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.

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

Retina development in atp7a−/− mutant treated with copper. A TEM analysis of retinal cells in copper-stressed atp7a−/− mutant embryos at 96 hpf. A1-A3, sagittal slides of retina, red color indicating mitochondria and green color indicating ER. B Quantitative PCR of ROS related genes (B1; cox4i2, hmox2, lox, and nrf2), UPR related genes (B2; ire1a, perk, and atf6), and retinal genes (B3; opn1wl1, opn1sw1, opn1sw2 and rhodopsin) in atp7a−/− mutants. C WISH assays of ER stress marker bip and chop in atp7a−/− mutants. D WISH assays of retinal marker (opn1wl1 and opn1sw1) in atp7a−/− mutants. C1-C8, lateral view, anterior to the left, D1-D8, dorsal view, anterior to the up. Scale bar: A1-A3, 1 μm; C1-C8 and D1-D4, 100 μm; **, P < 0.01; *, P < 0.05

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.

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

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

cx43 null zebrafish have reduced motile cilia in ECs.a Electropherograms of the target sequences of the cx43 gDNA in WT and cx43−/− zebrafish. Arrow indicates the deletion of the T nucleotide. b Zebrafish (n = 71) at 2 months post-fertilization (mpf) from mating of cx43+/− zebrafish were genotyped for cx43. c Images of 3 mpf zebrafish with the indicated cx43 genotype. d WT or cx43−/− embryos at 2 dpf were immunostained with anti-acetylated-α-tubulin antibody, imaged with a confocal microscope and genotyped for cx43. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. e Larvae at 8 dpf from mating of cx43+/− zebrafish were cut into cranial and caudal halves. The cranial half was used for cx43 genotyping, and the caudal half was coronally sectioned at a thickness of 14 μm and then processed for IF staining with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 30 μm. f Quantification of the number of cilia per frame in embryos in e. Mean ± SD. ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (cx43+/+ = 7 embryos; cx43+/− = 7 embryos; cx43−/− = 13 embryos; one frame per embryo). g Qdots were microinjected into the hindbrain ventricles of WT or cx43−/− zebrafish larvae at 10 dpf and then imaged at 5 and 60 min after the microinjection. Dashed lines indicate passive migration of Qdots. Arrowheads mark the caudal end of Qdot flow. Lateral view anterior to the left. Scale bar = 1 mm. Insets represent magnifications of the dotted areas.

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

Rescue of retinal defects with the addition of ROS scavenger and ER stress scavenger. ROS scavengers GSH & NAC and ER scavenger PBA recovered the copper induced small eye defects (A) and the expression of retinal markers opn1sw2 (B1-B5) & opn1lw1 (C1-C5 and D1-D5) in copper-stressed embryos. E ROS scavengers GSH significantly neutralized copper induced cell apoptosis in embryos. Average number of apoptotic cells of retinal sections in each group (E10) (n > 3, 3–5 sections from each embryo were used for counting the red positive apoptotic cells). B1-B5, C1-C5, and D1-D5, dorsal view, anterior to the up; E1- E9, sagittal slides in eyes domain. Scale bar, A, B1-B5, C1-C5, and D1-D5, 100 μm; E1-E9, 50 μm. **, P < 0.01; *, P < 0.05