Versican, a CS proteoglycan, is required for semicircular canal morphogenesis. (A,C) 3D-rendered OVs at 50 or 72 hpf from membrane-NeonGreen-expressing embryos injected with tyrosinase (left) or versican double (right) gRNA (A), and control (left) or versican double MO (right) (C). Scale bars: 50 μm. (B,D) Quantification of bud length at 50 hpf in embryos injected with tyrosinase or versican double gRNA (B), and control or versican double MO (D). Data are mean±s.e.m. In the absence of buds, bud lengths correspond to cell lengths. n denotes the number of buds from individual embryos measured from two independent experiments. P-values as labeled (unpaired, two-tailed Student's t-test). (E) Target sites for sgRNAs at the vcana or vcanb locus used to generate stable versican double-knockout mutants. (F) Brightfield images of sibling control and versican double-knockout mutant embryos at 72 hpf obtained from vcana−/+; vcanb−/−parents. The arrow indicates epicardial edema in the versican double-knockout mutant embryo. The dashed lines in the magnified images indicate the outlines of pillars or buds in the OV. Scale bars: 50 (right-hand panels); 500 μm (left-hand panels). (G) 3D-rendered OVs at 50 hpf from membrane-NeonGreen-mRNA injected embryos of sibling control (left) or versican double-knockout mutant (right). Representative images from two independent experiments are shown. Scale bar: 50 μm. (H) Quantification of bud length in embryos of sibling control or versican double knockout at 50 hpf. Data are mean±s.e.m. In the absence of buds, bud lengths correspond to cell lengths. n denotes the number of buds from individual embryos measured from two independent experiments. P-values as labeled (unpaired, two-tailed Student's t-test). a/A, anterior; al/AL; anterior-lateral; l, lateral; p/P, posterior; pl/PL, posterior-lateral; v, ventral. Representative images from two independent experiments are shown.
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