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Fig. 3 of Zhang et al., 2020
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.
Fig. 5 of Wang et al., 2020
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.
Fig. 2 of Marshall-Phelps et al., 2020
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.
Fig. 4 of Marshall-Phelps et al., 2020
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.
Fig. 2 of Zhang et al., 2020
plcδ3a is a target gene of Wnt signaling and plays an important role in the maintenance of ependymal motile cilia.a DMSO (vehicle control) or thapsigargin (4 nl at 5 μM) was microinjected into the hindbrain ventricles of zebrafish larvae at 4 dpf and the larvae were double immunostained with anti-acetylated α-tubulin antibody (red) and anti-γ-tubulin antibody (green) 4 h after injection. Arrowheads indicate motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. b Quantification of the number of cilia and basal bodies per frame in embryos in a. Mean ± SD. ****P < 0.0001 by two-tailed unpaired Student’s t test (n = 4 embryos per group; one frame per embryo). ns, not significant. c Embryos at 2 dpf were probed with plcβ4 or plcδ3a riboprobes and their SCs were cross-sectioned. Images are oriented ventral to the bottom. Arrowheads represent ECs. Scale bar = 20 μm. d DMSO (vehicle control) or U-73122 (4 nl at 10 μM) was microinjected into the hindbrain ventricles of zebrafish larvae at 4 dpf and the larvae were IF stained with anti-acetylated α-tubulin antibody 4 h after microinjection. Dorsal view anterior to the left. Scale bar = 20 μm. e Control morphants or wnt4b/11 double morphants were microinjected with plcβ4 or plcδ3a mRNA and immunostained with anti-acetylated α-tubulin antibody at 2 dpf. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. f Quantification of the number of motile cilia per frame in embryos in e. Mean ± SD. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (control morphants: n = 12 embryos; wnt4b/11 double morphants: n = 9 embryos; control morphants + plcβ4 mRNA: n = 4 embryos; control morphants + plcδ3a mRNA: n = 3 embryos; wnt4b/11 double morphants + plcβ4 mRNA: n = 9 embryos; wnt4b/11 double morphants + plcδ3a mRNA: n = 5 embryos; one frame per embryo). ns: not significant. g Control morphants or wnt4b/11 double morphants at 2 dpf were probed with plcδ3a riboprobes and their SCs were cross-sectioned. Images are oriented ventral to the bottom. Arrowheads point to ECs. Scale bar = 20 μm. h RNAs were extracted from each group (20 embryos in g) at 2 dpf and levels of plcδ3a mRNA were assessed by qPCR. Mean ± SD. *P < 0.05 by two-tailed unpaired Student’s t test from four biological replicates (three technical replicates each). CO: Control. i Embryos at 45 hpf were treated with DMSO or BIO (5 μM) for 1–3 h and probed with plcδ3a riboprobes, and their SCs were sectioned. Images are oriented ventral to the bottom. hpt: hours post-treatment. Arrowhead indicates ECs. Scale bar = 20 μm. j Upon BIO treatment, RNAs were extracted from each group (20 embryos in (i)) and levels of plcδ3a mRNAs were assessed by qPCR. Mean ± SD. *P < 0.05 and ****P < 0.0001 by two-tailed unpaired Student’s t test from three biological replicates (eight technical replicates each). k Schematic of the plcδ3a promoter-firefly luciferase construct used in the luciferase reporter assay. Black rectangles represent three (1–3) Tcf binding elements (TBEs) and red rectangles TBEs with deletions. l HEK 293T cells were transfected with WT or mutant plcδ3a promoter-firefly luciferase constructs and Renilla luciferase plasmid with or without β-catenin plasmid, and processed for dual luciferase assay. Relative Light Units: firefly luciferase activity/Renilla luciferase activity. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (n = 3 culture replicates per group; each culture was assayed three times).
Fig. 5 of Zhang et al., 2020
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.
