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

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).

ue58 mutant zebrafish have a severe, peripheral nerve myelin pathology. (A) Confocal images of the spinal cord of Tg(mbp:EGFP-CAAX) control (left) and ue58 mutant (right) at 7 dpf showing disruption to CNS myelin (region within brackets). Scale bar, 10 µm. (B) Confocal images of the pLLn in Tg(mbp:EGFP-CAAX) control (top) and ue58 mutant (bottom) animals at 5 dpf showing major disruption to myelin. Scale bar, 10 µm. (C) Higher magnification images of areas demarcated in B showing myelin in control (left) and ue58 mutant (right) animals. Scale bar, 10 µm. (D) DIC images of Tg(mbp:EGFP-CAAX) control (left) and ue58 mutants (right) at 6 dpf showing appearance of tissue edema. Scale bar, 10 µm. (E) Brightfield images of control (left) and ue58 mutants (right) at 6 dpf showing generally normal morphological development. Scale bar, 0.5 mm. (F) Genomic structure of the zebrafish slc12a2b gene, showing exons (boxes) and introns (lines). White boxes denote untranslated regions. Exons in black were annotated in partial genomic sequences LOC100537771 and LOC100329477 and matched homologous exons in the orthologue slc12a2a. Exons in blue did not align with any annotated genomic sequence, and their limits were inferred by homology with slc12a2a genomic structure. Exons are drawn to scale relative to each other; introns in pink contain unknown bases (N) and are of unknown size. The start (ATG) and stop (TGA) codons are indicated in green and red, respectively. The ue58 allele has a T>A mutation in exon 26 leading to a premature stop codon. (G) Alignment of the 40 most C-terminal amino acids of NKCC1b shows high similarity between species in this domain. Arrowhead indicates the position of the premature stop codon introduced by ue58. (H) Protein structural prediction algorithms, using CCTOP, indicate that NKCC1b in zebrafish is likely to have intracellular N and C termini and 12 transmembrane domains. (I) Sequence similarities of the protein products of zebrafish NKCC1a, NKCC1b, and murine and human NKCC1 homologues.

WISHto analyse the effect of chronic β-casomorphin exposure on insulin domain of expression. (A) Insulin domain of exposure (black arrowheads) in WT zebrafish embryos exposed to 50 µg/ml of bBCM-5, bBCM-7, hBCM-5 and hBCM-7 from 3–6 dpf. (B) Statistical analysis of insulin domain of expression using Student's t-test. Fold change (compared to untreated control zebrafish embryos) shows insulin domain of expression significantly decreased following bBCM-5 or -7 exposure and significantly increased following hBCM-5 or -7 exposure. Data represent mean±s.e.m.; *P<0.05, **P<0.005; Student's t-test (n=20).

Defective development of capn3b mutant zebrafish under environment stress. a, b WISH using the fabp10a probe on 3dpf- and 5dpf-old WT and capn3bΔ19Δ14 embryos grown at the low (60 embryo/dish) (left panel) and high (120cm2/dish) (right panel) rearing density in a 9 cm-diameter dish (a). In (b), the data of liver sizes were compared between 3dpf- and 5dpf-old WT or between 3dpf- and 5dpf-old capn3bΔ19Δ14 mutant embryos under low (L) and high (H) rearing intensity. c WISH using the fabp10a and trypsin probes on WT and capn3bΔ19Δ14 embryos at 2dpf and 3dpf. Embryos were shifted to 34.5 °C at 12 hpf till the time of sample harvesting. Numerator/denominator: number of embryos displayed the shown phenotype over total number of genotyped embryos. d Photo images showing the curved body phenotype displayed by the 3dpf-old capn3bΔ19Δ14 mutant but not WT embryos grown at 34.5 °C. e Body lengths of 6-, 8- and 12-months-old WT and capn3bΔ19Δ14 fish. f LBR of 6- and 12-months-old WT and capn3bΔ19Δ14 fish. Student’s T-test for statistical analyses, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001

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).

