WS patients with 7q11.23 microdeletions and RFC2 intragenic deletions. A: Five WS patients (P1–P5) with typical 7q11.23 microdeletions. Chromosomal microarray test detected 1.4 Mb–1.5 Mb microdeletions including RFC2. We also identified one patient (P6) with a smaller 167 kb deletion, comprising RFC2 but not ELN. B: Patients (P7–P10) with RFC2 intragenic microdeletions. RFC2 consists of 11 exons (1–11), spanning ∼23 kb. C: Comparison of human RFC2 (NP_852136.1) with zebrafish Rfc2 (NP_001013344.2) and human RFC5 (NP_031396.1) with zebrafish Rfc5 (NP_001003862.1). All four have an AAA domain and a Repl C domain in common. The zebrafish rfc2 encodes 353-aa protein, with a high level of similarity (90%) to the human RFC2 protein and the zebrafish rfc5 encodes 334-aa protein, with a high level of similarity (91%) to the human RFC5 protein. aa, amino-acid; Repl C, Replication factor C C-terminal domain; AAA, AAA⁺ ATPase domain.

Generation of rfc2 KO and rfc5 KO zebrafish using CRISPR-Cas9. A: Expression of rfc2 or rfc5 mRNAs in zebrafish embryos at 2 dpf. Lateral and dorsal view, anterior is to the left. Both genes are expressed in mb, mhb, hb, and eyes. B: Schematic gene structure of zebrafish rfc2 and rfc5 with KO target sites (arrows), exon 1 of rfc2 and exon 3 of rfc5. Indel mutations include 20-bp insertion (+22 bp, −2 bp) in rfc2 (named rfc2^ck179a) and 8-bp deletion in rfc5 (named rfc5^ck171a). C: Predicted protein structure of each KO mutation, resulting in truncated proteins caused by frameshift. D: Loss of rfc2 or rfc5 mRNA expression in each KO zebrafish at 2 dpf. Dorsal view. n = 12 for rfc2 WT, n = 17 for rfc2 KO, n = 12 for rfc5 WT, and n = 9 for rfc5 KO. Scale bar, 200 μm (A and D). mb, midbrain; mhb, midbrain-hindbrain boundary; hb, hindbrain; WT, wild-type; KO, knockout; dpf, days post fertilization; AAA, AAA⁺ ATPase domain; Repl C, Replication factor C C-terminal domain; pa, pharyngeal arches; in, intestine.

Reduced head and brain size in rfc2 KO and rfc5 KO zebrafish. A: Reduced head size in KO zebrafish is prominent at 5 dpf, whereas relatively comparable at 2 dpf. Eye size is also reduced in both KO zebrafish, compared to WT zebrafish at 5 dpf. n = 5 for rfc2 KO and n = 4 for rfc5 KO at 2 dpf. n = 13 for rfc2 KO and n = 25 for rfc5 KO at 5 dpf. Lateral view. B: Quantification of eye size in rfc2 KO and rfc5 KO zebrafish. The planar area of eye was measured by Image J software at 3 dpf and 5 dpf, respectively. C: Reduced brain size in rfc2 KO zebrafish at 5 dpf. Dorsal view. D: Quantification of reduced brain size (bordered area in C) in rfc2 KO zebrafish at 5 dpf. n = 18 for WT and n = 31 for KO. E: Representative fluorescent image of rfc5 KO zebrafish crossed with neuron-specific GFP transgenic line Tg(huc:egfp) at 5 dpf. Dorsal view, anterior is to the left. F: Quantification of brain size in rfc5 KO at 5 dpf. Relative brain size was obtained by multiplying the length of (a) and (b) marked in the brain image of (E). n = 7 for WT and n = 34 for KO. Data are presented as mean ± SEM. Statistical significance was determined by the Kruskal–Wallis tests with post-hoc Dunn's multiple comparisons tests. ns, not significant, P > 0.05; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001. Scale bar, 100 μm (A, C, E). dpf, days post fertilization; WT, wild-type; KO, knockout; SEM, standard error of the mean.

