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Growth

The Life of a Skeletal Muscle. Specification. Morphogenesis. Growth. Diseases. Genes Exogenous or Autonomous ? . Cell shape changes, migration - Identify positional cues. Stem cell origin -Proliferation Fusion. Genes Therapies. MyoD. S I. Initiation de la myogenèse. D.

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Growth

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  1. The Life of a Skeletal Muscle Specification Morphogenesis Growth Diseases • Genes • Exogenous or Autonomous ? • Cell shape changes, • migration • - Identify positional cues • Stem cell origin • -Proliferation • Fusion • Genes • Therapies

  2. MyoD S I Initiation de la myogenèse. D dermomyotome myotome sclerotome Neural tube medial lip sclerotome epithelial somite

  3. Initiation de la myogenèse: modèle inductif • Arguments expérimentaux: • Micromanipulations chez le poulet : rotation de somites > myogenèse normale • Culture de PSM souris et poulet: • + rien > rien • + Wnt > un peu • + Wnt + SHH > beaucoup Wnt-6 Wnt-1 -3a, -4 MyoD SHH

  4. WT Shh-/- Smo-/- Mais: Myogenèse normale sans signal Hh Myf5 E9.5 Zhang et al. 2001 Cell 105: 781-792

  5. 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?

  6. Les gènes de détermination myogénique Famille des bHLH Myogenic determination MyoD Myf5 Muscle differentiation Myogenin MRF4 Tous ont la capacité d’induire la myogenèse de cellules non myogéniques (gènes « maîtres ») MyoD -/- et Myf5 -/- sont viables et fertiles MyoD:Myf5 -/- létal, pas de muscle Myogenin -/- et MRF4 -/- possèdent des myoblastes Myf5 ou MyoD-positifs, mais la différenciation ultérieure est arrêtée. MyoD/Myf5: gènes de spécification Myogenin/MRF4: gènes de différentiation

  7. 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)

  8. 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

  9. Alveolar Rhabdomyosarcomas • Pediatric solid tumors of skeletal muscle in Human. • Chromosomal translocation t(2;13)(q35;q14) PAX3-FKHR PD L HD 1 479 PAX3 (PAX7) (weak transactivation/repression) 655 1 FKHR AD FH 1 837 PAX3-FKHR (strong transactivation) (PAX7-FKHR) AD PD L HD

  10. Muscle satellite cells in Pax7 mutant mice Pax7: phénotype chez l’adulte cellules souches (satellites) apoptotiques Pax3 : Pax 7 -/- pas de progéniteurs musculaires

  11. Early muscle morphogenesis Pax-7 MHC Dermomyotome Myotome

  12. The Electroporation Technique Lineage studies - GFP reporter Functional Studies - GOF - LOF (DN molecules, siRNAs) + - E 2.5

  13. MEDIAL LATERAL POSTERIEUR ANTERIEUR NT

  14. ANTERIOR POSTERIOR MEDIAL LATERAL NT NT NT NT 48h

  15. E 4.5 The formation of the myotome Medial Caudal + Rostral Lateral

  16. Questions -Primary myotome morphogenesis: -organized cellular movements -positional information -Mechanisms regulating muscle growth: -Origin of muscle stem cells? -Proliferation and differentiation (common mechanisms in the embryo & adult)?

  17. Origine embryonnaire des cellules souches du muscle squelettique de l’adulte Cellules satellites: Somites? Aorte dorsale? Moelle osseuse? Sat. C. SP Cellules « SP »

  18. Cellules souches du muscle squelettique de l’adulte: cellules satellites 5-12/fibre

  19. +24h mGFP / Ncadh +12h My +36h +48h mGFP / Ncadh Dermis ? Phalloidin / F-actin staining The EMT of the dermomyotome …

  20. 24 hours … triggers the entry of a population of muscle progenitors in the primary myotome... 36 hours 48 hours Actin / GFP

  21. « Parachutage » observé in vivo

  22. Long term lineage: Quail-chick grafting

  23. A somitic origin for satellite cells

  24. Morphogenesis and growth of the myotome

  25. Pax3-/-; Pax7-/-: NO progenitors Partie centrale bordures EMT Myotome primaire (MyHC, MyoD, Myf5)

  26. Fusion des myoblastes chez la Drosophile

  27. Fusion des myoblastes chez la Drosophile

  28. Regulation of the fusion of muscle cells ? 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 3. Functional analyses in the chick (GOF, LOF). In progress…

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