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First published online 13 August 2003
doi: 10.1242/dev.00688

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Intrinsic signals regulate the initial steps of myogenesis in vertebrates

Claudia Linker^1,*,, Cynthia Lesbros^1,*, Michael R. Stark² and Christophe Marcelle^1,

¹ Laboratoire de Génétique et de Physiologie du Développement (LGPD), Developmental Biology Institute of Marseille (IBDM), CNRS UMR 6545, University Aix-Marseille II, Campus de Luminy, case 907, 13288 Marseille Cedex 09, France
² Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602, USA

View larger version (112K): [in a new window]   Fig. 1. MyoD expression is independent from neural tube and ectodermal signals. (A,D,G,J) Schematic representation of the experimental procedures. Whole-mount in situ hybridization (B,E,H,K) and transversal sections (C,F,I). (B,C) Wnt4 (red) and MyoD (blue) expression after dorsal neural tube ablation. In these conditions, 91% of the embryos showed normal MyoD expression (n=10/11). (E,F) MyoD expression after ectoderm isolation. All of the embryos showed normal MyoD expression (n=11), compared with the contralateral, unoperated side, or with MyoD expression observed rostral and caudal to the membrane (indicated by arrowheads). (H,I) Wnt4 (red) and MyoD (blue) expression after both, dorsal neural tube and ectoderm ablation. Ninety-three percent of the embryos showed normal MyoD expression (n=14/15). (K) MyoD expression 1 hour after separation of somites from axial structures. All of the embryos failed to show MyoD expression (n=8). Broken lines indicate the level of the sections. n, notochord; nt, neural tube; e, ectoderm; s, somite.

View larger version (121K): [in a new window]   Fig. 2. Fz receptors and Wnt5b expression patterns. Whole-mount in situ hybridization of (A) Fz1; (B) Fz2; (C) Fz7 and (E) Wnt5b. (D) Section of C at the level of the first somite. (F) Section of Wnt5b (blue) and MyoD (red) double in situ hybridization at the level of the first somite. Sections levels are indicated by broken lines. Somite numbers are indicated by roman numerals. s, somite; nt, neural tube.

View larger version (82K): [in a new window]   Fig. 3. Fz7 signaling is required for MyoD, but not for Myf5 initiation. (A) MyoD expression in an embryo injected with control alkaline phosphatase-transfected cells. Eighty-eight percent of the embryos presented normal MyoD expression (n=23/26). (B) MyoD expression in an embryo injected with S-Fz7-transfected cells. S-Fz7-secreting cells were injected at the caudal, middle and rostral presomitic level, as well as in the third or fourth newly formed somite. Seventy-seven percent of the embryos presented an inhibition of MyoD expression in somites formed in the presence of S-Fz7-transfected cells (n=23/30). Inhibition was more pronounced around S-Fz7-expressing cells injected in the caudal and middle presomitic mesoderm (shown here). MyoD inhibition was sometimes observed when cells were injected in the rostral presomitic mesoderm, although this effect was seldom observed when they were injected in somites which already expressed MyoD. (C) In sharp contrast, embryos (87%) injected with S-Fz7-transfected cells presented normal Myf5 expression in injected somites (n=20/23). (D) Transverse section of B. (E) Transverse section of C. Section levels are indicated by broken lines. Injected cells were stained with the fluorescent dye DiI and observed under UV illumination. (D,E) Composites of a bright-field image of transverse sections and a fluorescent image showing the DiI-labeled S-Fz7-expressing cells. Arrowheads indicate the position of the injected cells.

View larger version (66K): [in a new window]   Fig. 4. Wnt5b signal regulates MyoD expression. (A,B) Rescue of the MyoD-inhibitory effect of S-Fz7 by Wnt5b co-injection. (A) MyoD expression in an embryo co-injected with S-Fz7 transfected cells mixed with control cells at a ratio of 1:5. Seventy-one percent of these embryos present an inhibition of MyoD expression (n=27/38). (B) Rescue of MyoD expression by co-injection of S-Fz7 transfected cells mixed with Wnt5b-expressing cells at a ratio of 1:5. In 57% of embryos, MyoD expression was rescued to normal levels (n=25/44). (C) Inhibition of MyoD initiation in DN-Wnt5b electroporated embryos. Mouse Wnt5b (red) and MyoD (blue) expression in embryos electroporated with DN-Wnt5b in the caudal neural tube. 80% of the embryos presented inhibition of MyoD initiation (n=15/18). (D) GFP (red) and MyoD (blue) expression in embryos electroporated with a GFP cDNA in the caudal neural tube. All of the embryos had normal MyoD expression (n=12).

View larger version (94K): [in a new window]   Fig. 5. MyoD expression is repressed by a Bmp signal in the presomitic mesoderm. (A) MyoD expression after injection of control cells. All of the embryos showed normal MyoD expression (n=25). (B) MyoD expression after injection of Noggin-expressing cells. Ninety-one percent of the embryos showed ectopic expression of MyoD in the presomitic mesoderm and first somites (n=42/46). (C) Sections at the level of newly formed somite. (D) Sections at the level of presomitic mesoderm. Arrowheads in C and D indicate the ectopic expression of MyoD, on the injected and contralateral sides of the embryo. The broken line indicates the position of nogginexpressing cells. Sections levels are indicated by arrows in B. (A,B) Composite of a bright-field image of whole-mount or transverse section and a fluorescent image showing the DiI-labeled control and noggin-expressing cells. Somite numbers are indicated by roman numerals.

View larger version (123K): [in a new window]   Fig. 6. Initiation of MyoD expression in vitro. (A,D) Culture of isolated presomitic mesoderm for 3-4 hours. (A) Delta1 (n=30), (B) Myf5 (n=55), (C) Wnt5b (n=67) and (D) Fz7 (n=8) were autonomously expressed in these conditions. (E,P). Culture of dissociated rostral presomitic mesoderm cells. Note that the same results were obtained in six independent experiments. In these experimental conditions, MyoD (F), Wnt5b (G), Fz7 (H), Paraxis (O) and Myf5 (P) are expressed. Pax1 (E) and Shh (M) serve as negative controls. When dissociated presomitic mesoderm cells are cultured in the presence of supernatant from Bmp4-expressing cells, initiation of MyoD expression is not observed (N). When Shh is added to dissociated presomitic cells, expression of MyoD (J), Wnt5b (K) and Fz7 (L) is strongly enhanced, while Pax1 (I) expression is activated.

View larger version (19K): [in a new window]   Fig. 7. A model for myogenesis initiation. The expression of Myf5 is autonomously initiated in the presomitic mesoderm. Soon after, Wnt5b, which is autonomously expressed in the presumptive caudal border of somite –I and 0, triggers the activation of MyoD expression. However, this effect is inhibited by the action of Bmp. Noggin, expressed in the lateral region of the rostral presomitic mesoderm and the newly formed somites, is likely to release Bmp inhibition. This leads to the expression of MyoD. Shh (and maybe Wnts), secreted by axial structures, amplifies and/or stabilizes MyoD expression in older somites.