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. 2015 Jan 7;35(1):234-44.
doi: 10.1523/JNEUROSCI.3353-14.2015.

Fgf-signaling-dependent Sox9a and Atoh1a Regulate Otic Neural Development in Zebrafish

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Free PMC article

Fgf-signaling-dependent Sox9a and Atoh1a Regulate Otic Neural Development in Zebrafish

Jialiang Wang et al. J Neurosci. .
Free PMC article

Abstract

Fibroblast growth factors (Fgfs) play important roles in developmental processes of the inner ear, including the ontogeny of the statoacoustic ganglia (SAG) and hair cells. However, the detailed genetic mechanism(s) underlying Fgf/Fgfr-dependent otic neural development remains elusive. Using conditional genetic approaches and inhibitory small molecules, we have revealed that Fgfr-PI3K/Akt signaling is mainly responsible for zebrafish SAG development and have determined that Sox9a and Atoh1a act downstream of Fgfr-Akt signaling to specify and/or maintain the otic neuron fate during the early segmentation stage. Sox9a and Atoh1a coregulate numerous downstream factors identified through our ChIP-seq analyses, including Tlx2 and Eya2. Fgfr-Erk1/2 signaling contributes to ultricular hair cell development during a critical period between 9 and 15 hours postfertilization. Our work reveals that a genetic network of the previously known sensory determinant Atoh1 and the neural crest determinant Sox9 plays critical roles in SAG development. These newly uncovered roles for Atoh1and Sox9 in zebrafish otic development may be relevant to study in other species.

Keywords: Fgf signaling; Fgfr-Erk1/2; Fgfr-PI3K/Akt; hair cells; statoacoustic ganglia.

