myo1d is required for Kupffer’s vesicle morphogenesis. a myo1d is ubiquitously expression. White arrow demarcates the region where KV is formed. b TALEN mediated genome editing of myo1d. TALENs were designed to target myo1d exon 2 (red arrow) encoding the myosin motor domain (blue). Calmodulin binding IQ motif (green) and Tail Homology 1 (TH1, red) domains are also indicated. Three F1 alleles were isolated, pt31a-c, with predicted amino acid sequence indicated. c–e myo1d MZ mutants showed KV lumen formation defects. Representative DIC images showing KV of myo1d MZ mutants were either smaller and dysmorphic lumens (c). ZO-1 staining at 8S confirmed that the apical surface of KVs were smaller or dysmorphic in myo1d mutants when compared to controls. myo1d MZ;Tg(dusp6:GFP-MA) injected with H2BmCherry mRNA showing dysmorphic KV epithelial cell morphology and decreased lumen area, when compared to controls. Lumen marked by dotted circular lines were quantified for area (WT: n = 44, myo1d MZ: n = 46) (d) and circularity (WT: n = 20, myo1d MZ: n = 21) (e). f–h Dysmorphic otic vesicle (OV) observed in myo1d MZ embryos. Tg(dusp6:GFP-MA) and myo1d MZ;Tg(dusp6:GFP-MA) embryos (f) or stained with aPKC (green) and acetylated tubulin (red) (g) at 24 hpf show smaller OVs and quantified (WT: n = 8, myo1d MZ: n = 12) (h). KV lumen was considered small when it was below threshold lumen size (1000 µm2). Unpaired t test was used with, ***p < 0.001, ****p < 0.0001 indicated. Data shown in the graphs are mean ± SEM. Scale bar, 50 µm
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