Induced Alternative Reading Frame (ARF) expression in hs:ARF fish suppresses cardiac regeneration. (a) Representative polymerase chain reaction (PCR) product of ARF expression from three experimental replicates of wild type (WT) and hs:ARF heart RNA on 15 dpi. The product run on gel electrophoresis shows ARF expression present in hs:ARF fish while absent in WT fish. (b) Acid Fuchsin Orange G (AFOG) and troponin staining of WT and hs:ARF heart cryosections on 15 days post-injury (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. (c) Myocardial recovery measured by troponin infiltration into the injury site. Infiltration quantified by imaging software shows decreased troponin in the injury site of hs:ARF fish compared to WT fish. N = 8 hearts. Results are shown as mean ± standard error. The * represents a statistically significant difference.

Spatial and temporal expression of zebrafish atp11b in wild-type embryos. Gene specific atp11b expression (antisense) at 0–2 hpf (4 cells) illustrating maternal deposition (A: lateral) and at 24 hpf (B: lateral and C: dorsal head) in the ventricle epithelial lining. Expression of atp11 b at 48 hpf (D and E; lateral) is ubiquitous in the head with concentrated expression in the brain tissue behind the eye and possibly within the eye itself. 72 hpf (F: lateral) and 96 hpf (G: lateral) expression is found in the ear and swim bladder. Non-specific staining (sense) is shown at the same timepoints for comparison (H–L).

Disruption to NKCC1b leads to swelling of the periaxonal space, dysmyelination, and axonal disorganization. (A) Electron micrographs of high-pressure–frozen pLLn in control (left) and slc12a2bue58 mutant (right) at 5 dpf. slc12a2bue58 mutants show significant enlargement of the periaxonal space, highlighted in blue and enlarged axons (asterisk). Insets show a higher magnification to highlight the periaxonal space in controls and slc12a2bue58 mutants. White scale bar, 1 µm. Black scale bars, 50 nm. (B) Quantification of periaxonal area in control and slc12a2bue58 mutants (control 0.05 ± 0.02 µm2 vs. slc12a2bue58 4 ± 3.5 µm2, P = 0.0065). Error bars represent mean ± SD. A two-tailed Student’s t test was used to assess statistical significance. Each point represents an individual myelinated axon from three control and five slc12a2bue58 mutant animals. **, P < 0.01. (C) Quantification of the diameter of myelinated axons in control and slc12a2bue58 mutants. Bracket indicates axons in the mutant with greater than normal diameter. (D) Confocal images of live Tg(cntn1b:mCherry), Tg(mbp:EGFP-CAAX) double-transgenic control (left) and slc12a2bue58 mutant (right) animals at 5 dpf indicates axonal defasciculation and derangement of myelin. Scale bar, 10 µm. (E) Confocal images of individual mosaically labeled Schwann cells in control (top left panel) and slc12a2bue58 mutants (panels 1–5) highlighting the variable morphological manifestation of the mutant phenotype. Scale bar, 10 µm. Arrows point to regions of normal appearing myelin and arrowheads to dysmyelination. (F) Quantitation of mean Schwann cell diameter in maximum intensity projection images of single Schwann cells at 6 dpf (control 2.6 ± 0.4 µm vs. slc12a2bue58 4.3 ± 1.3 µm, P = 0.0003). Error bars represent mean ± SD. A two-tailed Student’s t test was used to assess statistical significance. Each point represents a single cell from 11 control and 10 slc12a2bue58 mutant animals. Scale bar, 10 µm. ***, P < 0.001. (G) Quantitation of mean Schwann cell length in maximum intensity projection images of single Schwann cells at 6 dpf (control 72.1 ± 15.7 µm vs. slc12a2bue58 54.7 ± 13.8 µm, P = 0.011). Error bars represent mean ± SD. A two-tailed Student’s t test was used to assess statistical significance. Each point represents a single cell from 11 control and 10 slc12a2bue58 mutant animals. *, P < 0.05.

