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Figure 1. Differentiation of the median muscle fibers is initiated at the end of gastrulation. A: Expression of Myogenin, Mrf4, and muscle differentiation marker genes, Actc, Des, MyhE3, and Ckm determined by semi-quantitative polymerase chain reaction and Southern blotting. No variation of the internal standard control of quantification (Odc) was observed. B: Expression of myogenic regulatory factor (MRF) and muscle-marker genes determined by whole-mount in situ hybridization at stage 12.5 and during the first part of neurulation. Brackets indicate the posterolateral paraxial mesoderm. Vertical lines define the limit between anterior and trunk region. Dorsal views; the anterior side of the embryos is on the left; St, stage. Download figure to PowerPoint.
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Figure 2. A lateral myogenic population differentiates at the trunk level during neurulation. A: Whole-mount in situ hybridization of myogenic regulatory factor (MRF) and muscle-marker genes during the second part of neurulation. Brackets indicate the lateral myogenic population. Dorsal views. Vertical lines define the limit between anterior and trunk region. The anterior side of the embryos is on the left. B: Transverse sections at the trunk level of embryos submitted to in situ hybridization for Myod, Des (stage 16 and 17/18) or Myogenin mRNA detection (stage 17/18). 12/101 antibody detects differentiated muscle cells at stage 16 and 17/18 (blue). C: Localization of Myod mRNA on dorsal view and on transverse section at stage 21/22. Arrows and arrowheads indicate the dorsomedian and ventrolateral Myod expression domains, respectively. Dotted lines define the median differentiated muscle fibers. Nc, notochord. Download figure to PowerPoint.
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Figure 3. Expression of myogenic regulatory factor (MRF) at tailbud stages. Expression of the four MRFs at stages 22–23 in comparison with Des (A) and at stages 26-27 (B) determined by whole-mount in situ hybridization with labeled antisense probes against Myf5, Myod, Mrf4, Myogenin, and Des mRNAs. The anterior side of the embryos is on the left. Lateral views. Arrowheads in A: Myf5 mRNA staining at the dorsomedian and ventrolateral somitic borders. The probe is indicated in each panel. Time for Myogenin mRNA revelation was longer than for the other MRF at these stages.Download figure to PowerPoint
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Figure 4. The presomitic lateral cells extend to dorso- and ventromedian positions in somites. A: Gfp mRNA expression in the Myf5 transgenic embryos at stages 13, 15, and 19. B: Transverse sections at the trunk level of transgenic Myf5 embryos submitted to whole-mount immunohistochemistry with anti-gfp (purple) or 12/101 antibodies (blue) and compared with Myod mRNA expression in transgenic embryos at stages 21, 23, and 26. C: Lineage tracing experiments: the lateral and the axial/median paraxial cells of the same embryo (a) were injected, respectively, with WGA (green) and DiI (red) at stage 13. Serial transverse sections of the injected embryo (b, c, and d) at the trunk level and at stage 23. Dotted lines indicate the bilateral symmetry plan. In Ca: Vertical lines define the limit between anterior and trunk region. Nc, notochord.Download figure to PowerPoint
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Figure 5. The full myogenic program is expressed during the second myogenic wave. A: Comparison of each myogenic regulatory factor (MRF) mRNA expression from stage 30 to 40. Myf5 and Myogenin are easily detectable in the dorsal and ventral borders of the dermomyotome at stage 30 and 34, respectively. The four MRFs are successively expressed during hypaxial myogenesis (arrows), which gives rise to the ventrolateral musculature. B: The four MRFs are expressed at the epaxial region of the somites (arrowheads) during the second myogenic wave. C: Myf5 and Myod genes are expressed in the presomitic mesoderm of the caudal region. Myogenin expression is restricted to the dorsomedian and ventrolateral regions of the somites and Mrf4 accumulates in the differentiated muscle cells. D: Myf5 staining appears as dots at stage 40, probably reflecting the formation of a third wave of myoblasts. Myf5 is expressed in the anterior and posterior region of the somites (arrows) at stage 45, whereas Mrf4 mRNA staining is concentrated in the central region of the elongated cells which span entirely the somite. Lateral views, the anterior side of the embryos is on the left. The probe is indicated in each panel.Download figure to PowerPoint
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Figure 6. Formation and localization of the anterior muscle tissue. A: Double in situ hybridization with single or double color detection allowed us to localize the anterior myogenic cells which coexpressed Myod (blue) and Myf5 (purple) at stage 12.5. These myogenic cells (stained with Myod or Actc, blue) are positioned posterior to the prechordal mesoderm at both sides of the notochord (stained with Chrd, purple) at stage 13. Myogenic cells (stained with Des) are at a slightly more anterior level than the neural crest precursors (stained with Snai2, arrow), the preotic vesicle (arrows), and the pronephros anlagen (brackets), the latter stained with Pax8. The myogenic cells express Mespb (arrowheads), a gene involved in somitogenesis. At stages 14 and 16, these Myod-positive cells (blue) are localized at the level of rhombomeres 3 and 5, (stained with Egr2, purple, arrows). Dorsal views, the anterior side of the embryos is on the left. B: Double whole-mount in situ hybridization with Snai2 and muscle-markers: Myf5, Myod, Mrf4, and Des. Dorsal views. C: Comparison of Des mRNA expression with a muscle epitope staining by the 12/101 antibody in the anterior somites. Embryos were cleared in benzyl benzoate solution at