Transcriptome analysis identifies genes regulated by Dlx3b/4b. (A) Expression of pax8:DsRed in the otic-epibranchial progenitor domain (OEPD) and the nephric anlagen (NA) at the eight-somite stage (13 hpf). Lateral view. Scale bar: 250 µm. (B) Schematic illustration of the experimental procedures. Following dlx3b/4b morpholino injection at the one-cell stage, wild-type and dlx3b/4b morpholino-injected embryos were dissociated at late OEPD stages (12-13.5 hpf) followed by fluorescence-activated cell sorting. Subsequently, isolated transcripts from sorted, pax8:DsRed-positive cells were reverse transcribed into cDNA and used for transcriptome analysis (RNAseq). (C) Comparison of wild-type versus Dlx3b/4b-depleted samples with a false discovery rate of 10% revealed 3015 differentially expressed genes. 1486 and 1529 genes were up- and downregulated respectively. (D) Gene ontology (GO) analysis of the differentially expressed genes using the GOrilla gene ontology analysis tool (Eden et al., 2009). For more information see Material and Methods.

Dlx3b/4b controls the expression of numerous known and novel transcripts in the OEPD. In comparison to wild-type siblings, RNAseq and in situ hybridization show a downregulation of atoh1b (A), ptchd3a (B), stc2a (C), robo4 (D), sox9b (E), pcdh7b (F), si:ch211-137a8.2 (G), zgc:194210 (H), rsph9 (I), ccdc103 (J), fam20cb (K), klhl14 (L), mcf2lb (M), irx4b (N) and agr2 (O) in the OEPD of dlx3b/4b mutants at the six-somite stage (12 hpf). Dorsal views, anterior to the top. Scale bar: 100 µm. Except for atoh1b, plots show the read counts of the individual genes including standard deviation. Expression levels of atoh1b are given in transcripts per million (TPM) because it is annotated to belong to chrUn_KN150642v1 and mapping was done only against known chromosomes. Significance was based on the padj values which were calculated to control the false discovery rate and was assigned to the following ranges: ***: 0-0.001; **: 0.001-0.01; *: 0.01-0.01; not significant: 0.1-1.0.

Dlx3b/4b controls the expression of genes mimicking the expression pattern of atoh1b. Similar to atoh1b, foxj1b (A), mns1 (B), ulk1a (C), cdr2l (D), has3 (E), si:dkey-222f2.1 (F), zgc:158291 (G), cxcl14 (H) and gfi1ab (I) show a restricted expression in the OEPD. In comparison to wild-type siblings, RNAseq and in situ hybridization at the six-somite stage (12 hpf) show downregulation of all aforementioned genes in the OEPD of dlx3b/4b mutants. Dorsal views, anterior to the top. Scale bar: 100 µm. Plots show the read counts of the individual genes including standard deviation. Significance was based on the padj values which were calculated to control the false discovery rate with the following ranges: ***: 0-0.001; **: 0.001-0.01; *: 0.01-0.01; not significant: 0.1-1.0.

Atoh1b controls the expression of numerous genes in the OEPD. In comparison to wild-type siblings, RNAseq and in situ hybridization show downregulation of foxj1b (A), mns1 (B), ulk1a (C), cdr2l (D), has3 (E), si:dkey-222f2.1 (F) and zgc:158291 (G) in the OEPD of atoh1b mutants. Expression of cxcl14 (H) and gfi1ab (I) is also absent in the OEPD of atoh1b mutants although RNAseq indicates a trend but no significant difference. Expression of ptchd3a (J), fam20cb (K) and irx4b (L) are not affected in atoh1b mutants neither based on the RNAseq data nor by in situ hybridization. Dorsal views, anterior to the top. Scale bar: 100 µm. Plots show the read counts of the individual genes including standard deviation. Significance was based on the padj values which were calculated to control the false discovery rate with the following ranges: ***: 0-0.001; **: 0.001-0.01; *: 0.01-0.01; not significant: 0.1-1.0.

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.

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.

Schematic summary of the genetic events controlled by Dlx3b/4b during early otic development in zebrafish. Dlx3b/4b are expressed already during formation of the preplacodal region (PPR). After induction of the otic-epibranchial progenitor domain (OEPD), Dlx3b/4b control the onset of various genes which might govern various morphogenetic processes at subsequent placodal and vesicles stages. Among the genes showing an OEPD-wide expression (ptchd3, pcdh7b, si:ch211-137a8.2, robo4, sox9b, stc2a and zgc:194210), we also find klhl14; mcf2lb; irx4b and agr2 which have been reported to be expressed within the chicken OEPD (Chen et al., 2017). Hence, they might be members of a conserved, vertebrate core module regulating subsequent otic morphogenesis as well as otic versus epibranchial fate decisions. In addition, we find foxj1b, mns1, ulk1a, cxcl14, zgc:158291, si:dkey-222f2.1, gfi1ab, has3 and cdr2l, which display an atoh1b-like expression and might play a role in tether cell formation. Except has3 and cdr2l (grey), all genes of this module can be activated via ectopic expression of Atoh1b, even in the absence of Dlx3b/4b. Finally, we find that Dlx3b/4b controls the expression of fam20cb a gene associated with biomineral tissue development which might be important for subsequent otolith formation.