Fig. 3. Increased proliferation in the preoptic region and hypothalamus of cavefish embryos is Shh-dependent. (A-H) PCNA immunoreactivity in transverse sections (surface fish, left; cavefish, right) at 60 hpf (A-D) and 5 days (E-H, high magnification) through the preoptic region (A,B,E,F) and the hypothalamus (C,D,G,H). Note that proliferation in the dorsal diencephalon and mesencephalon is identical in both populations. (I,J) Cavefish embryos were treated with 20 µmol/l cyclopamine from 15 to 24 hpf, allowed to develop until 48 hpf, and proliferation was assessed with anti-phosphohistone H3 immunostaining (see Fig. S1 in the supplementary material for pictures). The results are expressed as the density of phospho-H3-positive cells (number of cells per surface area in arbitrary units). See methods for details. ^* indicates a significant effect (P<0.05, n=4 embryos; Student's t-test) when compared with the ethanol-treated controls. Scale bars: 50 µm. ddi, diencephalon; hyp, hypothalamus; mes, mesencephalon; po, preoptic region.