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

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.

The phenotype of dkc1elu1/elu1 larvae recapitulates the human phenotype. (A) Histological analysis of dkc1elu1/elu1 mutant larvae shows microphthalmia and cataracts. Both the eyes and the optic tectum of the mutants are abnormal and contain a high prevalence of cells with neuroepithelial character. Expression of cell-cycle markers ccnD1 and PH3 in the retinae and the tecta of 2-dpf and 3-dpf larvae, respectively, can be observed throughout these tissues instead of being restricted to the proliferative regions of the ciliary marginal zone and the mediolateral edges, suggesting a defective cell cycle. (All pictures show coronal sections.) (B) Further histological analysis shows 1) deformed semicircular canals, 2) undifferentiated gut, and 3) hypoplastic pronephros with a reduced number of Wt1-positive podocytes in the mutant animals. (Scale bars: 10 μm.) (C) When Dkc1 function is abrogated in Tg(foxd3:EGFP) animals using a synthetic MO oligo, parapineal migration is impaired, and the pineal–parapineal complex appears immature at 3 dpf. (White arrows denote the parapineal.) (D) Markers of tissue differentiation demonstrate a lack of differentiation in the intestines (ifbp), pancreas (try), and the major blood lineages (gata1 and rag1). (Black arrows denote area of expression.) (E) Injection of 1) human WT DKC1 mRNA resulted in phenotypic rescue of the mutant larvae, as shown by the genotyping of larvae showing a WT phenotype. In contrast, injection of 2) human Glu206Lys DKC1 mRNA elicited a much milder rescue, demonstrating the hypomorphic nature of this allele.

Intestinal inflammation induced by soybean meal alters intestinal physiology and is independent of the presence of microbiota. (A,B) Lateral view of the mid-intestine of 9 dpf larvae showing the diffusion of dextran in control (A) and inflamed (B) larvae. Scale bar, 200 um. (C) Normalized dextran fluorescence quantification in the trunk of control and inflamed larvae. (D) Relative mRNA expression of several tight junction proteins. All data was normalized against rpl13a and compared to the control condition (dotted line). (E–L) Transversal cryosection of the midintestine of 9 dpf control and inflamed larvae. (E–J) Immunofluorescence labeling the brush border (E,F), mucus (G,H), and Claudin 15 (I,J); nuclei were stained with DAPI (blue). (K,L) Merge of the four channels in control and inflamed larvae. Scale bar, 5 um. (M,N) Quantification of brush border interruptions (white arrowheads in E,F) and goblet cells (white asterisks in G and H). (O,P) Representative images of the area of the midintestine showing HRP absorption in control and inflamed larvae. Scale bar, 100 um. (Q) Quantification of the area covered by HRP in the midintestine of control and inflamed larvae. (R,S) Representative images showing the number of cell that proliferate during 16 h in control and inflamed larvae. Scale bar, 100 um. (T) Quantification of EdU+ cells in the midintestine per larva. (U–X) Lateral view of the midintestine showing immunohistochemistry against the neutrophil marker Mpx on those conventionally raised. The area quantified is delimited by the red dotted rectangle (U,V) and germ-free larvae (W,X). Scale bar, 100 um. (Y) Quantification of the amount of neutrophils present in the midintestine in conventionally raised and germ-free larvae. Permeability, immunofluorescence, protein absorption, proliferation assay, and immunohistochemistry were performed at least in three biological replicates with 15 larvae of 9 dpf per condition. Statistical analysis was performed using the Mann–Whitney U-test, ***p < 0.001, ****p > 0.0001. RT-qPCR was performed at least in three biological replicates with 100 intestines from 9 dpf larvae per condition. Statistical analysis was analyzed using one-way ANOVA and Tukey multiple comparison test. ns, non-significant, *p < 0.05, **p < 0.01, and ****p < 0.0001.

Hepatic accumulation of Chk1 and Wee1 in capn3bΔ19Δ14 at 3dpH. a Elevation of p53 protein level in capn3bΔ19Δ14 after PH. Western blot analysis of p53 at different time point after PH as indicated. Total proteins were extracted from the liver tissues of sham, 3dpH, 5dpH, 7dpH, 10dpH and 14dpH, respectively. GAPDH, loading control. b Western blot of Capn3b and Chk1 in WT and capn3bΔ19Δ14 mutant embryos at 3.5dpf and 5dpf grown at 30 °C and 34.5 °C, respectively. The embryos were shifted to 34.5 °C at 12hpf till the time of harvesting. β-Actin: loading control. c Western blot of Chk1 in WT and capn3bΔ19Δ14 sham and PH groups at 3dpH group. Fibrillarin: loading control. d Co-immunostaining of Wee1 and Fibrillarin in the liver of WT and capn3bΔ19Δ14 mutant fish at 3dpH. DAPI: staining nuclei

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.

ARF expressed under control of the endogenous human ARF promoter in ARF:ARF fish suppresses cardiac regeneration over time. (a) AFOG and troponin staining of WT and ARF:ARF heart cryosections at 1, 7, 15, and 30 dpi. The dotted lines demarcate the injury site. Red stains are for fibrin. Blue stains are for collagen. Brown stains are for muscle. Green stains are for troponin. (b) Myocardial recovery measured by troponin infiltration into the injury site. Infiltration quantified by imaging software shows decreased troponin in the injury site of ARF:ARF fish compared to WT fish from 7 dpi onward. N = 49 hearts. The grey and black lines are logarithmic approximations of the WT and ARF:ARF heart recovery trends respectively. Results are shown as mean ± standard error. The * represents a statistically significant difference.

Chk1 and Wee1 are substrates of the Def-Capn3b complex. a Diagram showing the Capn3 recognition motif (left panel) and the locations of this motif in zebrafish, human and mouse Chk1 (middle panel) and Wee1 (right panel) proteins. b, c Western blot of Def, HA-p53R143H, HA-Chk1 and HA-Wee1S44A in WT embryos injected with HA-p53R143H, HA-Chk1 or HA-Wee1S44A mRNA combined with or without def mRNA (b). Western blot of HA-Chk1 and HA-Chk1Δ in WT embryos injected with HA-Chek1or HA-Chek1Δ mRNA combined with or without def mRNA (c). Total proteins were extracted at 8-h post-injection. β-Actin: loading control. d Western blot of the endogenous Chk1 and Wee1 proteins in WT and def−/− mutant embryos at 5dpf. Tubulin: loading control. e, f Co-immunostaining of Chk1 (e) or Wee1 (F) with Fibrillarin in the liver of WT with def−/− at 5dpf. DAPI: stain nuclei

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