Functional validation of candidate genes in zebrafish. a Phenotype of controls and morphants. Single genes and combinations of genes targeted by splicing morpholinos are indicated on the left. Upper panels: gross appearance of 48 hpf zebrafish embryos. Lower panel: examples of cardiac phenotypes of 48 hpf wt, control, and morphant embryos. Hearts were visualized by ISH with a probe against the cardiac marker myl7. b Quantification of phenotypes in wt, controls, morphants, and mRNA rescued morphants. Note the combinatorial effects on cardiac phenotypes when more than one gene is affected. c, d Expression of mef2cb in 10 somite stage zebrafish embryos, analyzed by qRT-PCR (c) and ISH (d). ISH staining intensity was quantified and analyzed using Student’s T test. *p < 0.05, **p < 0.01

Knockdown of mondoa affects microtubule stability.(A) Confocal image of the blastoderm margin of an uninjected embryo at sphere stage; microtubules are stained with a mouse α-tubulin antibody followed by an anti-mouse Alexa Fluor 488-coupled antibody. DEL; deep cell layer; MT, microtubules; MTOC, microtubule organization center; YCL, yolk cytoplasmic layer; YSL, yolk syncytial layer; YSN, yolk syncytial layer nuclei. (B) Quantification of effects on YSL microtubule organization in uninjected (n = 9), mondoa-mo injected (n = 16), and mondoa-mo injected + pregnenolone (P5) treated (n = 9) embryos. Presence of stellar structures as shown in (F) = ‘affected’, normal appearance as in (A) = ‘unaffected’. (C–H) Overview (C, E, G) and close-up (D, F, H) images of uninjected embryos (C, D), mondoa-mo morphants (E, F) and P5 treated mondoa-mo morphants (G, H). Arrowheads indicate long parallel MT in uninjected and P5 rescued embryos (D, H) and short MT asters in untreated morphants (F). Scale bar: 90 µm; for higher magnifications, 30 µm.

Activation of Hif1a signaling impairs biliary morphogenesis. (A) Epifluorescence images showing PED6 accumulation in the gallbladder (arrows). (B) Confocal projection images showing BODIPY C5 staining (green) and Tp1:mCherry-CAAX expression (red; BEC membrane) in the liver (dotted lines). Scale bars: 200 (A), 50 μm (B).

Disruption of arl13b impairs the development of cerebellar granule cells. A Expression of the granule cell progenitor marker, zic1, in the cerebellum of wild-type embryos at 48 hpf revealed by in situ hybridization. B, C Expression of zic1 is reduced in the URL of both arl13b mutant and morphant embryos compared with wild-type embryos. Note that zic1 expression is severely reduced in the dorsomedial subregions of the URL (arrows). D The expression of the differentiated granule cell marker, reelin, in the cerebellum of wild-type embryos at 3 dpf. E, F Expression of reelin is reduced in the cerebellum of both arl13b mutant and morphant embryos. Note that in some embryos reelin expression is almost absent in the dorsal medial subregions of the cerebellum (colored ovals). G–G’’ In Tg(neurod1:eGFP) transgenic embryos, GFP+ granule cells are grouped into three clusters, the dorsomedial (dashed ovals), dorsoposterior, and ventrolateral subdivisions. H–H’’ The pattern of GFP+ granule cells is dramatically altered in the cerebellum, and particularly in the dorsomedial cerebellar subdivisions (dashed ovals) are severely affected in arl13b morphants. The parallel fibers connecting the two hemispheres are disrupted in arl13b morphants. Dorsal views of the embryos are shown. I–L Malformations of the dorsomedial cerebellar subdivisions (dashed ovals) and parallel fibers are also present in arl13b mutants both at 3 dpf and 4 dpf. cb, cerebellum; PF, parallel fiber. A–F, Scale bar 100 μm. G–L, Scale bar 50 μm.

The expression of markers of cerebellar granule cell progenitors is impaired in arl13b-deficient embryos. A–I’ Representative images of in situ hybridization illustrate that the three paralogues of atoh1 (atoh1a, 1b and 1c) are expressed in distinct populations of cerebellar granule cell progenitors. atoh1a is expressed in the URL and LRL. Similar expression patterns of atoh1a occur in wild-type embryos (A) and arl13b mutants (B) while its expression is absent from the oral dorsomedial URL (dashed box) in arl13b morphants (C) at 36 hpf and 48 hpf (A’–C’). The expression patterns of atoh1b remained unaffected in arl13b mutants and morphants (D–F’). The expression level of atoh1c was decreased in the URL in arl13b morphants (I and I’) (cb, cerebellum; URL, upper rhombic lip; LRL, lower rhombic lip). A–I’, Scale bar 100 μm.