Figure 1. Excessive exercise leads to pathological cardiac hypertrophy. (A,B) Representative images from control (A) and excessively-exercised zebrafish hearts (B) after 4 weeks of static and excessive-exercise treatment. (C–F) Representative pictures of Masson’s trichrome-stained ventricular tissues of control (C,D) and excessively-exercised zebrafish hearts (E,F). Blue area indicates fibrosis. (D,F) Are enlargements of the rectangle area in (C,E), respectively. Scale bar = 100 μm. (G) Quantitative analysis of Masson staining positive myocardial fibrosis area using Image J. ∗p < 0.05 by unpaired Student’s t-test. n = 3. (H,I) TEM images of control (H) and excessively exercised heart tissue (I). Scale bar = 5 μm. (J) RT-qPCR analysis of the expression of mitochondrial functional markers. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student’s t-test. (K) Echocardiographic analysis of zebrafish after excessive exercise and in the static control (LVPW, LV posterior wall thickness). Error bars indicate SEM. n = 4. (L) The maximal oxygen consumption (MO2) of zebrafish after excessive exercise and in the control group was analyzed. n = 8. (M) RT-qPCR analysis of the expression of pathologic and physiological hypotrophy-related marker genes. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student’s t-test.

Neuronal activity drives peripheral nerve pathology in slc12a2b mutants. (A) Schematic overview of when, where and for how long TTX was applied to slc12a2bue58 mutants. (B) Confocal images of a Tg(mbp:EGFP-CAAX) control (top), slc12a2bue58 mutant (middle), and slc12a2bue58 mutant injected with TTX (bottom). Scale bar, 20 µm. (C) Quantitation of mean myelinated nerve diameter in controls, slc12a2bue58 mutants and slc12a2bue58 mutants injected with TTX (control 7.1 ± 0.5 µm vs. slc12a2bue58 13.4 ± 1.9 µm vs. slc12a2bue58+ TTX 6.1 ± 0.9 µm). Error bars represent mean ± SD. One-way ANOVA followed by Tukey’s multiple comparison test was used to assess statistical significance (ANOVA F(2,27) = 97, P < 0.0001). Each point represents an individual animal. ****, P < 0.0001. (D) Schematic overview of when, where, and for how long TTX was applied to either constitutive or glial-specific slc12a2b mutants. (E) Confocal images of 6 dpf slc12a2bue58 mutant larvae. Top and bottom panels show the same region of the pLLn before and 4–6 h after injection with either a control solution (left), or TTX (right). Scale bar, 20 µm. (F) Confocal images of 6 dpf Tg(mbp:EGFP-CAAX) larvae, in which slc12a2b has been targeted in myelinating glial cells. Top and bottom panels show the same region of the pLLn before and 4–6 h after injection with either a control solution (left) or TTX (right). Scale bar, 20 µm. (G) Quantitation of the change in mean myelinated nerve diameter following injection with either a control solution or TTX in slc12a2b mutants (G; sham injected −0.6 ± 1.1 µm vs. TTX −3.2 ± 1.6 µm, P < 0.0001) or animals in which slc12a2b has been disrupted specifically in myelinating glial cells (H; sham injected −0.4 ± 1 µm vs. TTX −2.5 ± 1.7 µm, P < 0.0001). Two-tailed Student’s t test was used to assess statistical significance. Each point represents an individual animal. ****, P < 0.0001.

Neuropathology of Atp13a2 deficient zebrafish. (A) Axial sections of the posterior tuberculum containing dopaminergic (DA) neurons of zebrafish brain at 4 and 12 months. Right figures in each section are the enlarged images of the left figures. (B) Axial sections of the locus coeruleus containing norepinephrine (NE) neurons of zebrafish brain at 4 and 12 months. Right figures in each section are enlarged images of the left figures. (C) The number of TH + neurons in the posterior tuberculum and the locus coeruleus. (D) The amount of cathepsin D protein in zebrafish brain. WT indicates wild type and KO indicates Atp13a2 deficient zebrafish. (E) Transmission electron microscopy analysis of the middle diencephalon samples from Atp13a2 deficient zebrafish. Atp13a2 deficiency induces lysosome-like bodies that contain granular deposits (arrowheads) and fingerprint-like structures (arrows). Right figures in each section are the enlarged images of the left figures. Scale bars are indicated in A, B and E. *p < 0.05 and ***p < 0.001.