   0.0000303   -­‐1.57   5033414K04Rik   0.0000965   -­‐1.57   Pde2a   0.0009307   -­‐1.57   Slc24a3   0.0002559   -­‐1.57   Spire2   0.0012376   -­‐1.57   Ralgps2   0.0007975   -­‐1.56   Ssx2ip   0.0000207   -­‐1.56   Jam3   0.0001360   -­‐1.56   Sox8   0.0000811   -­‐1.56   Stx11   0.0002393   -­‐1.55   Gpr68   0.0012135   -­‐1.55   St3gal4   0.0010419   -­‐1.55   Anpep   0.0008426   -­‐1.55   Rilpl1   0.0009444   -­‐1.54   Cd72   0.0006599   -­‐1.54   Gpt2   0.0002384   -­‐1.54   Camk2a   0.0000476   -­‐1.54   Spg21   0.0008725   -­‐1.54   Klhl30   0.0005874   -­‐1.54   Map2k6   0.0007294   -­‐1.53   Sema4c   0.0006704   -­‐1.53   Myoz3   0.0000697   -­‐1.53   Nup210   0.0001144   -­‐1.52   Gpd1l   0.0003795   -­‐1.52   Frem1   0.0011749   -­‐1.52   Ccl2   0.0010132   -­‐1.52   Cdc42ep2   0.0010561   -­‐1.52   Inpp4a   0.0003303   -­‐1.51   Slc41a1   0.0004059   -­‐1.51   Pdlim2   0.0001386   -­‐1.51   Sema3e   0.0001987   -­‐1.51   286   Gpr126   0.0000224   -­‐1.51   Zfp503   0.0003160   -­‐1.50   Slco5a1   0.0002747   -­‐1.50   Tcea3   0.0001091   -­‐1.50   Rgma   0.0005848   -­‐1.50   Gan   0.0003136   -­‐1.49   Rnf150   0.0003691   -­‐1.49   C1qtnf1   0.0005087   -­‐1.49   Glis2   0.0005919   -­‐1.49   Ttyh2   0.0010319   -­‐1.49   Gprc5c   0.0009785   -­‐1.49   Zdhhc14   0.0000648   -­‐1.48   D830046C22Rik   0.0007857   -­‐1.48   Mcf2l   0.0002461   -­‐1.48   Garnl4   0.0000157   -­‐1.48   4833442J19Rik   0.0005980   -­‐1.48   6530418L21Rik   0.0011071   -­‐1.47   Fsd2   0.0004613   -­‐1.47   Pdp1   0.0005220   -­‐1.47   Wfs1   0.0005797   -­‐1.47       0.0000663   -­‐1.47   Atp7a   0.0009846   -­‐1.46   Gfra1   0.0009387   -­‐1.46   Acsl6   0.0006204   -­‐1.46   Synpo2   0.0001918   -­‐1.46   Rbm47   0.0007276   -­‐1.46   Schip1   0.0000378   -­‐1.46   Rev3l   0.0007159   -­‐1.45   Afap1l1   0.0002174   -­‐1.45   Gpr155   0.0009251   -­‐1.45   Sepp1   0.0002459   -­‐1.44   Cldn15   0.0011745   -­‐1.44   Pfkfb4   0.0003285   -­‐1.43   Gprc5c   0.0009910   -­‐1.43   Ip6k3   0.0006022   -­‐1.43   Gatm   0.0000508   -­‐1.43   Gm10035   0.0003891   -­‐1.42   Adamts5   0.0004502   -­‐1.42   Sort1   0.0000860   -­‐1.42   Ddah1   0.0000785   -­‐1.41   Atp2b4   0.0005385   -­‐1.41   Cnr1   0.0001792   -­‐1.41       0.0001502   -­‐1.41   Itga11   0.0004874   -­‐1.41   287   Matn2   0.0001063   -­‐1.41   Cabc1   0.0000122   -­‐1.41   Slc25a26   0.0000946   -­‐1.40   Hdac11   0.0005091   -­‐1.40   Necab1   0.0008027   -­‐1.40   Plk2   0.0003242   -­‐1.40   Cd200   0.0000151   -­‐1.40   Tnk2   0.0006440   -­‐1.39   Abr   0.0000219   -­‐1.39   Pip4k2a   0.0002004   -­‐1.39   Gm672   0.0006084   -­‐1.38   Arhgef3   0.0008949   -­‐1.38   Rassf7   0.0001721   -­‐1.38   Oasl2   0.0010275   -­‐1.38   Plscr4   0.0010023   -­‐1.38   Klhl8   0.0011902   -­‐1.38   Ebf3   0.0003617   -­‐1.38   Antxr1   0.0002091   -­‐1.38   Cdkn2c   0.0003739   -­‐1.37   