Sox8 remodels the cranial ectoderm to generate the ear

Abstract

The vertebrate inner ear arises from a pool of progenitors with the potential to contribute to all the sense organs and cranial ganglia in the head. Here, we explore the molecular mechanisms that control ear specification from these precursors. Using a multiomics approach combined with loss-of-function experiments, we identify a core transcriptional circuit that imparts ear identity, along with a genome-wide characterization of noncoding elements that integrate this information. This analysis places the transcription factor Sox8 at the top of the ear determination network. Introducing Sox8 into the cranial ectoderm not only converts non-ear cells into ear progenitors but also activates the cellular programs for ear morphogenesis and neurogenesis. Thus, Sox8 has the unique ability to remodel transcriptional networks in the cranial ectoderm toward ear identity.

Keywords: ear; ectoderm; gene regulatory network; sensory placode; transcription factor.

Fig. 1.

Transcriptomic characterization of ear development. (A–C) In ovo electroporation (A) was used to label and collect otic (B; Lmx1aE1-EGFP⁺) and epibranchial (C; Sox3U3-EGFP⁺) cells for bulk RNAseq. (D) Volcano plot showing genes differentially expressed (absolute log2 fold change > 1.5 and adjusted P value < 0.05) in otic (orange) and epibranchial cells (green). (E, F) Cells expressing the Pax2E1-EGFP reporter active in OEPs and otic and epibranchial placodes were collected for scRNAseq at the stages indicated (F). (G–L) t-distributed stochastic neighbor embedding (tSNE) representation of unsupervised hierarchical clustering of all cells (G–I) and of the placodal cell subset (J–L); cells are color-coded according to the stage collected (G, J; OEP gray, ss11-12 blue, ss14-15 black), clusters (H, K), and placodal marker expression: Six1 (I) and Pax2 (L). (M) Heatmap showing partitioning of the placodal subset (C1, C2 in H) into five clusters (PC1–5) based on gene modules (GM). Expression profiles reveal that PC1 largely contains OEP-like cells, while PC2/3 and PC4/5 are composed of otic-like and epibranchial-like cells. (N–O) Pseudotime ordering using Monocle2 shows trajectories between stages (N) and clusters (O). Note that otic- and epibranchial-like cells segregate into two branches: otic-like in orange, epibranchial-like in green, and OEP-like in pink. (P) RNA velocity vector field verifies the directional trajectories predicted by Monocle2.

Fig. 2.

Dynamic gene expression as OEPs segregate into otic and epibranchial fates. (A) BEAM identifies genes regulated in a branch-dependent manner. (B) Histogram showing the proportion of cells coexpressing genes that are expressed before the branching point but later segregate to the otic or epibranchial branch. Significantly more cells coexpress such genes before the branching point than thereafter (Error bars, ± 1 SD). **P value < 0.01; ***P value < 0.001. (C) Dot plot for OEPs and otic and epibranchial markers based on scRNAseq data. Expression level is indicated by color intensity and gene expression frequency by dot size. (D, E) A proportion of cells coexpresses otic (Sox8, Lmx1a) and epibranchial (Foxi3, Ap2e) markers prior to the branching point. (F–I) In situ HCR (F, H) and intensity measurements (G, I) along the medial (M) to lateral (L) axis of the OEP domain indicated by a dashed line in F, H, confirms gradual segregation of OEP gene expression as distinct otic and epibranchial cell populations emerge.

Fig. 3.

Identification of putative regulatory regions in OEPs. (A) Genome browser view of ATAC, H3K27ac, and H3K27me3 profiles in OEPs at the Lmx1a locus. Cloned putative enhancer is shown in green. (B) Coelectroporation of Lmx1aE1-EGFP reporter (green) and constitutive mCherry (magenta) reveals in vivo enhancer activity in the otic placode. Sections reveal that the enhancer activity is restricted to the ectoderm at the level of the otic placode: Lmx1aE1 in green, constitutive mCherry in magenta, and DAPI in blue. (C) Motif enrichment analysis of all identified enhancers. (D) Diagram showing the onset of expression of potential otic regulators during specification and commitment stages. (E) Unilateral knockdown of selected TFs using fluorescein-labeled aON (green) leads to down-regulation of otic markers as shown by in situ hybridization (purple) on the targeted side of the embryo. Note: fluorescent images were taken prior to in situ hybridization for Sox8 aON and controls. (F) Unilateral coelectroporation of Sox8 aON (green) and Sox8-mCherry construct (magenta) on the right side of the embryo restores Soho1 expression in the otic territory. Sox8 OE, Sox8 overexpression.

Fig. 4.

Sox8 converts non-otic cells into otic vesicles with associated neurons. (A) Coelectroporation of mCherry-tagged vectors containing the full-length sequence of Sox8/Pax2/Lmx1a (magenta) generates ectopic vesicles across the cranial ectoderm associated with neurofilament-positive neurons (green). (A’, A’’) Transverse sections at the level indicated by arrows in A show neurofilament/mCherry-positive neuronal projections from the ectopic vesicles. (B) Overexpression of Sox8 (Sox8 OE; magenta) alone activates Lmx1aE1-EGFP (green). (C–E) Overexpression of Sox8 (Sox8 OE) induces otic markers on the electroporated side, but not on the control side (Ctrl) as shown by HCR (C) and in situ hybridization (D, E) of otic markers. (F) RNAseq of Sox8 and control electroporated cranial ectoderm; volcano plot shows enrichment of otic genes after Sox8OE (absolute log2 fold change > 1.5 and adjusted P value < 0.05). (G) Sox8-mCherry overexpression (magenta) generates ectopic otic vesicles with neuronal projections (green), while controls do not (H); transverse sections of ectopic otic vesicles (G’, G’’) and control ectoderm (H’, H’’) show neurofilament-positive neurons within the ectopic vesicle. (I) BioTapestry model showing the minimal transcriptional circuit for otic specification with Sox8 at the top of the hierarchy. Asterisks indicate enhancers with predicted Sox8 binding motifs.