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Molecular myogenic program Bentzinger, C. F., Wang, Y. X. and Rudnicki, M. A. (2012). Building muscle: molecular regulation of myogenesis. Cold Spring.

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Presentation on theme: "Molecular myogenic program Bentzinger, C. F., Wang, Y. X. and Rudnicki, M. A. (2012). Building muscle: molecular regulation of myogenesis. Cold Spring."— Presentation transcript:

1 Molecular myogenic program Bentzinger, C. F., Wang, Y. X. and Rudnicki, M. A. (2012). Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol 4. Morphogen gradients (myocyte fate arises external to somite) Myogenic regulatory factors Satellite cells Proliferation-differentiation competition

2 Myogenic program Six 1/4 Pax 3/7 MyoD/myf5 Myogenin/ Mrf4 Myosin and other phenotypic SpecificationCommitmentDifferentiation

3 Body pattern begins in the oocyte Interaction with follicle – Cadherin – Microtubule structure Interaction with sperm – Centrosome/microtubule organization Nonmammalian systems – Microtubule-anchored translation/transcription repressors: bicoid, gurken, dorsal – Probably analogs in mammals

4 Gastrulation Alberts & Johnson MBoC 2002

5 Neurulation Stage 20Stage 13Stage 16Stage 18 Schroeder, 1970 Proliferation of neural plate ectoderm Formation of neural tube

6 Somitogenesis Segmentation of mesoderm Temporal oscillation of hairy/Fringe Origin of myocytes, but myocyte determination is extrinsic to the somite Forsberg & al., 1998 L-fng waves: -2, -1, 0, 1, 2 Time  Rostral  Somite 1 Somite 2 Mouse embryo (dorsal)

7 Molecular progression of muscle Six 1/4 sine oculis-related homeobox Pax paired-homeobox MyoD/Myf5: commitment factors Myogenin/MRF4: differentiation factors Phenotypic functional proteins – Desmin – Myosin – Troponin

8 Discovery of MyoD Deciphering of differentiation works backward Stephen Konieczny & Charles Emerson (1984) 5- Azacytidine poisoned limb bud cells (chick) – Methytransferase inhibitor – DNA methylation  deacetylation – Deacetylation  gene inactivation – ie: 5-aza  gene activation

9 5-Aza induces differentiation Normal 10T1/2 cellsChondrocyte (~1%) Adipocyte (7%) Myocyte (25%) 10T1/2 cells have limited ability to differentiate: pluripotent not omnipotent Cells in limb bud are not fully committed to a terminal phenotype

10 Protein identification Isoelectric focusing SDS-PAGE Untransformed 10T1/2Myogenic 10T1/2 Some proteins lost One gained  pH gradient   Mass gradient 

11 mRNA identification Stephen Tapscott, Andrew Lassar & al. Subtractive cDNA hybridization – Present in proliferating myogenic 10T1/2 – Absent in differentiated myotubes – Absent in unmodified 10T1/2 3 products, One of which caused conversion

12 MRF knockout animals Michael Rudnicki & al. Homologous recombination If MyoD makes muscle, then no MyoD should mean no muscle – Or at least, no specific-subset-of-muscles +/- cross litters – Normal, Mendelian (25, 25, 50) ratios – Muscle is normal in every way

13 Myogenic regulatory factors Basic, Helix-loop-helix transcription factor – Myf5, MRF4, myogennin (muscle) – Neurogenin (neurons), twist (chondrocyte)... Induction of MRF causes myogenic transformation in other undifferentiated cells ie: commitment marker

14 Genetic disruption of myogenesis MyoD or myf5 individually: normal MyoD and myf5: lethal, no muscles MRF4: disrupted axial musculature Myogenin: lethal, failure of muscle expansion

15 Mutant mice lead to pax Splotch (1954) – -/- lethal e13: no neural tube – -/+ spotted – Pax 3 identified 1991 Pax3 required for migration of hypaxial myoblasts Not for epaxial muscle Tremblay & al., 1998 Desmin positive cells Wild type Splotch Limb bud

16 Pax 3/7 Pax 3/7 necessary for myogenic commitment Pax 3/7 blocks myogenesis – Cells that remain pax+ do not become myofibers Relaix et al., 2005

17 Sine oculis SIX-1/SIX-4 dko – Fails muscle formation – MEF3 cofactor – Required for pax, MRFs Grifone et al., 2005 WTSIX-/-

18 Myogenic lineages Ventro-lateral somite – Six1/4  pax3  MyoD  hypaxial muscle – Pax3  MyoD  (primary myoblasts)  myoG – Pax3/7  (secondary myoblasts)  MRF4 Dorso-medial somite – ???  myf5  epaxial muscle Position and time matter: external cues

19 Pre-somitic mitogen gradients wnt3a FGF-8 Raldh (retinoic acid) Ligand synthesis Effector activity Phenotype expression Rostral Caudal Aulehla and Pourquié, 2010

20 Somitic gradients

21 Wnt FGF signaling Wnt 1/3  fzd1/6  dsh  GSK3  pax3  ?  myf5 Wnt6/7a  fzd7  PKC  ?  myoD FGF8  FGFR  ras  raf  MEK1/2  ERK1/2  cyclin A

22 Wnt signaling map=map04310

23 SHH signaling Shh  Patched  smoothened  GLI  ?  myf5 TCF+GLI  myf5 (differentiation)

24 TGF-b signaling BMP4  SMAD  pax3--|myf5 (pool expansion) Myostatin  SMAD2/3--|MuRF1/MafBx Wnt/shh  noggin--|BMP Delta1  Notch  CSL  Hes1--|myoD (pool expansion)

25 Pool expansion vs Differentiation Canonical model – myoD/MYf5  myogenin/MRF4  phenotype Pathway crosstalk – Pax7  Wdr5-Ash2L-MLL2  H3K4 trimethylation  epigenetic activation – MyoD  miR206--|pax3/7 – Pax3/7  TCF-->myf5

26 Summary Myogenic regulatory factors – MyoD/myf5: commitment – Myogenin/MRF4: differentiation Diffusion gradients – Retinoic acid: rostral/caudal; FGF caudal/rostral – Wnt1/3 dorsal/ventral; SHH ventral/dorsal Signaling pathways – Wnt, shh, TGF


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