Fig. 9 of Chen et al., 2020
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
Fig 6 of Liu et al., 2020
Disruption of TBC1D23-golgin-97/245 interaction impairs neuronal growth and brain development in zebrafish.(A) HuC (elavl3) spatial expression in zebrafish at 48 hpf, determined by in situ hybridization. MO: MO injection; MO+ FL: MO and human FL TBC1D23 mRNA co-injection; MO+278/281/282: MO and human TBC1D23 L278A/Y281A/Y282A mutant mRNA co-injection; MO+236/237/239: MO and human TBC1D23 I236A/I237A/V239A mutant mRNA co-injection; MO+399/405: MO and human TBC1D23 C399S/R405A mutant mRNA co-injection. All injections are performed at the one-cell stage Top, lateral view; bottom, dorsal view. (B) The relative size of zebrafish midbrain. The size of the midbrain was measured from lateral view, and 5 to 10 embryos from each group were used for comparison. Data are presented as mean ± S.E.M. ****P < 0.0001. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. (C) Relative transcription level of HuC at 48 hpf by semiquantitative RT-PCR analysis. Mean ± S.E.M. *P < 0.05. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. (D) HuC (green) expression in Tg[HuC: GFP] transgenic zebrafish at 48 hpf, determined by immunofluorescence. Top, lateral view; bottom, dorsal view. (E) Morphology of CaP axons from embryos at 48 hpf that were injected MO and/or different mRNA. All injections are performed at the one-cell stage of the Tg[hb9: GFP]ml2 transgenic zebrafish embryos. Arrows indicate abnormal branches. Lateral views and enlarged views are shown. (F) Statistical results of the branch number of CaP axons in embryos treated as in (E). For each group, approximately 45 axons from nine Tg[hb9: GFP]ml2 transgenic zebrafish embryos are scored. Mean ± S.E.M. ****P < 0.0001,*P < 0.05. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. FL, full-length; hpf, hours post fertilization; MO, morpholino oligonucleotide; ns, not significant; RT-PCR, reverse transcription PCR; WT, wild type.
FIGURE 6 of Min et al., 2020
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).
Fig. 2 of Chen et al., 2020
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
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Fig. 3 of Zhang et al., 2020
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.
Fig. 5 of Wang et al., 2020
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.
Fig. 2 of Marshall-Phelps et al., 2020
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.
Fig. 4 of Marshall-Phelps et al., 2020
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.
Fig. 2 of Zhang et al., 2020
plcδ3a is a target gene of Wnt signaling and plays an important role in the maintenance of ependymal motile cilia.a DMSO (vehicle control) or thapsigargin (4 nl at 5 μM) was microinjected into the hindbrain ventricles of zebrafish larvae at 4 dpf and the larvae were double immunostained with anti-acetylated α-tubulin antibody (red) and anti-γ-tubulin antibody (green) 4 h after injection. Arrowheads indicate motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. b Quantification of the number of cilia and basal bodies per frame in embryos in a. Mean ± SD. ****P < 0.0001 by two-tailed unpaired Student’s t test (n = 4 embryos per group; one frame per embryo). ns, not significant. c Embryos at 2 dpf were probed with plcβ4 or plcδ3a riboprobes and their SCs were cross-sectioned. Images are oriented ventral to the bottom. Arrowheads represent ECs. Scale bar = 20 μm. d DMSO (vehicle control) or U-73122 (4 nl at 10 μM) was microinjected into the hindbrain ventricles of zebrafish larvae at 4 dpf and the larvae were IF stained with anti-acetylated α-tubulin antibody 4 h after microinjection. Dorsal view anterior to the left. Scale bar = 20 μm. e Control morphants or wnt4b/11 double morphants were microinjected with plcβ4 or plcδ3a mRNA and immunostained with anti-acetylated α-tubulin antibody at 2 dpf. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. f Quantification of the number of motile cilia per frame in embryos in e. Mean ± SD. