Figure 4. Wnt signaling regulates OV expansion by maintaining epithelial barrier function during canal morphogenesis.

A. UMAP dimensionality reduction of OV cells in the published embryo-wide Daniocell scRNAseq atlas, with dorsal OV cells highlighted in blue, defined by counts of the transcription factor gene LIM homeobox transcription factor 1, beta b (lmx1bb). B. Volcano plot showing differential gene expression between dorsal OV cells and all other OV cells. Genes meeting the significance criteria (adjusted P < 10⁻⁹ and log2 fold change > 1, blue; log2 fold change < −1, yellow) are colored by direction of change; non-significant genes are shown in black. Several genes related to Wnt signaling are highlighted (red), including wntless (wls). C. Representative images of 3D-rendered OVs at the developmental stages indicated, stained with multiplex in situ probes against wls. Illustrations show typical progression of OV morphogenesis, highlighting the projections that fuse into the three pillars: anterior, posterior, and ventral. D. Representative images of OVs from membrane-mNeonGreen-expressing embryos at 72 hpf after Crispr-Cas9 knockdown of tyrosinase (control) or wls. Prior to imaging, embryos were injected periotically with Dextran conjugated with Texas Red to visualize the outer surface of the OV. E. Quantification of whole OV volume (pL), OV lumen volume (pL), epithelial tissue volume (pL), and tissue fraction (%), defined as the tissue volume divided by the whole OV volume in embryos at 72 hpf after Crispr-Cas9 knockdown of either tyrosinase (control, white) or wls (grey). P-values as labeled (Tissue fraction and OV volume: unpaired, two-tailed Student’s t-test; Tissue volume and lumen volume: MannWhitney U test). F. UMAP dimensionality reduction of scRNAseq data from cells isolated from the micro-dissected inner ear region of embryos after Crispr-Cas9 knockdown of either tyrosinase (control) or wls. Upper-left panel: all cells. Upper-right panel: all cells with expression of the OV marker stm overlain as a heatmap. Lower-left panel: all cells in the stm OV cluster, colored by subcluster. Lower-right panel: all cells in the OV cluster, with expression of the dorsal OV marker lmx1bb overlain as a heatmap, highlighting the pillar-forming cell clusters. G. Pseudobulk analyses of notch1b in cells from embryos upon knockdown of tyrosinase (white) or wls (grey) in the whole OV or in the dorsal OV clusters. n denotes the number of biological replicates. Statistical significance was determined using DESeq2, and adjusted P values (Benjamini–Hochberg correction) are indicated. H. Representative images of 3D-rendered OVs from membrane-mNeonGreen-expressing embryos at 72 hpf after soaking for 24 hours in either DMSO or the notch inhibitor RO4929097, with pillar region outlined in yellow. Right plot: quantification of OV lumen volume (pL) after 24 hrs of soaking in either DMSO (white) or RO4929097 (grey). P-values as labeled (unpaired, two-tailed Student’s t-test). I. Representative images of OVs from membrane-mNeonGreen-expression embryos at the developmental stages indicated, upon knockdown of either tyrosinase or wls. Prior to imaging at 52 hpf, embryos were injected pericardially with α-Bungarotoxin conjugated to Alexa Fluor 647 to visualize permeabilization of the OV epithelium barrier from 52 to 56 hpf. Right upper plot: quantification of OV lumen volume (pL) at 52 and 56 hpf after knockdown of either tyrosinase or wls. Right lower plot: normalized intensity (arbitrary units, A.U.) of α-Bungarotoxin-Alexa Fluor 647 in OVs upon wls knockdown from 52 hpf (blue) to 56 hpf (red), demonstrating increased permeability of the OV epithelial barrier. P-values as labeled (two-tailed paired t-test and unpaired, two-tailed Student’s t-test). J. Representative images of the dorsal OV region of Tight Junction Protein 1a, or ZO-1a (Tjp1a)-tdTomato-expressing embryos after treatment by soaking in either DMSO or the porcupine inhibitor C59. Right upper plot: change in mean intensity (A.U.) of Tjp1a-tdTomato at cell vertices after 24 hours of soaking in either DMSO (white) or C59 (grey), quantified at 48 and 72 hpf. Right-hand lower plot: change in mean intensity (A.U.) of Tjp1a-tdTomato along cell edges after 24 hours of soaking in either DMSO (white) or C59 (grey), quantified at 48 and 72 hpf. P-values as labeled (unpaired, two-tailed Student’s t-test). “n” denotes the number of embryos unless otherwise specified. Scale bar: 50 μm. Data are mean±s.d.