Itga9   0.0005712   -­‐1.37   Srgap3   0.0010084   -­‐1.37   1110003O08Rik   0.0009932   -­‐1.37   Dcn   0.0004499   -­‐1.37       0.0002832   -­‐1.37   Klhl5   0.0009788   -­‐1.36   Mef2d   0.0002491   -­‐1.36   Rdh5   0.0010769   -­‐1.36   Arhgef6   0.0000996   -­‐1.36   Spg20   0.0005115   -­‐1.35   4930523C07Rik   0.0000658   -­‐1.35   Iffo1   0.0001819   -­‐1.35   Igf1   0.0012483   -­‐1.35   Fhad1   0.0003061   -­‐1.35   Robo1   0.0008355   -­‐1.34       0.0000033   -­‐1.34   2210011C24Rik   0.0005836   -­‐1.34   Smad3   0.0007121   -­‐1.34   9-­‐Sep   0.0010350   -­‐1.34   Gpsm2   0.0003465   -­‐1.34   Cnot6l   0.0001278   -­‐1.34   Golm1   0.0000001   -­‐1.34   Gpc4   0.0000629   -­‐1.34   Tmem62   0.0000380   -­‐1.33   Prmt2   0.0012170   -­‐1.33   288   Mfhas1   0.0005969   -­‐1.32   Nfia   0.0001630   -­‐1.32   Ube2e2   0.0008489   -­‐1.32   Hspa12a   0.0008942   -­‐1.32   Clec2d   0.0005843   -­‐1.32   Morc4   0.0007879   -­‐1.32   1700116B05Rik   0.0001667   -­‐1.32   Arhgap22   0.0005609   -­‐1.32   Cadm1   0.0010777   -­‐1.31   Gpr56   0.0009049   -­‐1.31   Pbx3   0.0000377   -­‐1.31   Lgals9   0.0000945   -­‐1.31   Ccdc8   0.0001870   -­‐1.31   Zfp367   0.0007592   -­‐1.31   Plagl1   0.0000215   -­‐1.31   6-­‐Sep   0.0001069   -­‐1.31   Nes   0.0003728   -­‐1.31   Svil   0.0000221   -­‐1.30   Pxdn   0.0002950   -­‐1.30   Pitpnc1   0.0003048   -­‐1.30   Clasp1   0.0009174   -­‐1.30   Macc1   0.0009132   -­‐1.30   Myo18a   0.0003121   -­‐1.29   Rsph1   0.0012212   -­‐1.29   Rin2   0.0010779   -­‐1.29   Tmem177   0.0005345   -­‐1.29   Fnbp1   0.0000288   -­‐1.29   Cntln   0.0003720   -­‐1.29   Rptor   0.0002238   -­‐1.29       0.0004727   -­‐1.29   5730559C18Rik   0.0006805   -­‐1.29   Pipox   0.0008969   -­‐1.29   Sfxn5   0.0000959   -­‐1.28   Ptgds   0.0011799   -­‐1.28   Ypel3   0.0002675   -­‐1.28       0.0001266   -­‐1.28   Parp3   0.0011653   -­‐1.28   Zfp422   0.0003814   -­‐1.28   Nfib   0.0006320   -­‐1.28   Il22   0.0004058   -­‐1.28   Adamts14   0.0011272   -­‐1.27       0.0006693   -­‐1.27   Mef2d   0.0000398   -­‐1.27   Gm16492   0.0007891   -­‐1.27   289       0.0002167   -­‐1.27   Cnn2   0.0003997   -­‐1.26   Sgsm2   0.0012080   -­‐1.26       0.0000010   -­‐1.26   Klf5   0.0007842   -­‐1.26   Btrc   0.0006194   -­‐1.26   Slc9a3r1   0.0002137   -­‐1.26   Il20rb   0.0011864   -­‐1.26   Hrh3   0.0004856   -­‐1.26   Arsb   0.0003754   -­‐1.26   Dysf   0.0001191   -­‐1.25   AI464131   0.0000589   -­‐1.25   Klhdc10   0.0011130   -­‐1.25   Bicd2   0.0005959   -­‐1.25   4933426M11Rik   0.0002758   -­‐1.24   Sulf2   0.0006144   -­‐1.24   Pmaip1   0.0000253   -­‐1.24   Chst10   0.0003443   -­‐1.24   Ehbp1   0.0010043   -­‐1.24       0.0009202   -­‐1.24   Acpp   0.0004499   -­‐1.24   Ppap2a   0.0008828   -­‐1.24   Sox9   0.0007794   -­‐1.24   Pabpc1l   0.0009369   -­‐1.24   Zfp473   0.0009668   -­‐1.23   Lrrfip1   0.0010260   -­‐1.23   