Transcript Molecular myogenic program - Georgia Institute of Technology

Molecular myogenic program
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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
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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
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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 methylationdeacetylation
– Deacetylationgene inactivation
– ie: 5-azagene 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
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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
– Six1/4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
Pre-somitic mitogen gradients
Rostral
wnt3a
Caudal
FGF-8
Raldh
(retinoic
acid)
Aulehla and Pourquié, 2010
Somitic gradients
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
Wnt signaling
http://www.kegg.jp/kegg-bin/highlight_pathway? map=map04310
SHH signaling
• ShhPatchedsmoothenedGLI?myf5
• TCF+GLImyf5 (differentiation)
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)
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
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