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Fig. 4. Incubation of embryo explants with GoH3 perturbs myotome formation. (A-D) Transverse cryostat sections of E8.0 (A,B) and E9.5 (C,D) wild-type embryo explants cultured for 14 hours under control conditions (A,C) or with GoH3 (B,D). In E8.0 (+14 hours) control explants, a few Myf5-positive cells are localised under the dermomyotome in a typical myotomal position (arrow in A). In E8.0 (+14 hours) explants cultured with GoH3, Myf5-positive cells are found near the neural tube (arrows in B). Myf5 expression is present in the dermomyotome (asterisks in B). Laminin immunoreactivity at the myotome-sclerotome interface is discontinuous in GoH3-treated embryos (arrows in D; also see amplification in inserts), and laminin staining is interrupted at the epaxial lip (arrowheads). (E,F) X-gal staining of Myf5^nlacZ/+ embryos (E) shows normal distribution of reporter-expressing cells in the myotome, while in Myf5^nlacZ/nlacZ embryos (F), these cells disperse. (G-L) Whole mount co-immunohistochemistry of E9.5 embryo explants cultured under control conditions for 24 hours show a normal expression pattern of Myf5 (G,I) and desmin (H,I), while the presence of GoH3 resulted in abnormal dispersion of Myf5-positive cells (J,L), particularly in interlimb-level somites. Furthermore, Myf5-positive cells fail to invade the myotome (absence in the area indicated by brackets). These effects are very similar to the defect observed in Myf5^nlacZ/nlacZ embryos (compare J with F). A disorganised pattern of desmin immunoreactivity (K,L) is also observed (area indicated by the brackets and patches among dispersed cells). Anterior is towards the right in E-L. ep, epaxial; hyp, hypaxial; nt, neural tube; fl, forelimb. Scale bars: 100 µm in A-D; 80 µm in G-L.

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