Fig. 5.

Fig. 5.

Ectopic Atoh1b rescues gene expression in the OEPD in the absence of Dlx3b/4b. (A) Scheme of the experimental outline. Progeny were obtained from heterozygous animals carrying the dlx3b/4b deletion allele (dlx3b/4b^+/−) crossed with animals carrying the dlx3b/4b deletion allele as well as the transgene to misexpress atoh1b conditionally [dlx3b/4b^+/−;Tg(hsp70l:mCherry-T2a-atoh1b)], both in heterozygosity. At 10 hpf, the clutch was split and either treated with heat or served as untreated control. At 13 hpf, corresponding to late OEPD stages, heat-treated embryos were sorted for mCherry fluorescence and subsequently fixed for analysis just as untreated controls. (B) In untreated controls (ctrl), foxj1b is robustly expressed in the majority of the embryos (wild-type) whereas one quarter displays a significant downregulation (dlx3b/4b^−/−). In contrast, all heat-treated embryos (heat), show a widespread foxj1b expression, even extending the common OEPD territory. (C) Multiplex PCR reveals the genotype of individual embryos following in situ hybridization. dlx3b/4b mutants are indicated with an asterisk. Two embryos of untreated controls with a strong foxj1b expression carry the wild-type allele detected with a 473 base pairs (bp) amplicon in homozygosity or in combination with the dlx3b/4b deletion allele detected with a 618 bp amplicon. In contrast, two embryos of untreated controls with strongly reduced foxj1b expression show only presence of the dlx3b/4b deletion allele (asterisk). Out of 12 heat-treated embryos with strong foxj1b expression, two embryos carry the dlx3b/4b deletion allele only (asterisk). M indicates marker for molecular size standard. (D-F) Expression of zgc:158291 (D), cxcl14 (E) and gfi1ab (F) behave similar to foxj1b in response to misexpression of atoh1b. (G,H) Expression of cdr2l (G) and has3 (H) is unchanged after misexpression of atoh1b. (I) Similarly, ectopic expression of atoh1b is not able to rescue fam20cb expression. (B, D-I) Dorsal views, anterior to the top. Scale bar: 100 µm.