Zebrafish modelling of the chromosome 4 CNV showing disruption in eye and midbrain development.A Comparative images of the eye in wild-type (mab21l2wt/wt), lrba homozygous knockout fish (lrbamw716/mw716), fish with a homozygous deletion of the CNV identified in Individual III.5 (Family 2) (mab21l2mw715/mw715) and fish compound heterozygous for the deletion and a p.Gln48Serfs*5 frameshift allele in mab21l2 (mab21l2mw715/mw702). Red arrows: misshapen eyecups with a visible gap between the neuroectodermal layers of the optic vesicle (ventral coloboma); white arrows: optic tectum; blue arrows: gap resulting from the reduced size of optic tectum; asterisks: small lens; R: retina; L: lens; OT: optic tectum. B, C Histogram of the lens diameter (B) and eye width (proximal to distal) (C) of wild-type and homozygous mab21l2mw715 fish at 24hpf (n = 17 and 18, correspondingly), 48hpf (n = 22 and 30) and 72hpf (n = 29 and 30). Lens diameter was significantly reduced in mutant fish at 24hpf (p < 0.000001) and 48hpf (p = 0.001282), but not at 72hpf (p = 0.121573). Eye width was subtly decreased in mutant fish at 72hpf (p = 0.028665), but not at 24hpf (p = 0.520299) or 48hpf (p = 0.300519). Statistical analyses: two-tailed t test for two independent samples with Welch correction for unequal variances. Dmab21l2 (yellow) and foxe3 (green) mRNA expression in whole mount wild-type and homozygous mab21l2mw715 fish. White arrows indicate the position of the midbrain (M), eye (E), and lens (L): the mutants exhibited a visibly reduced mab21l2 expression in the midbrain region, and a slightly reduced mab21l2 staining in the eyes at 20- and 24hpf; foxe3 expression (green) in the developing lens was detected at all stages, showing a smaller lens in 20- and 24hpf mutant embryos. Panels (B and C): Black bars: wild-type; greyscale bars: homozygous mab21l2mw715 embryos (24hpf: medium grey; 48hpf: grey; 72hpf: dark grey); * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Source data is provided in the Source Data file.

Cyclin D1 and Her9 control the proliferation of boundary cells. (A-D) Tg[BCP:GFP] embryos at the indicated stages were in situ hybridized with ccnd1 and cdkn1ca or her9 probes. (E,F) Tg[BCP:H2BGFP;ccnd1+/+] and Tg[BCP:H2BGFP;ccnd1pfu1/pfu1] embryos displaying boundary cells in green at 26?hpf . (G) Plot showing the number of boundary cells (87.3±16.5 in ccnd1+/+ n=4 versus 61.3±10.8 in ccnd1pfu1/pfu1 n=8; *P=0.04, Welch's test). (H-K) Tg[BCP:H2BGFP;ccnd1+/+] and Tg[BCP:H2BGFP;ccnd1pfu1/pfu1] embryos injected with control-MO (H,I) or her9-MO (J,K). (L) Plot showing the number of boundary cells in Tg[BCP:H2BGFP;ccnd1+/+] and Tg[BCP:H2BGFP;ccnd1pfu1/pfu1] embryos upon different conditions. (H) 75.7±15.9 in ccnd1+/+ control-MO n=3; (I) 44.7±19.4 in ccnd1pfu1/pfu1 control-MO n=3; (J) 46.8±7.7 in ccnd1+/+ her9-MO n=6; (K) 46.5±16.1 in ccnd1pfu1/pfu1 her9-MO n=6. H versus I, *P=0.04; H versus J, *P=0.03; H versus K, *P=0.03; I versus J, P=0.99; I versus K, P=0.99; J versus K, P>0.99. One-way ANOVA, Dunnett's multiple comparison test. Images are transverse views of the r4/r5 boundary (E,F,H-K,a-d) or dorsal maximum intensity projections of the hindbrain with anterior to the left (A-D). The plots show mean±s.d. Dotted lines delimitate the contour of the neural tube. Arrowheads indicate the position of the hindbrain boundaries. BCP, boundary cell population; hpf, hours post-fertilization; MO, morpholino; ns, not significant. Scale bars: 50?µm.

Myo1G regulates heart and brain LR asymmetry independent of the LRO flow. a, a? Quantification of cardiac jogging indicates that MZ myo1g mutants present laterality defects that are enhanced in MZ myo1d; MZ myo1g double mutants (a). Concomitant inactivation of the LRO flow (through dnaaf1 mutation) reveals that MZ myo1d/g mutations enhance the cardiac jogging defects of flow-deficient animals (a?). b Brain asymmetry is impaired in MZ myo1g single and MZ myo1d; MZ myo1g double mutants. Frontal views of pitx2 expression at 30 somites, dorsal up. c MZ myo1g mutants do not show visceral LR defects. L liver, G gut, P pancreas. Dorsal views of foxa1 expression at 48 h, anterior up. d MZ myo1d/g inactivation enhances the brain laterality phenotypes of LRO flow-deficient dnaaf1 mutants. e, f Visceral laterality phenotypes of dnaaf1 mutants are unaffected by myo1d/g inactivation. f Dorsal views of foxa1 expression at 48 h, anterior up. Pictures are derived from the data set quantified in (e). Scale bars: 50 µm. All p values were obtained using non-directional statistical tests. Complete numerical and statistical information for all experiments are provided in the Source Data files.

Red cones are transformed into hybrid red/UV cones in the larval samd7?/? retina. (A?D) Confocal images of 5-dpf WT and samd7?/? eyes show that UV opsin and UV-cone-specific opn1sw1:GFP is up-regulated in samd7?/? red cones (thrb:tdTomato+). (E) UV cones (opn1sw1:GFP+ cells per 1,600 µm2) are increased in the samd7?/? retina. The subset of UV cones that are not derived from red cones (opn1sw1:GFP+;thrb:tdTomato?) are also increased, suggesting that a small subset of supernumerary UV cones in the samd7?/? retina derive from another unknown cell type or precursor (mean ± SD; n = 4 WT, n = 6 samd7?/?). (F) Volcano plot from bulk RNA-seq comparing gene expression in isolated 5-dpf WT thrb:tdTomato+ cells (i.e., red cones) and samd7?/? thrb:tdTomato+ cells (i.e., transformed hybrid red/UV cones). UV-cone genes are up-regulated in samd7?/? hybrid red/UV cones including tbx2a, tbx2b, opn1sw1, arr3b, mir729, tgfa, and grk7b (5). Red-cone-specific changes in gene expression were also observed, including reduced expression of the thyroid-hormone-inactivating enzyme, dio3b; increased expression of the red-shifted red opsin paralog, opn1lw1 (?max = 558 nm); and decreased expression of the blue-shifted red opsin paralog, opn1lw2 (?max = 548 nm). Similar changes in red opsin paralog expression were previously observed in larval zebrafish treated with thyroid hormone (44). Red/green-cone-specific arr3a expression was absent, and exorh and samd7 expression were increased in samd7?/? hybrid red/UV cones. (Scale bar, 10 µm.) Statistical comparisons in E were performed using the two-tailed, unpaired t test assuming unequal variance. *P < 0.05 and ***P < 0.0005. SD, standard deviation.