ER stresses in copper-stressed and BFA-treated embryos. A Phenotypes of representative copper-stressed embryos with or without BFA. BFA treatment destroys the COPII function and induces ER stresses, A7, measurement of eye diameters of the embryos. A1-A6, lateral view, anterior to the left. B Protein levels of P­eIF-2α in copper-stressed embryos with or without BFA (B1) and the quantification of protein levels of P­eIF-2α in each sample (B2). C Western blot detection of XBP1-s in copper-stressed embryos (C1) and the quantification of protein level in each sample (C2). D Immunostaining of PDI (ER marker) in copper-treated embryos. D1-D3, merged images of immunostaining of PDI (green) and DAPI staining (blue); D4, D5 and D6 were magnified domains of white boxes marked in D1, D2 and D3, respectively. D1- D6, sagittal slides in eyes domain. Scale bar: A1-A6, 0.2 mm; D1-D9, 50 μm; D10-D12, 10 μm. **, P < 0.01; *, P < 0.05

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.

Cell proliferation, apoptosis, and oxidative & ER stresses in copper-stressed embryos. A Cell proliferation assay by PH3 staining (green dots). B Cell apoptosis assay by TUNEL (red dots) detection. (A, B) sections of embryonic eyes. A10, number of PH3 positive cells per section, B10, average number of apoptotic cells of retinal sections in each group (n > 3, 3–5 sections from each embryo were used for counting the red positive apoptotic cells). C Immunostaining of Caspase3 (green) in retina sections. D Western blot detection with antibody Bcl-2 in copper-stressed embryos. E TEM analysis of retinal cells in copper-stressed embryos at 96 hpf. E1-E6: sagittal slides of retina, red color indicating mitochondria and green color indicating ER. F DCHF-DA assay of embryonic retina. G qRT-PCR detection of ER-stressed genes in embryos at 96 hpf. Scale bars: A1-A9, B1-B9 and C1-C9, 50 μm; E1-E6, 0.5 μm; F1-F3, 100 μm; **, P < 0.01; *, P < 0.05

a In situ hybridization to examine the localization of MyolncR4 at different developmental stages of zebrafish embryos with a specific probe targeting zLEMP. hpf, hours post fertilization. Scale bar, 200 μm. b Diagram to show the targeting sequence of the LEMP-AMO. c Representative images of zebrafish at 24 or 48-hpf after injection of different amounts of LEMP-AMO at one-cell stage. Scale bar, 200 μm or 25 μm. d Representative images of zebrafish at 24 or 48-hpf after injection of LEMP-AMO in the presence or absence of the zebrafish LEMP mRNA at one-cell stage. Scale bar, 200 or 25 μm. e MHC immunostaining to examine myofiber alignment in 48-hpf embryos injected with Cntl-AMO, LEMP-AMO, LEMP-AMO with zLEMP mRNA, or LEMP-AMO with mLEMP mRNA, respectively. Scale bar, 10 μm.

Protein levels of retinal photoreceptor in copper stressed embryos. A Immunostaining of Rodopsin (labeling retinal rod cells) (A1-A9). B Double immunostaining of ZPR-1 (labeling retinal cone cells) and Opn1sw2 (blue opsin, labeling retinal rod cells) in embryos at 10 dpf. A10, B13, B14, Quantification of protein level in each sample. Scale bars: A1-A9 and B1-B12, 50 μm; **, P < 0.01; *, P < 0.05

Cx43 is implicated in the maintenance of ependymal motile cilia.a WISH of a WT embryo at 2 dpf with cx43 riboprobes (left). Lateral view anterior to the left. Right panel shows the cross-section image of the SC ventral to the bottom. Arrowhead indicates ECs. Scale bars = left: 650 μm; right: 20 μm. b Embryos at one-cell stage were microinjected with cx43 MO and imaged at 2 dpf. Lateral view anterior to the left. Scale bar = 650 μm. c Embryos at one-cell stage were microinjected with either control MO or cx43 MO or cx43 MO + mouse Cx43 mRNA, and IF stained with anti-acetylated α-tubulin antibody at 2 dpf. Arrowheads represent spinal motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. dTg(bactin2:Arl13b-GFP) embryos expressing GFP in motile cilia were microinjected at one-cell stage with either control (CO) MO or cx43 MO and subjected to time-lapse imaging for 2 min (6.7 frames s−1) with an intravital multiphoton microscope. Dorsal view anterior to the left. Scale bar = 7.5 μm. e Embryos at one-cell stage were microinjected with control MO or cx43 MO and Qdots were microinjected into the hindbrain ventricles of larvae at 4 dpf, which were then imaged at 10 min and 60 min after the microinjection. Dashed lines indicate migration of Qdots. Arrowheads mark the caudal end of Qdot flow. Lateral view anterior to the left. Scale bar = 650 μm. Insets represent magnifications of the dotted areas. CO: Control. f Quantification of Qdot migration distance in the larvae shown in d. Mean ± SD. ***P < 0.001 by two-tailed unpaired Student’s t test (n = 3 larvae per group). g Embryos at one-cell stage were microinjected with either control MO or cx43 MO and immunostained with anti-γ-tubulin antibody at 2 dpf. Arrowheads indicate motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. hTg(foxj1a:eGFP) embryos expressing GFP in ECs were microinjected with either control MO or cx43 MO and imaged at 2 dpf. Arrowheads represent spinal ECs. Dorsal view anterior to the left. Scale bar = 650 μm.

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

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

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.