Figures

Figure 1.
Figure 1.
SAG development is impaired when Fgf-PI3K/Akt signaling is blocked. AC, Heat-shocked controls (A), hs:dn-fgfr1 embryos (B), and hs:fgf8a embryos (C) are immunostained by HuC antibody at 48 hpf. The 40 min heat shock starts at 10 hpf. DF, HuC antibody staining in DMSO- (D), SU5402- (E), and wortmannin (F)-treated embryos at 48 hpf. The chemical treatment period is 10–14 hpf. GI, Expression of neurod in DMSO- (G), SU5402- (H), and wortmannin (I)-treated embryos at 30 hpf. The chemical treatment period is 10–14 hpf. J, Summary of SAG size after manipulating Fgf signaling at different heat shock time point (n = 10; 48 hpf). K, Summary of size of SAG populations after blocking the Fgf signaling (n = 10; 48 hpf). Allg, Anterior lateral line ganglion. White dotted lines indicate otic neurogenic region; black dotted lines indicate otic region. AI, lateral views with anterior to left and dorsal up. Results in J and K are presented as mean ± SEM. ***p < 0.001, one-way ANOVA. Scale bar, 30 μm.
Figure 2.
Figure 2.
The sox9a and atoh1a are two parallel targets downstream of Fgfr-PI3K/Akt signaling. AC, Expression of sox9a in heat-shocked controls (A), hs:dn-fgfr1 embryos (B), and hs:fgf8a embryos (C) at 12 hpf. The 40 min heat shock starts at 10 hpf. DF, Expression of atoh1a in heat-shocked controls (D), hs:dn-fgfr1 embryos (E), and hs:fgf8a embryos (F) at 12 hpf. The 40 min heat shock starts at 10 hpf. GI, Expression of sox9a in DMSO- (G), SU5402- (H), and wortmannin (I)-treated embryos at 12 hpf. The chemical treatment period is 10–12 hpf. JL, Expression of atoh1a in DMSO- (J), SU5402- (K), or wortmannin (L)-treated embryos at 12 hpf. The chemical treatment period is 10–12 hpf. MN, Analyzing the expression profiles of sox9a (M) or atoh1a (N) genes by qRT-PCR at 14 hpf. White dotted lines indicate the otic region. AL, Dorsal views with anterior to top. Results in M and N are presented as mean ± SEM (one-way ANOVA test). Scale bar, 30 μm.
Figure 3.
Figure 3.
sox9a and atoh1a both work in otic neurogenesis. AC, Expression of neurod in wild-type embryos (A), sox9a morphants (B), and atoh1a morphants (C) at 30 hpf. DF, HuC antibody staining in wild-type embryos (D), sox9a morphants (E), and atoh1a morphants (F) at 48 hpf. GJ, sox9a (I) and atoh1a (J) mRNA can individually and partially restore SAG areas of SU5402-treated (10–14 hpf) neurod:EGFP embryo (H) at 30 hpf. K, Summary of the restored SAG areas in mRNA-injected embryos. The chemical treatment period is 10–14 hpf (n = 10; 48 hpf). Allg, Anterior lateral line ganglion; Pllg, posterior lateral line ganglion. White dotted lines indicate the otic neurogenic region; black dotted lines indicate the otic region. AJ, Lateral views with anterior to left and dorsal up. Results in K are presented as mean ± SEM. **p < 0.01, ***p < 0.001 (one-way ANOVA). Scale bar, 30 μm.
Figure 4.
Figure 4.
Identifying direct targets of Sox9a and Atoh1a. A, Diagram of the recombinant plasmid pN3-X-Myc. B, Western blot analysis using a Myc antibody detects Sox9a-Myc and Atoh1a-Myc fusion proteins in embryos at 14 hpf. C, ChIP-PCR analysis detects known targets of Sox9a (col2a1a) and Atoh1a (hes6). D, Distribution of all sorts of Sox9a (left) or Atoh1a (right) genomic DNA-binding sites as revealed by ChIP-Seq analyses. E, Peaks of Sox9a (gray) or Atoh1a (dark gray) genomic DNA binding areas is largely mapped near Ensembl transcriptional starting sites (TSS). F, ChIP-PCR analysis confirms selected potential targets for both Sox9a and Atoh1a. G, qRT-PCR assay confirms selected targets for both Sox9a and Atoh1a at 16–22 hpf. The col2a1a and hes6 bars serve as positive controls. Results in G are presented as mean ± SEM. *p < 0.05 (one-way ANOVA).
Figure 5.
Figure 5.
tlx2 and eya2 contribute to otic neurogenesis downstream of Sox9a and Atoh1a. AD, Expression of tlx2 in wild-type embryos (A), sox9a morphants (B), atoh1a morphants (C), and double morphants (D) at 24 hpf. EH, Expression of eya2 in wild-type embryos (E), sox9a morphants (F), atoh1a morphants (G), and double morphants (H) at 14 hpf. IL, Otic neurons are reduced in wild-type embryos (I), tlx2 morphants (J), eya2 morphants (K), and tlx2/eya2 double morphants (L) at 30 hpf. M, Summary of the restored SAG areas in mRNA-injected embryos (n = 10; 48 hpf). Allg, Anterior lateral line ganglion. White dotted lines indicate the otic neurogenic region; black dotted lines indicate the otic region. AL, Lateral views with anterior to left and dorsal up. Results in M are presented as mean ± SEM. **p < 0.01, ***p < 0.001 (one-way ANOVA test). Scale bar, 30 μm.
Figure 6.
Figure 6.
Fgfr-Erk1/2 signaling contributes to otic sensorigenesis. AF, Expression of atoh1a, atoh1b, and sox9a in embryos treated by DMSO (A,C,E) and PD98059 (B,D,F) at 12 hpf. The chemical treatment period is 10–12 hpf. GJ, Utricular hair cells in 48 hpf embryos treated with DMSO (G), SU5402 (H), PD98059 (I), or Wortmannin (J). K, Utricular hair cell number is severely reduced by blocking Fgfr-Erk1/2 signaling starting from 10–14 hpf (n = 10; 48 hpf). L, Blocking Fgfr signaling using 100 μm SU5402 at different stages indicates the number of utricular hair cells is drastically reduced upon 9–11 hpf and 13–15 hpf treatments (n = 10; 48 hpf). M, Model of zebrafish otic neurogenesis and sensorigenesis is proposed. White dotted lines indicate the otic region. AF, Dorsal views with anterior to top. GJ, Lateral views with anterior to left and dorsal up. Results in K and L are presented as mean ± SEM. **p < 0.01, ***p < 0.001 (one-way ANOVA test). Scale bar, 30 μm.

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