Fig. 2

Pax3/7 protein accumulates in nuclei on the somite surface. Immunodetection of slow muscle (B, C, green; E, red; F-H, purple), Pax7 (C, red), Pax3/7 (B, D, red) or H3P (D, E, green; F-H, brown). Wholemount 24 hpf confocal images (B-E) in transverse reconstruction (B, E left), lateral view (C, D, anterior to left) or rendered 3D reconstruction (E right) or transverse cryosection at 20 som (F) or 24 hpf (G, H) are shown with dorsal to top. (A) Western blot of 24 hpf embryo proteins reveals a similar ∼ 50 kD band with both Pax7 and Pax3/7 antibodies. MyHC shows the specificity of the reagents. (B) Confocal images reveal two types of Pax3/7-expressing nuclei (red/purple). Left panel, transverse section of a yolk extension level somite showing superficial location of Pax-expressing cells. Right panel, rendered ¾ view showing infrequent intensely-labelled red nuclei located dorsal to the neural tube and lateral to the dorsal somite (blue arrowheads). More abundant weakly-labelled purple nuclei are evenly spread over the lateral somite surface (yellow arrows), but tend to congregate near somite borders (indicated by white arrows) and at the horizontal myoseptum (yellow bracket). Note weak Pax3/7 immunoreactivity in nuclei of dorsal neural tube, floor plate and between somite and neural tube. The latter may be medially-migrating neural crest. (C) A short confocal stack showing unlabelled slow muscle fibre nuclei (white arrow) and Pax7 immunoreactivity (red). (D) Pax3/7 (red) and phosphohistone H3 (green) dual stained nuclei (arrows). Inset plots distribution of Pax3/7 and H3P dual labelled nuclei on a schematised somite from analysis of somites 12 and 13 in 20 wild type fish. Nuclei with only H3P are generally in the epidermis. (E) The non-epidermal nature of an H3P-labelled nucleus (arrow) superficial to the slow fibre layer is demonstrated by the two layers of overlying epidermal cell nuclei (arrowheads). (F-I) At 20 s stage, abundant H3P is present in the lateral somite, which consists chiefly of undifferentiated fast muscle precursors (F). After fast fibre formation and slow fibre migration, H3P is generally restricted to the somite surface lateral to slow myosin (G). Note the presence of two dividing cells in adjacent cell layers, confirming that somitic external cells divide (H). Quantification of the fraction of DAPI-labelled nuclei in 80 somites 12-16 of 24 hpf embryos that express H3P in Pax3/7⁺ or Pax3/7^- cells. Total H3P⁺ nuclei counted were 114 and 38, respectively. Somites in this region and stage contain 271 nuclei ± 19 sem, n = 4, estimated from confocal stacks (I).