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.

In situ hybridization of adamts9 (A–D) Lightly-stained staged series from WT fish showing adamts9 expression in the head, cloaca, and at the tips of growing fins rays. (E–G) Extended staining at 13 dpf highlights specific adamts9 expression in the head in bilateral strips in the brain (red arrows; E, F), staining of the melanocytes (yellow arrows), in the ciliated marginal zone of the eyes (F) and in the lateral line (G).

Wnt1 is selectively down-regulated in the cerebellum of arl13b mutants. wnt1 is transiently expressed in the cerebellum of WT embryos (A, A’) (arrows) while it is dramatically reduced in arl13b mutants (B, B’). A and B, dorsal view; A’ and B’, lateral view. Note that the expression of wnt1 is selectively decreased in the cerebellum while its expression at the midbrain–hindbrain boundary is intact. A–B’, Scale bar 100 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).

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.

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

Oxamflatin and salermide have dose-dependent effects, independent of dystrophin expression. a Graph of average normalized pixel intensities for treatments of dmd mutants with doses of oxamflatin and salermide. Control treatment is 1% DMSO. Chemicals were used between 0.5 μM and 4 μM, over two separate experiments. For each treatment condition, n = 4 replicates, with 2-11 dmd−/− embryos in each replicate. Plot shows the average normalized pixel intensity for each of the 4 replicate pools for each treatment. The vertical line separates the treatment conditions from the two experiments, each of which has its own WT + DMSO and dmd + DMSO controls. The dashed lines represent the average normalized pixel intensity for all of the DMSO-treated dmd animals (n = 26 and n = 30). Error bars represent standard error. Significance was determined using a one-way ANOVA test comparing each treatment group to the dmd DMSO control group with Dunnett’s correction for multiple comparisons. *p ≤ 0.029, **p = 0.0029, ***p = 0.0007 compared to dmd DMSO control. b-e Confocal images of anti-dystrophin staining in the trunk musculature of 4 dpf b WT + DMSO, c WT + oxamflatin and salermide, d dmd + DMSO, and e dmd + oxamflatin and salermide larvae. Lateral views, anterior to the left. Arrow points to dystrophin expression in the vertical myoseptum. All dmd+/+ animals showed normal dystrophin expression (WT + DMSO, n = 16; WT + ox+sal, n = 14) and all dmd−/− animals lacked detectable dystrophin expression (dmd−/− + DMSO, n = 23; dmd−/− + ox+sal, n = 18). Scale bar = 50 μm. f-i Confocal images of anti-β-dystroglycan (βDG) and phalloidin staining in the trunk musculature of 4 dpf f WT + DMSO, g WT + oxamflatin and salermide, h dmd + DMSO, i dmd + oxamflatin and salermide. Lateral views, anterior to the left. Arrow points to βDG expression (white) in the vertical myoseptum. Phalloidin staining of filamentous actin (magenta) shows the disrupted muscle structure in dmd mutants (* in h). All wild type animals (+/+ and +/−) showed normal β-dystroglycan expression (WT + DMSO, n = 27; WT + ox+sal, n = 26), and dmd−/− animals showed largely maintained β-dystroglycan expression (dmd−/− + DMSO, n = 9; dmd−/− + ox+sal, n = 14). Scale bar = 50 μm

Valve morphology and hemodynamics are compromised in llgl1 mutants. (A) Representative images from videos acquired using light microscopy of 99 hpf hearts. A heart from an llgl1−/− embryo with slight pericardial effusion is displayed in the middle, whereas on the right, a heart with severe pericardial effusion is depicted. Above: blood flow was analyzed during diastasis using the particle image velocimetry in Fiji. Arrow vectors show directionality distance moved in pixels. Below: particle image velocimetry (PIV) data were analyzed using the PIV analyzer function in Fiji and qualified for anterograde or retrograde flow. Quantification of anterograde versus retrograde area within the heart chambers during diastasis is depicted in the graph (right). n=4 for llgl1+/+ embryos and n=5 for llgl1−/− embryos. (B) Blood flow velocity measurements analyzed from videos. n=10 for untreated embryos and n=5 for llgl1 morpholino-treated embryos. (C) Representative images from confocal z-stack micrographs of hearts of 2 dpf embryos carrying the tp1 transgenic Notch reporter. The area of cardiac GFP expression is depicted in the graph. n=9 for untreated embryos and n=12 for llgl1 morpholino-treated embryos. (D) Representative images of in situ hybridization of valve markers in 48 hpf fish. Ventral view focusing on the heart (red dashed line). Color model depicts the ventricle in red, atrium in blue, AVC in yellow and venous pole in purple. Two-tailed unpaired Student's t-test. Error bars indicate s.e.m. *P<0.05, **P<0.01, ***P<0.001. Scale bars: 100 µm.

(A) Plot of the moderated log2 fold change (mondoa-mo vs. mondoa-mis injected embryos) over averaged normalized counts. The most downregulated gene nsdhl is indicated. (B) The main steps in the conversion of acetyl coenzyme A (acetyl-CoA) to cholesterol and pregnenolone. (C, D) Transcript levels of nsdhl (C) and ef1a (D) of embryos injected with water (white bars) as a control or with the glucose analog 2-deoxy-D-glucose (2-DG; black bars) in the presence (+) or absence (-) of mondoa and mlx mRNA (n = 9). (E) Embryos injected with a splice-site MO against nsdhl (nsdhl-mo) show a severe developmental delay and arrest at ~50% epiboly, when uninjected controls were at bud stage (~10 hpf). (F) Quantification of the experiments in (E). Percentages of ‘affected’ embryos: uninjected (0/84, 0%), nsdhl-mo injected (n = 136/141, 96.5%). (G) Injection of nsdhl-mo into 1 k cell stage embryos led to arrest at 95% epiboly (n = 11/11, 100%), when uninjected control embryos (n = 15/15, 100%) were at bud stage (10 hpf). (H,I) Percentage of epiboly progression under different treatments determined via no tail (ntl) WISH. (H) Uninjected control embryos (n = 30) were fixed at the 75% epiboly stage along with the treated embryos. Treatment with 20 µM pregnenolone (P5) led to a partial rescue of the mondoa morphants (n = 19), which achieved 48% epiboly (untreated morphants: 38% epiboly; n = 25). Control and mondoa-mo morphant results are reproduced from Figure 2E, as these experiments were carried out in parallel. (I) P5 treatment also led to a partial rescue of the nsdhl morphants (n = 30, 74.6% epiboly compared to 59.0% in untreated morphants, n = 50). Asterisks label the blastoderm margin. Scale bar: 0.2 mm. Error bars represent SEM; *, p≤0.05; **, p≤0.01; ***, p≤0.001.