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Fig. 4 Pax3 marks the somitic external cell layer and is expanded when Fgf8 signalling is blocked. (A) Pax3 in situ mRNA hybridisation (blue/purple) with whole mounts showing expression in somites throughout the axis at 15 s, but more highly expressed in nascent somites by 20 s. Cryosections at the levels indicated reveal that somitic expression is in a discontinuous layer in the lateral region at 20 s (arrows), weakening further by 24 hpf and most prominent at the dorsal and ventral somitic extremes and the horizontal myoseptum level. There is persistent expression in dorsal neural tube. (B) Dorsal flat mount, anterior towards the top showing pax3 expression in all somites at 15 s. (C,D) Electron micrographs in transverse (C) and longitudinal (D) section of 24 hpf lateral somite at midbody level showing external cells (black arrows, ext) superficial to mononucleate slow muscle cells with pronounced medially located myofibrils (white arrows), which themselves overlie multinucleate fast fibres. The epidermis (epi) is separated from the external cells by amorphous material. Scale bar: 1.43 µm in C; 10 µm in D. (E) Oblique section through a heavily developed 24 hpf embryo showing pax3 expression in the region of the external cells (arrows) clearly superficial to slow MyHC (brown). (F) Compared with control 23 s siblings (con), SU5402-treated embryos show upregulation and maintenance of somitic pax3 both in whole mount and in cryosections. (G) Fgf8 MO reduces the downregulation of meox expression as somites mature (22/23 injected embryos). (H) wnt11r marks the dorsomedial somite edge (arrows) and neural tube. (Left) Dorsal flat mount at 15 s. (Right) Cryosections. (I) SU5402 upregulates wnt11r expression in both spinal cord and dorsomedial somite (56/56 treated embryos at 15 s).