Fig. 4. Homozygosity for a null allele of Shh results in closure of the neural tube by bending at DLHPs, at E8.5 (9-10 somites) when wild-type and heterozygous embryos of this strain exhibit bending only at the MHP (Mode 1 neurulation). Transverse sections (H and E) through the rostral end of the posterior neuropore of embryos from Shh^+/– x Shh^+/– litters. (A-D) MHP (arrows in A,B) but no DLHPs are present in wild-type (A; n=10) and Shh^+/– (B; n>20) embryos. In contrast, Shh^–/– embryos (C,D; n=7) exhibit DLHPs (black arrowheads). MHP is present in some Shh^–/– embryos (arrow in C) but not in others (white arrowhead in D). (E-G) E8.5 embryos from Shh^+/– x Shh^+/– litters, cultured for 6 hours in the presence of 0.05 µg/ml cytochalasin D, to reveal bending points in the neural plate, which are resistant to cytochalasin (Ybot-Gonzalez and Copp, 1999). Wild-type (E) and Shh^+/– (F) embryos exhibit a clear MHP (arrows in E,F) but rarely show DLHPs (1/6 and 1/13 respectively). Cytochalasin treatment causes the normally straight neural folds of Mode 1 to flare outwards. In contrast, cytochalasin D-treated Shh^–/– embryos exhibit DLHPs in 6/13 cases (arrowheads in G), and all have an MHP (arrow in G). Scale bar: 50 µm.