stage 18 to see deeper staining. Anterior views. Brackets indicate the anterior tip of the embryo. D: Whole-mount in situ hybridization of Pax7 or Myod mRNAs (purple) was followed by whole-mount immunohistochemistry with 12/101 antibody (blue) at stage 17 and 18. Embryos were cleared in benzyl benzoate solution. Arrows show the Pax7 or Myod mRNA staining at the level of the fourth somite. Dorsal views. E: 12/101 staining of a transverse section at the head level of stage 23 embryo. F: Comparison of Myogenin mRNA expression (purple) with 12/101 antibody staining (blue) indicates that the second myogenic wave takes place at the trunk level and not at the head level (brackets). G: Comparison of Des and Mrf4 mRNA expression with 12/101 antibody staining in the anterior somites at stage 30 (magnification indicated on the right panel). Double in situ hybridization with single color detection allowed us to position the anterior myogenic cells relative to the otic vesicle and the pronephros, both stained by Pax8 (arrows and brackets, respectively). The otic vesicle is visible without a specific staining (arrows) in embryos incubated with 12/101 antibody. Embryos were cleared in benzyl benzoate solution to see a deeper staining. Lateral views. A–G: numbers refer to the developmental stages. Probes and/or antibodies are indicated for each panel.Download figure to PowerPoint
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Figure 7. Expression of Pitx2 and Tbx1 in the embryo head. A: Pitx2 is detected in the cement gland and stomodeum-pituitary anlagen (bracket) at stage 16/17. It can also be detected in the prechordal mesoderm and in the most anterior part of the head paraxial mesoderm (arrows). At stage 20, the staining is observed in cells associated to the eye, in a region corresponding to the anlagen of extraocular muscles at stage 28–30 (arrowheads). B: At stage 15/16, Tbx1 expression is localized at the border of neurectoderm as two bilateral stripes. On slide section, at stage 15/16, the staining (arrows) appears to be strong at the border of ectoderm and mesoderm but equally present in mesoderm cells. The staining of mesodermal cells is stronger at the next stages. Dash lines define the level of slide sections. Numbers refer to the developmental stages. Anterior views for stages 15/16 and 20, lateral views for stage 22, 24, 28, and 30. Download figure to PowerPoint.
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Figure 8. Distinct myogenic programs are expressed during craniofacial myogenesis. A: Schematic representation of head muscle anlagen according to Ziermann and Olsson (2007). B: Expression of Myf5, Myod, Myogenin, and Mrf4 mRNAs in head muscles anlagen. Myf5 is expressed neither in the M. levatores mandibulae, nor in the M. quadrato-hyoangularis and orbitohyoideus anlagen (black arrows). Myod transcripts are not detected in the dorsal part of branchial muscle anlagen and in the intermandibularis anlage (brackets). Green arrows design extraocular muscle anlagen. C: Expression of Des, Actc, and Ckm, as well as MyhE3 and MyhE19 coding, respectively, for embryonic fast and slow myosin heavy chain (Radice and Malacinski, 1989). St, stage. For each probe, ventral view in the left panel, lateral view in the right panel with the anterior side of the embryos on the left. h, heart. Download figure to PowerPoint.
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Figure 9. Myf5 is a less efficient inducer of differentiation than Myod and Mrf4. A: Western blot with anti-flag antibody of late gastrula embryos bilaterally injected at the two-cell stage with 100 pg of Myf5F, MyodF, or 25 pg of Mrf4F mRNA. Mrf4 protein seems to be more stable than Myf5 and Myod as 4 times less mRNA were injected to obtain the same protein level. B: Embryos were unilaterally injected at the two-cell stage at the marginal zone level with the same amounts of synthetic mRNA as in (A). Embryos were fixed at stages 15–17 and submitted to whole-mount in situ hybridization with Actc, Des, MyhE3, or Ckm probes (indicated in each panel). Arrows designate sites of ectopic expression.Download figure to PowerPoint
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des.1 (desmin, gene 1) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 17, cross-section through trunk section view, anterior left.
Image extracted from XB-ART-44993
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des.1 (desmin, gene 1) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 22-23, lateral view, anterior left, dorsal up.
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des.1 (desmin, gene 1) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 17, dorsal view, anterior left.
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Schematic representation of head muscle anlagen according to Ziermann and Olsson (2007). The green displays the extrinsic eye muscles.
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Schematic representation of head muscle anlagen according to Ziermann and Olsson (2007). The orange displays the M. intermandibularis anlage.
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Schematic representation of head muscle anlagen according to Ziermann and Olsson (2007). The red displays the M. levatores mandibulae anlagen muscle.
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Schematic representation of head muscle anlagen according to Ziermann and Olsson (2007). The light blue displays the M. interhyoideus anlage muscle.
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Schematic representation of head muscle anlagen according to Ziermann and Olsson (2007). The dark blue displays the M. quadrato-hyoangularis and orbitohyoideus anlagen muscle.
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Schematic representation of head muscle anlagen according to Ziermann and Olsson (2007). The yellow displays the branchial muscles anlagen.
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