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (control morphants: n = 12 embryos; wnt4b/11 double morphants: n = 9 embryos; control morphants + plcβ4 mRNA: n = 4 embryos; control morphants + plcδ3a mRNA: n = 3 embryos; wnt4b/11 double morphants + plcβ4 mRNA: n = 9 embryos; wnt4b/11 double morphants + plcδ3a mRNA: n = 5 embryos; one frame per embryo). ns: not significant. g Control morphants or wnt4b/11 double morphants at 2 dpf were probed with plcδ3a riboprobes and their SCs were cross-sectioned. Images are oriented ventral to the bottom. Arrowheads point to ECs. Scale bar = 20 μm. h RNAs were extracted from each group (20 embryos in g) at 2 dpf and levels of plcδ3a mRNA were assessed by qPCR. Mean ± SD. *P < 0.05 by two-tailed unpaired Student’s t test from four biological replicates (three technical replicates each). CO: Control. i Embryos at 45 hpf were treated with DMSO or BIO (5 μM) for 1–3 h and probed with plcδ3a riboprobes, and their SCs were sectioned. Images are oriented ventral to the bottom. hpt: hours post-treatment. Arrowhead indicates ECs. Scale bar = 20 μm. j Upon BIO treatment, RNAs were extracted from each group (20 embryos in (i)) and levels of plcδ3a mRNAs were assessed by qPCR. Mean ± SD. *P < 0.05 and ****P < 0.0001 by two-tailed unpaired Student’s t test from three biological replicates (eight technical replicates each). k Schematic of the plcδ3a promoter-firefly luciferase construct used in the luciferase reporter assay. Black rectangles represent three (1–3) Tcf binding elements (TBEs) and red rectangles TBEs with deletions. l HEK 293T cells were transfected with WT or mutant plcδ3a promoter-firefly luciferase constructs and Renilla luciferase plasmid with or without β-catenin plasmid, and processed for dual luciferase assay. Relative Light Units: firefly luciferase activity/Renilla luciferase activity. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (n = 3 culture replicates per group; each culture was assayed three times).
Fig. 5 of Zhang et al., 2020
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.
Fig. 9 of Chen et al., 2020
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
Fig 6 of Liu et al., 2020
Disruption of TBC1D23-golgin-97/245 interaction impairs neuronal growth and brain development in zebrafish.(A) HuC (elavl3) spatial expression in zebrafish at 48 hpf, determined by in situ hybridization. MO: MO injection; MO+ FL: MO and human FL TBC1D23 mRNA co-injection; MO+278/281/282: MO and human TBC1D23 L278A/Y281A/Y282A mutant mRNA co-injection; MO+236/237/239: MO and human TBC1D23 I236A/I237A/V239A mutant mRNA co-injection; MO+399/405: MO and human TBC1D23 C399S/R405A mutant mRNA co-injection. All injections are performed at the one-cell stage Top, lateral view; bottom, dorsal view. (B) The relative size of zebrafish midbrain. The size of the midbrain was measured from lateral view, and 5 to 10 embryos from each group were used for comparison. Data are presented as mean ± S.E.M. ****P < 0.0001. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. (C) Relative transcription level of HuC at 48 hpf by semiquantitative RT-PCR analysis. Mean ± S.E.M. *P < 0.05. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. (D) HuC (green) expression in Tg[HuC: GFP] transgenic zebrafish at 48 hpf, determined by immunofluorescence. Top, lateral view; bottom, dorsal view. (E) Morphology of CaP axons from embryos at 48 hpf that were injected MO and/or different mRNA. All injections are performed at the one-cell stage of the Tg[hb9: GFP]ml2 transgenic zebrafish embryos. Arrows indicate abnormal branches. Lateral views and enlarged views are shown. (F) Statistical results of the branch number of CaP axons in embryos treated as in (E). For each group, approximately 45 axons from nine Tg[hb9: GFP]ml2 transgenic zebrafish embryos are scored. Mean ± S.E.M. ****P < 0.0001,*P < 0.05. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. FL, full-length; hpf, hours post fertilization; MO, morpholino oligonucleotide; ns, not significant; RT-PCR, reverse transcription PCR; WT, wild type.
FIGURE 6 of Min et al., 2020
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).
Fig. 2 of Chen et al., 2020
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