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Molecular myogenic program

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

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Molecular myogenic program

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  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 Specification Commitment Differentiation • Six 1/4 • Pax 3/7 • MyoD/myf5 • Myogenin/Mrf4 • Myosin and other phenotypic

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

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

  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 cells Chondrocyte (~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  pH gradient  Untransformed 10T1/2 Myogenic 10T1/2 Some proteins lost  Mass gradient  One gained

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

  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 WT SIX-/- Grifone et al., 2005

  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 Phenotype expression Ligand synthesis Effector activity Rostral wnt3a Caudal FGF-8 Raldh (retinoic acid) 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 http://www.kegg.jp/kegg-bin/highlight_pathway? 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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