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Fig. 3 Constitutive activation of low- and medium- but not high-level Hh targets in gpr161 mutants. (A) Prox1 (green)/Eng (purple) immunostaining of mutant embryos at 24 hpf. Scale bar: 50 µm. (B,C) Quantification of SSFs and MPs from experiments presented in A (gpr161b−/−; gpr161a−/−; smo+/+ and gpr161b+/−; gpr161a+/−; smo−/−: n=15 somites in five embryos; gpr161b−/−; gpr161a−/−; smo−/−: n=21 somites in seven embryos; all others: n=27 somites in nine embryos; ns, not significant; one-way ANOVA, Tukey's post-hoc test for pairwise comparisons). (D) RNA in situ hybridisation of ptch2, olig2 and nkx2.2a transcripts in gpr161b−/−; gpr161a−/−; smo+/+ and gpr161b−/−; gpr161a−/−; smo−/−embryos fixed at 24 hpf (lateral view). Scale bar: 100 µm. (E,F) Quantification of SSFs and MPs in wild-type embryos at 24 hpf after treatment with 0-15 µM cyclopamine (DMSO and 5 µM cyclopamine: n=27 somites in nine embryos; 10 µM cyclopamine: n=15 somites in five embryos; 15 mM cyclopamine: n=21 somites in seven embryos; ***P<0.001; ns, not significant; one-way ANOVA, Tukey's post-hoc test for pairwise comparisons). (G) Representative Prox1 (green)/Eng (purple) immunostaining of wild-type embryos at 24 hpf after treatment with 0-15 µM cyclopamine. Scale bar: 50 µm. (H) RNA in situ hybridisation of nkx2.2a and olig2 transcripts in wild-type embryos at 24 hpf after treatment with 0-15 µM cyclopamine (lateral view). Scale bar: 100 µm. (A,C,G) The high levels of Eng (purple) staining in the gpr161 double mutants (A) and DMSO-treated gpr161 mutants (G) are due to Hh-dependent Eng-positive, Prox1-negative medial fast fibres, which are not included in the quantification (C). Box plots show median values (centre lines) and the interquartile ranges (boxes); whiskers extend to the highest and lowest values within 1.5×IQR (inter-quartile range).