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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 Harb Perspect Biol 4. • Morphogen gradients (myocyte fate arises external to somite) • Myogenic regulatory factors • Satellite cells • Proliferation-differentiation competition Myogenic program Specification • • • • Six 1/4 Pax 3/7 MyoD/myf5 Myogenin/ Mrf4 • Myosin and other phenotypic Commitment Differentiation 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 Gastrulation Alberts & Johnson MBoC 2002 Neurulation Stage 13 Stage 16 Stage 18 Stage 20 Proliferation of neural plate ectoderm Formation of neural tube Schroeder, 1970 Somitogenesis Rostral • 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 Time L-fng waves: -2, -1, 0, 1, 2 Forsberg & al., 1998 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 Discovery of MyoD • Deciphering of differentiation works backward • Stephen Konieczny & Charles Emerson (1984) • 5- Azacytidine poisoned limb bud cells (chick) – Methytransferase inhibitor – DNA methylationdeacetylation – Deacetylationgene inactivation – ie: 5-azagene activation 5-Aza induces differentiation Normal 10T1/2 cells Adipocyte (7%) Chondrocyte (~1%) 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 Protein identification • Isoelectric focusing • SDS-PAGE pH gradient Mass gradient Untransformed 10T1/2 Myogenic 10T1/2 Some proteins lost One gained 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 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 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 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 Mutant mice lead to pax Desmin positive cells Wild type • Splotch (1954) Splotch – -/- 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 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 Sine oculis • SIX-1/SIX-4 dko – Fails muscle formation – MEF3 cofactor – Required for pax, MRFs WT SIX-/- Grifone et al., 2005 Myogenic lineages • Ventro-lateral somite – Six1/4pax3MyoDhypaxial muscle – Pax3MyoD(primary myoblasts)myoG – Pax3/7(secondary myoblasts)MRF4 • Dorso-medial somite – ???myf5epaxial muscle • Position and time matter: external cues Pre-somitic mitogen gradients Rostral wnt3a Caudal FGF-8 Raldh (retinoic acid) Aulehla and Pourquié, 2010 Somitic gradients Wnt FGF signaling • Wnt 1/3fzd1/6dshGSK3pax3?myf5 • Wnt6/7afzd7PKC?myoD • FGF8FGFRrasrafMEK1/2ERK1/2 cyclin A Wnt signaling http://www.kegg.jp/kegg-bin/highlight_pathway? map=map04310 SHH signaling • ShhPatchedsmoothenedGLI?myf5 • TCF+GLImyf5 (differentiation) TGF-b signaling • BMP4SMADpax3--|myf5 (pool expansion) • MyostatinSMAD2/3--|MuRF1/MafBx • Wnt/shhnoggin--|BMP • Delta1NotchCSLHes1--|myoD (pool expansion) Pool expansion vs Differentiation • Canonical model – myoD/MYf5myogenin/MRF4phenotype • Pathway crosstalk – Pax7Wdr5-Ash2L-MLL2H3K4 trimethylationepigenetic activation – MyoDmiR206--|pax3/7 – Pax3/7TCF-->myf5 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