Sulf2a restricts dorsal extent of the high-threshold Shh response without affecting expression of shh. Side views (a,c) and transverse sections (b,d,f,g) of 24 hpf embryos. a-d: Immunostaining of Sox2 in Tg(GBS-ptch2:EGFP) embryos injected with ctrlMO (a,b) or sulf2aMOATG (c,d). Dotted lines in b and d delineate the medial floor plate. Note the dorsal expansion of the GBS-ptch2:EGFP signal in sulf2a morphants. (e) Quantification of GBS-ptch2:EGFP + /Sox2 + cells in embryos injected with ctrlMO (n = 13), sulf2aMOATG (n = 12) or sulf2aMOsplice (n = 9) from two independent experiments. Datasets were compared with Mann Whitney’s test (two-tailed). Data are presented as mean number of cells along the dorso-ventral axis on each side of the lumen per embryo ± s.d (***p < 0.0005, ** p < 0.005). (f–h) Double detection of patched2 mRNA (red, f, g) and Sox2 (blue, f, g) in embryos injected with ctrlMO (f, n = 5) or sulf2aMOATG (g, n = 5). Patched2 relative fluorescence intensity in neural progenitors (white frame in f,g) was quantified along the dorso-ventral (DV) axis (grey line for ctrlMO and purple for sulf2aMOATG). Datasets were compared with two-way ANOVA test. Data are presented in arbitrary units of fluorescence (AU; mean values ± s.d) along the DV axis per section (*p < 0.005).