Ttpa signal localized throughout early embryo and brain ventricle borders regardless of VitE status. Ttpa expression in E+ and E− embryos is indicated with red fluorescence; dorsal direction is indicated by arrow (A–D). At 6 hpf (dorsal shield stage, A, B), Ttpa expression was present throughout the animal poles [E+ embryos, n = 5/5 (n = number of animals with the observed defect/total number of animals observed)], E− embryos, n = 6/6). At 12 hpf (90% epiboly, C, D), ttpa expression was present both in the embryo and in the yolk syncytial layer (E+ embryos, n = 6/6; E− embryos, n = 7/8 ), arrow indicates anterior region of the embryo. At 24 hpf (E, F), Ttpa expression was localized in the brain ventricle borders and within cells of the fore (f), mid- (m), and hindbrain (h). Arrows indicate the midbrain-hindbrain boundary where diencephalic ventricle expansion was altered; *Represent inflation in E+ embryos (E, n = 6/6) or lack thereof in E− embryos (F, n = 3/7). Scale bar represents 500 μm (A–D) and 50 μm (E, F); representative embryos are shown. Figure panels were generated with the BZ- × 700 microscope, processed with BZ-X Analyzer Software with image adjustments equally applied across time points in Adobe Photoshop v21.2.1.

Gastrulation marker goosecoid (gsc) is not affected by VitE deficiency. Gsc expression at 6 hpf in the neuroectoderm of the dorsal embryonic shield, as indicated by red fluorescence in (A) E+ embryos (n = 9/9) and (B) E− embryos (n = 8/8). Arrow indicates dorsal region of the embryo. Scale bar represents 500 μm; representative embryos are shown. Figure panels were generated with the BZ- × 700 microscope, processed with BZ-X Analyzer Software with image adjustments equally applied across time points in Adobe Photoshop v21.2.1.

Llgl1 promotes appropriate Yap protein levels in cardiomyocytes, and exogenous expression of Yap in cardiomyocytes ameliorates deleterious cardiac physiology associated with loss of Llgl1. (A) Representative images of 2 dpf zebrafish ventricles from single plane confocal micrographs (left). Boxes indicate area of enlargement. Arrows depict Yap within the cardiomyocytes. Quantification of the intensity of anti-Yap signature in tropomyosin+ cells normalized to the control morpholino group (right). n=8 for untreated embryos and n=15 for llgl1 morpholino-treated embryos. (B) qRT-PCR transcript analysis for llgl1, llgl2 and genes regulated by Yap activity in llgl1+/+ or llgl1−/− 3 dpf zebrafish hearts. n=10 for each group. (C) Representative images of 3 dpf embryos microinjected with llgl1 morpholino with or without Tol2-mediated integration of myl7:yap-myc (left). Arrows depict the region measured for pericardial effusion. Quantification of pericardial effusion in 3 dpf embryos (right). n=6 for uninjected control group, n=6 for the myl7:yap-myc control group, n=7 for llgl1 morphants and n=7 for llgl1 morphants with myl7:yap-myc expression. (D) Quantification of blood flow velocity in 2 dpf embryos. n=8 for the uninjected control group, n=5 for the transposase only control group, n=5 for the myl7:yap-myc control group, n=13 for llgl1 morphants and n=9 for llgl1 morphants with myl7:yap-myc expression. Two-tailed unpaired Student's t test (A,B) and One-way ANOVA, Tukey's multiple comparisons test (C,D). Error bars indicate s.e.m. *P<0.05, **P<0.01, ***P<0.001,****P<0.0001. Scale bars: 50 µm (A); 1 mm (C).

Disruption of arl13b reduces both precursor and differentiated cerebellar Purkinje cells. A–I Representative images of in situ hybridization using anti-sense probes against ptf1a and roraa to label Purkinje precursors and differentiated cells, respectively. The dorsomedial clusters of precursor of Purkinje cells and differentiated Purkinje neurons are selectively reduced (arrows). J–L’’ Immunostaining of mature Purkinje neurons using anti-parvalbumin antibody reveals that the dorsomedial Purkinje neurons are reduced in arl13b-deficient embryos. A–I, Scale bar 100 μm. J–L’’ Scale bar 50 μm.