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.

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.

WISH analysis of the opioid nature of the β-casomorphins. (A) WT zebrafish embryos were pre-treated with 10 μM naloxone for 2 h to block the μ-opioid receptors and exposed to naloxone and 50 µg/ml of bBCM-5, bBCM-7, hBCM-5 and hBCM-7 every 24 h from 3–6 dpf. (Black arrowheads represent the insulin domain of expression). (B) Statistical analysis of the 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, -7 exposure and significantly increased following hBCM-5, -7 exposure. WT embryos treated with µ-opioid receptor antagonist, naloxone and bBCM-5, -7 and hBCM-5, -7 showed no changes in the insulin domain of expression compared to untreated controls. Data represent mean±s.e.m.; *P<0.05, **P<0.005; Student's t-test. (n=20).

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

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.

Treating the arl13b mutants with lithium mitigates the morphological defects in the cerebellum. A–D Representative images of embryos treated with 50 mmol/L LiCl at 30–37 hpf, fixed at 3 dpf, and immunostained with anti-tubulin antibody to reveal cerebellar morphology. Treatment of wild-type embryos with Li+ does not affect the cerebellar morphology (A, B) (arrows). The morphological defects of the cerebellum in arl13b mutants treated with Li+ are partially rescued (C, D) (arrow). A–D, Scale bar 100 μm. E Statistics revealing that the proportion of arl13b mutant embryos with cerebellar defects is dramatically decreased by LiCl treatment. F–I’’ Representative images of Tg(neurod1:EGFP) transgenic embryos used to label granule cells. Treating wild-type transgenic embryos with Li+ causes no defect (F–G’’) (dashed ovals). Treating arl13b morphant transgenic embryos restores the dorsomedial cluster of granule cells (H–I’’) (dashed ovals). F–I’’, Scale bar 100 μm. J The proportion of arl13b morphant embryos with cerebellar defects in the dorsomedial clusters is dramatically reduced by LiCl treatment.

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.

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).

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