Stard13   0.0006472   -­‐1.23   Tmpo   0.0004364   -­‐1.23   Sync   0.0002768   -­‐1.23   Gamt   0.0000392   -­‐1.23   Ttll1   0.0004726   -­‐1.23   Brd3   0.0010180   -­‐1.22   Ly6e   0.0010958   -­‐1.22   Cdk5r1   0.0001534   -­‐1.22   Tmem182   0.0010704   -­‐1.22   Hddc3   0.0004282   -­‐1.22   Ccpg1   0.0007982   -­‐1.22   Trak1   0.0005620   -­‐1.22   Pcbd1   0.0002601   -­‐1.22   Hoxa7   0.0005522   -­‐1.22   1700025G04Rik   0.0000430   -­‐1.21   Ube2d1   0.0003390   -­‐1.21   Tspan9   0.0002975   -­‐1.21   Trim55   0.0006725   -­‐1.21   290   Akap1   0.0005634   -­‐1.21   Prune   0.0007284   -­‐1.20   Cdkn1c   0.0002044   -­‐1.20   Osgin2   0.0001631   -­‐1.20   Ccdc111   0.0003039   -­‐1.20   Tspan13   0.0006857   -­‐1.20   Tbx18   0.0004987   -­‐1.20   Ssbp3   0.0000117   -­‐1.20   St6gal1   0.0008657   -­‐1.20   Dlgap4   0.0009546   -­‐1.20   Myh1   0.0005541   -­‐1.19   Copg2   0.0000081   -­‐1.19   Traf3ip2   0.0010664   -­‐1.19   Rasgrp2   0.0004989   -­‐1.19   Cds2   0.0003658   -­‐1.19   Crat   0.0010853   -­‐1.19   Samd4   0.0003362   -­‐1.19   Nedd9   0.0000466   -­‐1.18   Myom2   0.0002899   -­‐1.18   Enthd1   0.0007770   -­‐1.18   Mgam   0.0011734   -­‐1.18   Dock8   0.0010771   -­‐1.18   Lmo7   0.0006554   -­‐1.18   Ubap2   0.0002069   -­‐1.18   Igfbp5   0.0001033   -­‐1.18   Parp10   0.0012029   -­‐1.18   Gm10570   0.0004120   -­‐1.18   Gys1   0.0008782   -­‐1.17   Ahnak   0.0001294   -­‐1.17   Pira2   0.0001695   -­‐1.17   Cplx1   0.0005145   -­‐1.17   Scarb2   0.0005859   -­‐1.17   Nr4a1   0.0003467   -­‐1.17   Sh2d3c   0.0002684   -­‐1.17   Hmcn1   0.0000792   -­‐1.17   Pygm   0.0009729   -­‐1.17   Vdr   0.0010799   -­‐1.16   Olfr419   0.0009757   -­‐1.16   Ankrd23   0.0001329   -­‐1.16       0.0008378   -­‐1.16   Mup4   0.0010978   -­‐1.16   Entpd4   0.0001356   -­‐1.16   Acap2   0.0007870   -­‐1.16   Fbln1   0.0000918   -­‐1.16   291   Psme1   0.0003794   -­‐1.15   Mtus1   0.0009074   -­‐1.15   Rnaset2a   0.0000983   -­‐1.15   Scrn1   0.0002679   -­‐1.15   Lmod3   0.0003488   -­‐1.15   Hsd17b4   0.0002664   -­‐1.15   Chi3l1   0.0007822   -­‐1.15   Rnaset2a   0.0002168   -­‐1.15   Ablim2   0.0009087   -­‐1.14   Cmya5   0.0005178   -­‐1.14   Myh8   0.0009070   -­‐1.14   Fcer2a   0.0001455   -­‐1.13   Chd7   0.0010807   -­‐1.13   Pgm5   0.0005868   -­‐1.12   Ano6   0.0000693   -­‐1.12   Isca1   0.0006159   -­‐1.11   Prkar1a   0.0002074   -­‐1.11   C1galt1c1   0.0012239   -­‐1.11       0.0009858   -­‐1.10   Srl   0.0005447   -­‐1.10   Gpr125   0.0000306   -­‐1.10       0.0000057   -­‐1.09   1700047I17Rik1   0.0008674   -­‐1.09   1700047I17Rik1   0.0008674   -­‐1.09   Kbtbd10   0.0007341   -­‐1.09   Nnt   0.0002018   -­‐1.08       0.0010791   -­‐1.08   Ddit4   0.0009305   -­‐1.02   Rpl32   0.0012127   -­‐1.02     Appendix  Table  7.  Transcripts  downregulated  by  BMP4-­‐treatment  in  MuSK  null   myotubes.       292