Fig. S1

Fig. S1 The smo^s294 allele acts as a dominant-negative. In situ hybridisation for the endodermal marker gene sox17 (A-D,J,K) and the Hh target gene ptc1 (E-I) at 9-10 hpf. (A,B) smo^hi1640 mutant embryos showed normal expression of sox17 during gastrulation (compare A with B), in contrast to smo^s294 mutant embryos (see Fig. 1B). This suggests that smo^s294 acts differently to the hi1640 null allele. (C) To confirm that the s294 allele affects Smo function during gastrulation, we also inhibited Smo function using cyclopamine, and found that cyclopamine treatment phenocopies smo^s294 mutant embryos. (E-G) To confirm that the endodermal defects we observed in smo^s294 mutants correlate with defects in Hh pathway target genes, we performed in situ hybridisation for ptc1 and found that smo^s294 (F), but not smo^hi1640 (G), showed reduced ptc1 expression at the end of gastrulation (compare with E). These results indicate attenuated Hh signalling in smo^s294 but not in smo^hi1640 mutant embryos during gastrula stages. (D,H,I) As smo is maternally provided in zebrafish, this suggests that the smo^s294 allele might act as a dominant-negative. To test this, we injected mRNA encoding wt smo or smo^s294 into wt embryos and assessed sox17 and ptc1 expression. Injection of wt smo mRNA does not affect the expression of either sox17 (data not shown) or ptc1 (H). However, injection of smo^s294 mRNA strongly reduces expression of both sox17 (D) and ptc1 (I). These results suggest that s294 acts as a dominant-negative allele of smo. (J) dtr^ts269 mutant embryos showed a slight reduction in sox17-positive endodermal cells at the end of gastrulation, suggesting that loss of Gli1 causes a reduction in endodermal cell numbers. (K) yot^ty119 mutant embryos showed a similar reduction in the number of endodermal cells at the end of gastrulation. As the yot^ty119 allele acts as a dominant repressor, these results suggest that Gli2/3 is required for gli1 expression and normal endodermal cell number in the gastrula stage embryo.