Midbrain-hindbrain boundary formation shown by pax2a expression is dysregulated by VitE deficiency. Pax2a expression in early optic stalk (os), midbrain-hindbrain boundary (mhb) and otic placode (op) in 12 hpf embryos, lateral views (A, B); arrow indicates dorsal region. At 12 hpf, E+ embryos (C) had defined mhb and op borders (n = 8/8), while E− embryos (D) had diffuse mhb and op borders (n = 8/12 assessed). Shown are representative E+ embryo with mhb 25 μm wide and op 49 μm apart, while representative E− embryo measurements were mhb 42 μm wide and op 63 μm apart. At 24 hpf, in E+ (E) and E− embryos (F) pax2a was expressed in the os, mhb, otic vesicles (ov) and spinal cord neurons (sn). Distance between os and mhb, a measure of first brain ventricle inflation, were greater in a representative E+ (91 μm) relative to an E− (80 μm) embryo. E+ embryo (E) spinal cord neurons at the same fluorescence exposure had significantly increased pax2a signal (n = 9/9), as compared with E− embryos (n = 6/9). Scale bar represents 100 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.

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.

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.

Knockdown of arl13b impairs posture, locomotion, and cerebellar morphology in zebrafish larvae. A Wild-type sibling larvae remain vertically oriented at 5 days post-fertilization (dpf), with both eyes visible from a top view (arrows). B In contrast, larvae injected with arl13b MO (subthreshold dose, 7.3 ng) often lie on their side at the bottom of the dish, with only one eye visible (arrows). Note that the body of the subthreshold-dose arl13b morphants are relatively straight and only slightly curved. C Statistics of the posture of zebrafish larvae at 5 dpf. D Wild-type larvae perform stereotyped spontaneous swimming with small bending angles. E The arl13b morphants (subthreshold dose, 7.3 ng) swim slower and exhibit greater bending angles. F–H Immunostaining with acetylated tubulin antibody outlines the cerebellum of larvae at 3 dpf. Comparing the dorsal view of wild-type embryos (F) with arl13b mutants (G) reveals morphological defects of the cerebellum (arrows). The cerebellar defects were also present in embryos injected with arl13b MO (H) (cb, cerebellum). I Statistics of the embryos with morphological defects of the cerebellum. The number of embryos examined in each condition is indicated above each column. A–E, Scale bar 1 cm. F–H, Scale bar 100 μm.

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

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.

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.

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).

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.