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Staining for chondroitin sulphate in phenotypically wild-type zebrafish ears marks sites of epithelial projection outgrowth. (A–F) Confocal images of Alexa-phalloidin (green) and anti-CS antibody (magenta) whole-mount stains of phenotypically wild-type ears (Section 2). Blue arrowheads mark foci of chondroitin sulphate (CS) staining associated with the emergence (evagination) of epithelial projections from the otic epithelium; blue bracket marks the invaginating endolymphatic duct (ed); blue asterisk marks staining in the otolithic membrane overlying hair cells of the anterior (utricular) macula. Boxed areas highlight regions of interest (ROI), enlarged in the bottom row of panels: (i), apical focus of CS staining at 45 hpf prefiguring site of emergence of the anterior projection; (ii,iii), foci of CS staining associated with the anterior and posterior projections, respectively, at 50 hpf; (iv), basal focus of CS staining prefiguring site of emergence of the ventral projection; (v), CS staining in the utricular otolithic membrane; (vi), absence of CS stain in the endolymphatic duct. All panels are lateral views with anterior to left, and dorsal to top (orientation shown in (A): A, anterior; D, dorsal). Scale bar in (C), 50 µm (applies to (A–F)). Abbreviations: CS, chondroitin sulphate; ed, endolymphatic duct; hc, stereociliary bundles on the apices of hair cells in the anterior (utricular) macula. Additional examples and developmental stages are shown in Figure 4; Supplementary Figure S1; Supplementary Movie S1.
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