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. 2014 Oct;243(10):1317-27.
doi: 10.1002/dvdy.24148. Epub 2014 Jun 12.

Compensatory regulation of the size of the inner ear in response to excess induction of otic progenitors by fibroblast growth factor signaling

Jian Zhang  1 , Kevin D Wright, Amanda A Mahoney Rogers, Molly M Barrett, Katherine Shim
Affiliations

Compensatory regulation of the size of the inner ear in response to excess induction of otic progenitors by fibroblast growth factor signaling

Jian Zhang et al. Dev Dyn. 2014 Oct.

Abstract

Background: The otic placode comprises the progenitors of the inner ear and the neurons that convey hearing and balance information to the brain. Transplantation studies in birds and amphibians demonstrate that when the otic placode is morphologically visible as a thickened patch of ectoderm, it is first committed to an otic fate. Fibroblast growth factor (FGF) signaling initiates induction of the otic placode, and levels of FGF signaling are fine-tuned by the Sprouty family of antagonists of receptor tyrosine kinase signaling.

Results: Here, we examined the size of the otic placode and cup by combinatorial inactivation of the Sprouty1 and Sprouty2 genes. Interestingly, in a Sprouty gene dosage series, early enlargement of the otic placode was progressively restored to normal. Restoration of otic size was preceded by normal levels of FGF signaling, reduced cell proliferation and reduced cell death.

Conclusions: Our study demonstrates that excess otic placode cells, which form in response to increased FGF signaling, are not maintained in mammals. This suggests that growth plasticity exists in the mammalian otic placode and cup, and that FGF signaling may not be sufficient to induce the genetic program that maintains otic fate.

Keywords: FGF; Sprouty1; Sprouty2; cell death; cell proliferation; gene dosage; induction; inner ear; otic placode.

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Figures

Figure 1
Figure 1. Reduction of Spry1 and Spry2 gene dosage increases otic placode size
In situ hybridization analysis of Pax8, Dlx5, and Hmx3 in 8 – 11 s embryos with the genotypes indicated (A – L). Lateral views are shown with anterior to the left. Otic placode staining of Pax8 (A – D) and Hmx3 (I – L) is outlined (white dashed line) and of Dlx5 (E – H) is bracketed. Epibranchial placode (“e”) staining adjacent to the otic domain is indicated. Expansions of gene expression domains are highlighted (asterisk). (M, N) Mid-placodal, sagittal sections of 11 s DHet and DKO embryos stained for Hmx3 (brackets). Otic placode is outlined (black dashed line). (O) Average anterior to posterior (A-P) lengths of the otic placode in 9 – 11 s embryos with the genotypes indicated. Yellow line in (M) depicts where the A-P length of the otic placode was measured. **, p < 0.01 by ANOVA in comparisons between DHet and the genotype indicated by color coding; error bars represent standard error of sample means. Scale bars (A – L), 50 μm and (M, N), 100 μm.
Figure 2
Figure 2. The otic placode remains larger in Spry1−/+; Spry2−/− and DKO embryos during early invagination
In situ hybridization analysis of Pax8, Dlx5, Hmx3, and Foxi2 in 12 – 14 s embryos with the genotypes indicated. Otic placode staining of Pax8 (A – D), Dlx5 (E – H), and Hmx3 (I – L) is bracketed. Epibranchial placode (“e”) staining of Pax8, adjacent to the otic domain, is labeled (A). Absence of Foxi2 staining in the otic placode (M – P) is outlined (white dashed line). *, expansion of the otic domain. Lateral views are shown with anterior to the left. Scale bar, 50 μm.
Figure 3
Figure 3. Restoration of otic size in Spry1−/+; Spry2−/− and DKO embryos
(A – D) Mid-placodal, sagittal sections of 17 – 19 s embryos with the genotypes indicated. Sections are located between 41% and 60% along the medial to lateral axis. Anterior is to the left. (E) Average anterior to posterior lengths of the otic placode in 17 – 20 s embryos with the genotypes indicated. Yellow line in (A) depicts where the A-P length of the otic placode was measured. *, p < 0.05 by ANOVA in comparisons between DHet and DKO embryos; error bars represent standard error of sample means. (F, G) Paint fills of DHet and DKO otic vesicles to visualize the shape and size of the lumenal space. Axial orientation is indicated: D, dorsal; A, anterior; L, lateral. (H, I) Mid-otic, transverse sections of DHet and DKO embryos. Basal surface of the otic epithelium and outline of the nuclei in the cochleovestibular ganglion are traced with yellow and blue dashed lines respectively. Scale bar (A – D, H, I), 50 μm.
Figure 4
Figure 4. Transcriptional readouts of FGF signaling in Spry1−/+; Spry2−/− and DKO embryos
(A, B) In situ hybridization for Spry1 transcript in 14 – 15 s embryos. Otic region is bracketed. Lateral views are shown, with anterior to the left. (C) Real-time PCR analysis of Spry1 and Dusp6 transcript levels in RNA collected from 13 – 16 s otic cup-containing tissue. (D – L) In situ hybridization analysis of Etv4 expression. Embryos are oriented laterally, with anterior to the left. The OEPD (D – F), otic cup during size restoration in Spry1−/+;Spry2−/− embryos (G – I), and otic cup after size restoration in Spry1−/+;Spry2−/− embryos (J – L) are bracketed. Expanded Etv4 expression domains are highlighted (asterisk). (M) Real-time PCR analysis of Etv4 and Etv5 transcript levels in RNA collected from 13 – 16 s otic cup-containing tissue. (C, M) Tissue from two embryos was pooled into one sample; error bars represent standard error of sample means. *, p < 0.05; **, p < 0.001. Scale bar, (A, B, D – L), 50 μm.
Figure 5
Figure 5. Cell proliferation and death in a Sprouty gene dosage series
Immunolabeling to detect activated caspase 3 (A – D) and phosphorylated Histone H3 (F – I) in transverse sections of 13 – 16 s embryos with the genotypes indicated. (E) Average number of activated-caspase-3+ cells normalized by volume of the otic epithelium. (J) Average number of phosphorylated-Histone-H3+ cells normalized by volume of the otic epithelium. Decreases in average numbers of caspase-3+ and pH3+ cells were observed in Spry1−/−;Spry2−/+ embryos, but were not statistically significant (caspase-3+ cells, p = 0.11; pH3+ cells, p = 0.09). *, p < 0.05 by ANOVA in comparisons with DHet measurements; error bars represent standard error of sample means. Scale bar, 50 μm.
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