Fig. S2

CRISPR/Cas9 mediated nlz1 knockout alters heart laterality and motile cilia formation (A) Wild-type embryo at 48h. After putative F0 founders (3 male and 3 female) were bred in one tank, the F1 nlz1 mutants (B, n=20/150) had bent tail, hydrocephaly, ocular coloboma, and three otoliths in the otic vesicle. (C) in situ hybridization using cmlc2 ribo probe showed different heart looping ( normal looping, reverse looping, no looing) phenotypes in zebrafish embryos at 48h. Bar graph represent distribution of different heart looping phenotypes in wild-type and in nlz1^-/- mutant embryos. (D) Motile cilia in the KV were labelled with acetylated -α-tubulin antibody (green), nuclei were counterstained with DAPI (blue). In nlz1^-/-mutant embryos (n=5/5) the number of KV cilia were dramatically reduced compared to nlz1^+/+ wild-type embryos (n=10/10). (E) Sequences of the nlz1 target region in eight F1 putative mutant progeny. All sequences had indels close to nlz1 target region (highlighted in yellow), and 5 different nlz1 mutant alleles were recovered. The sequence variations are indicated in the right (+ insertion, - deletion), the wild-type reference allele sequence is underlined, and the protospacer- adjacent motif (PAM) is highlighted in blue. (A,B) Lateral view, anterior to left; (C) anterior view; (D) flat mount of posterior region; a, atrium; v, ventricle; hy, hydrocephaly; 1 ov, otic vesicle;KV, Kupffer’s Vesicle; scale bar 100 µm.