Fig. 6.

Ectopic Atoh1b rescues otic sensorigenesis 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 15 or 21 hpf, corresponding to placodal or vesicle stages, heat-treated embryos were sorted for mCherry fluorescence and subsequently fixed for analysis just as untreated controls. (B) In untreated controls (ctrl) at 15 hpf, atoh1a is expressed in the majority of the embryos in discrete anterior and posterior domains of the otic placode (arrows in wild-type) whereas one quarter display a complete absence of atoh1a (dlx3b/4b^−/−). In heat-treated embryos (heat), otic atoh1a expression is less regular but present in the majority of embryos. (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 proper atoh1b expression carry the wild-type allele (473 bp amplicon) in homozygosity or in combination with the dlx3b/4b deletion allele (618 bp amplicon). Two embryos of the same, untreated controls with no atoh1b expression show only presence of the dlx3b/4b deletion allele (asterisk). Out of 12 heat-treated embryos with atoh1b expression, two embryos carried the dlx3b/4b deletion allele only (asterisk). M indicates marker for molecular size standard. (D) Expression of the hair-cell marker myo7aa is present anteriorly and posteriorly in the otic vesicle of untreated, wild-type controls at 21 hpf. In contrast, myo7aa expression is absent in dlx3b/4b mutant embryos, which can be recognized based on smaller otic vesicles. In the heat-treated sample, myo7aa expression is found in several embryos with reduced otic vesicles. (E) Multiplex PCR corroborates the finding following in situ hybridization. dlx3b/4b mutant embryos are indicated with an asterisk. Two embryos of untreated controls with proper myo7aa expression carry the wild-type allele in combination with the dlx3b/4b deletion allele. Two embryos of the same, untreated control sample with no myo7aa expression show only presence of the dlx3b/4b deletion allele (asterisk). All six embryos with a reduced otic vesicle size but detectable myo7aa expression carry the dlx3b/4b deletion allele only (asterisk). M indicates marker for molecular size standard. (B,D) Dorsal views, anterior to the top. Scale bar: 75 µm.