Staining for chondroitin sulphate in the ext2 mutant ear is delayed at early stages, but accumulates abnormally in unfused epithelial projections by 65 hpf. (A–H’) Confocal images of Alexa-phalloidin (green) and anti-CS antibody (magenta) whole-mount stains of phenotypically wild-type sibling and ext2 mutant 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 asterisks mark 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, v), apical focus of CS staining at 40 hpf, absent in the ext2 mutant, prefiguring site of emergence of the anterior projection in the wild-type ear; (ii, vi), foci of CS staining associated with the ventral projection at 48 hpf; (iii, vii), CS staining in the emerging ventral projection, delayed in the ext2 mutant; (iv, viii) no detectable staining in the wild-type posterior projection (iv) contrasts with strong staining in the unfused posterior projection in the ext2 mutant (viii). The boxed area in the lower left of (H, H’) is taken from a different focal plane in the stack to show the anterior (utricular) macula and otolithic membrane. All images are maximum intensity projections (MIPs) of between 3 and 10 selected z-slices, with the exception of the images at 40 hpf, which are single z-slices. All images are lateral views with anterior to left and dorsal to top (orientation shown in (A): A, anterior; D, dorsal). Scale bars, 50 µm. Abbreviation: CS, chondroitin sulphate.
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