growthspecificationmorphogenesisdiseases the life of a skeletal muscle -genes -exogenous or...
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GrowthSpecification Morphogenesis Diseases
The Life of a Skeletal Muscle
-Genes-Exogenous or Autonomous ?
-Cell shape changes, migration- Identify positional cues
-Stem cell origin-Proliferation - Fusion
-Genes-Therapies
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Initiation de la myogenèse.
D dermomyotome
sclerotome
myotome
epithelial somite
sclerotome
Neural tubemedial lip
S I
MyoD
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Initiation de la myogenèse: modèle inductif
MyoD
Wnt-1 -3a, -4
SHH
Wnt-6Arguments expérimentaux:
-Micromanipulations chez le poulet : rotation de somites > myogenèse normale-Culture de PSM souris et poulet:+ rien > rien+ Wnt > un peu+ Wnt + SHH > beaucoup
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Mais:Mais:Myogenèse normale sans signal Hh
WT Shh-/- Smo-/-
Myf5E9.5
Zhang et al. 2001 Cell 105: 781-792
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George-Weinstein M, Gerhart J, Reed R, Flynn J, Callihan B, Mattiacci M,
Miehle C, Foti G, Lash JW, Weintraub H. Skeletal myogenesis: the preferred Pathway of chick embryo epiblast cells in vitro. Dev Biol. 1996 Jan 10;173(1):279-91.
Induction de la myogenèse: modèle permissif?
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Les gènes de détermination myogénique
Famille des bHLH Myogenic determinationMyoDMyf5Muscle differentiationMyogeninMRF4
Tous ont la capacité d’induire la myogenèse de cellules non myogéniques (gènes « maîtres »)
MyoD -/- et Myf5 -/- sont viables et fertilesMyoD:Myf5 -/- létal, pas de muscleMyogenin -/- et MRF4 -/- possèdent des myoblastes Myf5 ou MyoD-positifs, mais la différenciation ultérieure est arrêtée.
MyoD/Myf5: gènes de spécificationMyogenin/MRF4: gènes de différentiation
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Et avant les gènes de détermination myogénique?
Poulet 3 jours, section transversale, anticorps anti Pax3. Remarquer les cellules migrant dans le mésenchyme de l’aile.
Pax3 et Pax7, exprimés de manière similaire (TN et somites ou muscles)
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Skeletal muscles are ablated in the body of Sp/Myf5 double homozygous embryos
Tajbakhsh et al., 1996 Cell 89: 127-138
Pax3: rôle au cours de la migration des progéniteurs musculaires (membres)Collaboration de Myf5 et Pax3 pour la myogenèse des muscles du corps
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• Pediatric solid tumors of skeletal muscle in Human.
• Chromosomal translocation t(2;13)(q35;q14) PAX3-FKHR
PD L HDPAX3 (PAX7) (weak transactivation/repression)
1 479
PD LHD AD
8371PAX3-FKHR (strong transactivation)(PAX7-FKHR)
AD
1 655
FKHRFH
Alveolar Rhabdomyosarcomas
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Muscle satellite cells in Pax7 mutant mice
Pax7: phénotype chez l’adulte cellules souches (satellites) apoptotiquesPax3 : Pax 7 -/- pas de progéniteurs musculaires
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Pax-7MHC
Early muscle morphogenesis
Dermomyotome
Myotome
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E 2.5
The Electroporation Technique
+ -
Lineage studies
- GFP reporter
Functional Studies
- GOF
- LOF (DN molecules, siRNAs)
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MEDIAL
LATERAL
ANTERIEUR
POSTERIEUR
NT
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MEDIAL LATERAL ANTERIOR POSTERIOR
NT NT NT NT
48h
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E 4.5
Medial
Caudal +Rostral
Lateral
The formation of the myotome
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Questions
-Mechanisms regulating muscle growth:-Origin of muscle stem cells?-Proliferation and differentiation (common mechanisms in the embryo & adult)?
-Primary myotome morphogenesis:-organized cellular movements-positional information
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Origine embryonnaire des cellules souches du muscle squelettique de l’adulte
Cellules satellites: Somites?Aorte dorsale?Moelle osseuse?Cellules « SP » SP
Sat. C.
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Cellules souches du muscle squelettique de l’adulte: cellules satellites
5-12/fibre
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The EMT of the dermomyotome …
Phalloidin / F-actin staining
+12h +24h
+36h +48h
Dermis ?
mGFP / Ncadh
mGFP / Ncadh
MyMy
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Actin / GFP
24 hours 36 hours 48 hours
… triggers the entry of a population of muscle progenitors in the primary myotome...
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« Parachutage » observé in vivo
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Long term lineage: Quail-chick grafting
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A somitic origin for satellite cells
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Morphogenesis and growth of the myotome
QuickTime™ et undécompresseur h264
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Myotome primaire
bordures
(MyHC, MyoD, Myf5)
Partie centrale
EMT
Pax3-/-; Pax7-/-:NO progenitors
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Fusion des myoblastes chez la Drosophile
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Fusion des myoblastes chez la Drosophile
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Regulation of the fusion of muscle cells
?
1. Descriptive analysis of fusion in the chick embryo: When does fusion start?Determine which cell types fuse?
2. Identify chick homologs of Drosophila genes, perform ISH
In progress…
3. Functional analyses in the chick (GOF, LOF).