Increased cell death in rfc2 KO and rfc5 KO zebrafish. A: Detection of cell death by AO staining in KO zebrafish at 3 dpf. Apoptotic cells were mainly detected in midbrain, mhb, and retina. Earlier apoptotic cells were confirmed by the apoptosis marker tp53 expression at 2 dpf. Left panels are dorsal view, middle and right panels are lateral view, anterior is to the left. B: Quantification of AO-positive cells within the brain region in rfc2 KO or rfc5 KO zebrafish. n = 16 for rfc2 WT, n = 14 for rfc2 KO, n = 15 for rfc5 WT, and n = 14 for rfc5 KO. Data are presented as mean ± SEM. Statistical significance was determined by the unpaired two-tailed t-tests with Welch's correction. ∗∗∗∗, P < 0.0001. C: Visualization of RNA-seq analysis with volcano plot. Blue dots represent down-regulated, red dots for up-regulated, gray dots for not significantly changed, and purple dots for labeled genes in rfc2 KO, compared to WT. D: List of representative gene sets involved in eye development or pancreas function. Scale bar, 100 μm (A). AO, acridine orange; mhb, midbrain-hindbrain boundary; WT, wild-type; KO, knockout; SEM, standard error of the mean.

Skeletal and vascular phenotypes in rfc2 KO and rfc5 KO zebrafish. A: Defects in head cartilage formation in KO zebrafish at 5 dpf. Alcian blue staining, ventral view, anterior is to the left. B: Quantification of cartilage structures in rfc2 KO zebrafish. n = 14 for WT and n = 18 for KO. C: Defects in bone structures in KO zebrafish. Alizarin red S staining at 9 dpf. Lower panels are magnifications of upper panels, showing defects in bone and teeth development (arrows). n = 8 for rfc2 WT, n = 25 for rfc2 KO, n = 10 for rfc5 WT, and n = 23 for rfc5 KO. Ventral view, anterior is to the left. D: Vascular defects in KO zebrafish crossed with Tg(kdrl:gfp) at 7 dpf. Aortic arches are indicated by arrows. n = 9 for rfc2 WT, n = 27 for rfc2 KO, n = 14 for rfc5 WT, and n = 18 for rfc5 KO. Ventral view, anterior is to the left. E: Lateral view of aortic arches. Blood vessels were narrowed and hypo-branched in rfc2 KO, compared to WT and Het. F: Quantification of vessel diameter of the most posterior aortic arch (arrow in E) was measured using Image J software. n = 9 for WT, n = 18 for Het, and n = 9 for KO. Data are presented as mean ± SEM. Statistical significance was determined by the Kruskal–Wallis tests with post-hoc Dunn's multiple comparisons tests. ns, not significant, P > 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001. Scale bar, 100 μm (A, C, D, E). dpf, days post fertilization; AW, articulation width; PL, Palatoquadrate length; M, Meckel's cartilage; m, maxilla; o, opercle; en, entopterygoid; br, branchiostegal ray 1; cb, ceratobranchial 5; WT, wild-type; KO, knockout; Het, heterozygous; SEM, standard error of the mean.

Increased social cohesion and anxiety-like behavior in Het adult rfc2 KO zebrafish. A: Representative image of the shoaling bowl assay. Each arrow indicates a single zebrafish. B: Quantification of DM of zebrafish during shoaling bowl assay. Compared to WT siblings, rfc2 KO Het zebrafish showed a significant reduction in their movement during the test. n = 5 for WT and n = 9 for Het fish in 15 times of tests. C: Quantification of MID between three subjects. Compared to WT siblings, rfc2 KO Het tend to stay closer each other. D: Heatmap of scototaxis test. The red color indicates increased dwell time and blue indicates decreased. E: Quantification of duration time in the light zone during scototaxis test. Compared to WT siblings, rfc2 KO Het zebrafish stayed more time in the dark zone than the light zone. n = 5 for WT and n = 9 for Het fish in total 13 times of tests. Data are presented as mean ± SEM. Statistical significance was determined by the Mann Whitney U test. ns, not significant, P > 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001. MID, mean individual distance; Het, heterozygous; DM, distance moved; LZD, light zone duration; WT, wild-type; KO, knockout; Het, heterozygous; SEM, standard error of the mean.

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