Wnt signaling is involved in the maintenance of ependymal motile cilia in zebrafish embryos.a Transmission electron microscopy (TEM) of the spinal cords (SCs) of zebrafish embryos at 2 dpf. Arrowhead indicates a motile cilium with the 9 + 2 microtubule configuration, which is magnified to the right. Scale bar = 1 μm. b Immunofluorescence (IF) staining of an embryo at 1 dpf with anti-acetylated-α-tubulin antibody. Dorsal view anterior to the left. Arrowheads represent motile cilia. Scale bar = 20 μm. c, d IF staining of sonic-you (syut4) mutant embryos at 2 dpf with anti-acetylated-α-tubulin antibody. Dorsal view anterior to the left (c). d The cross-section image of the SC ventral to the bottom. Arrowheads represent motile cilia. Scale bars = 20 μm. e Embryos were treated with DAPT (50 μM) at 34–48 hpf and IF stained with anti-acetylated-α-tubulin antibody. Dorsal view anterior to the left. Arrowheads represent motile cilia. Scale bar = 20 μm. f Embryos were co-microinjected with wnt4b MO and wnt11 MO (wnt4b/11 MO) alone or along with wnt4b mRNA and wnt11 mRNA (wnt4b/11 mRNA), and IF stained at 2 dpf with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. CO: Control. g Quantification of the number of cilia per frame in embryos in f. Data are presented as mean ± SD. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s honest significant difference (HSD) post hoc test (control morphants: n = 12 embryos; wnt4b/11 double morphants: n = 9 embryos; wnt4b/11 double morphants + wnt4b/11 mRNA: n = 9 embryos; one frame per embryo). h A cross-section images of the SCs of control morphants and wnt4b/11 double morphants at 2 dpf probed with foxj1a riboprobes ventral to the bottom. Arrowheads represent ECs. Scale bar = 20 μm. CO: Control. i RNAs were extracted from each group (20 embryos in h) at 2 dpf and levels of foxj1a mRNAs were assessed by qPCR. Mean ± SD. ****P < 0.0001 by two-tailed unpaired Student’s t test from four biological replicates (three technical replicates each). j A cross-section image of the SC of a WT embryo at 2 dpf probed with fzd7b riboprobes ventral to the bottom. Arrowhead represents ECs. Scale bar = 15 μm. k Embryos were microinjected with control MO, fzd7b MO or fzd7b MO + fzd7b mRNA, and IF stained at 2 dpf with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. CO: Control. l Quantification of the number of cilia per frame in embryos in k. Mean ± SD. ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (control morphants: n = 21 embryos; fzd7b morphants: n = 21 embryos; fzd7b morphants + fzd7b mRNA: n = 18 embryos; one frame per embryo). mTg(hsp70l:dkk1b-GFP) embryos at 24 hpf were subjected to heat shock at 39 °C for 1 h, and IF stained at 2 dpf with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. n, o Embryos were treated with IWR-1 (10 μM) at 8–48 hpf and IF stained with anti-acetylated-α-tubulin antibody only (n) or double immunostained with anti-acetylated-α-tubulin antibody and anti-γ-tubulin antibody (o) at 2 dpf. Arrowheads represent motile cilia. Dorsal view anterior to the left. The boxed areas in o are magnified in the respective lower panels. Scale bar = 20 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).

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).

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.

Spatial and temporal expression of zebrafish atp11c in wild-type embryos. Gene specific atp11c expression (antisense) at the 8-cell stage showing maternal deposition (A), 24 hpf (B: lateral, C: lateral tail, and D: dorsal) and 48 hpf (E: lateral, F: dorsal, G: lateral tail) showing expression in the mid-hindbrain boundary (MHB), anterior rhombomeres (rh), pharyngeal arches (PA), pectoral fin buds, olfactory pits (OP) and in a neuronal pattern along the tail. At 72 hpf (H: lateral and I: dorsal) and 96 hpf (J: lateral and K: ventral) there is ubiquitous expression in the head with increased expression in the jaw and OP. Both time points show a specific expression of atp11c in the liver. Non-specific staining (sense) is shown at the same timepoints for comparison (L–P).

Generation of capn3b null mutant allele. a Generation of the capn3bΔ19Δ14 mutant allele. Upper panel: diagram showing the genomic structure of the zebrafish capn3b gene and the two mutated sites in capn3bΔ19Δ14 generated by TALEN and CRISPR-Cas9 approaches, respectively. Vertical bar: exon; line connecting vertical bar: intron. Lower panel: highlighting the 19 bp deletion (red letters) in 1st exon generated by the TALEN approach (on the left) and 14 bps deletion (red letters) in 3rd exon by the CRISPR-Cas9 approach (on the right), respectively. The position of nucleotide in the capn3b open reading frame (ORF) from the translation start codon ATG is provided. C120, Capn3b activity center. b Predicted peptide encoded by the capn3bΔ19 (∆19) and capn3bΔ19Δ14 (Δ19Δ14) mutant mRNA, respectively. The Δ19 mutation leads to an early stop codon in the capn3b ORF and resulted in a 30 amino acids (aa) long polypeptide, however, the Δ19 mutation potentially allows the ATG codon encoding M94 residue of WT Capn3b to be used as an alternative translation start codon to translate an N-terminus truncated peptide which harbors the activity center C120. The Δ19Δ14 mutation creates a new early stop codon in the presumed variant initiated from M94 after C120, therefore, capn3bΔ19Δ14 is likely a null allele. c Western blotting analysis of Capn3b in WT, capn3b capn3bΔ19Δ14, capn3bΔ19 at 5dpf. β-Actin: loading control. d Immunostaining of Capn3b in WT and capn3bΔ19Δ14 mutant embryos at 5dpf. Scale bar: 50 μm