Fig. 3. Cell proliferation defects in Shh mutants. (A-G) BrdU incorporation analysis at the 15- to 16-somite stages. (A,B) The plane of sections examined. BrdU was detected by immunostaining with Alexa 568-conjugated secondary antibody [yellow because of overlap with YoPro1 (green) labeled nuclei]. In wild type, the telencephalon (C), diencephalon (D) and midbrain (E) showed similar incorporation rates (G, black square). In Shh mutants (F), only the telencephalon showed a comparable rate with that of wild-type embryos, while the diencephalon and midbrain showed significantly decreased rates of BrdU incorporation (G, circles). (H,I) Ccnd1 (cyclin D1) expression at the 17-somite stage. In wild type (H), Ccnd1 expression was detected in all three brain regions. The anterior midbrain showed a higher level of expression than others. In Shh mutants (I), its expression was absent from the diencephalon and weaker in the anterior midbrain. Note that the strong expression in the tail was maintained in Shh mutants (I, asterisk). Arrowhead in H and I indicates the diencephalon-midbrain boundary. t, telencephalon; d, diencephalon; m, midbrain.