Figure S2.
Generation and characterization of cavin4a and cavin4b zebrafish mutant lines. (A) Alignment of nucleotide and amino acid sequences for cavin4auq5rp (cavin4a−/−) and cavin4buq6rp (cavin4b−/−) mutant lines in comparison to WT sequences. The CRISPR-generated cavin4a−/− line harbors a single base pair insertion (underlined), resulting in a frameshift (truncating stop codon indicated by asterisk) within the helical region 1 (HR1) domain of Cavin4a (arrow). Cavin4a protein domains include DR1 (residues 1–15), DR2 (residues 124–260), and DR3 (residues 270 to C’ end) shaded gray; helical regions HR1 (residues 16–123) and helical region 2 (HR2; residues 160–270) shaded yellow. The cavin4b−/− line, identified by a TILLING mutagenesis screen, harbors a single point mutation (underlined) resulting in a nonsense mutation within the HR1 domain of Cavin4b (arrow). Cavin4b protein domains: Disordered regions DR1 (residues 1–11), DR2 (residues 120–169) and DR3 (residues 256–319) shaded gray; helical regions HR1 (residues 12–119) and HR2 (residues 170–255) shaded yellow. (B) qRT-PCR of caveolae-associated genes (relative to β-actin) in WT and cavin4a−/− 5-dpf embryos (n = 3 WT clutches and n = 4 cavin4a−/− clutches, performed in triplicate, two-tailed t test). **, P ≤ 0.01 for cavin4a expression; the remaining comparisons were not significant. (C) qRT-PCR of caveolae-associated genes (relative to β-actin) in WT and cavin4b−/− 5-dpf embryos (n = 4 clutches from each, performed in triplicate, two-tailed t test). ****, P ≤ 0.0001 for cavin4b expression; the remaining comparisons were not significant. (D) Embryo length (millimeters) for 72-hpf cavin4a−/− and cavin4b−/− zebrafish embryos in comparison to WT. Quantitation from n = 40 WT and n = 40 cavin4a−/− embryos from two clutches, and n = 67 WT and n = 50 cavin4b−/− embryos from four and three clutches, respectively (two-tailed t test). Colored circles represent individual embryos. ****, P ≤ 0.0001. (E) Mean intensity of birefringence (measured as average gray value of pixels per area) in 5-dpf cavin4a−/− and cavin4b−/− zebrafish embryos in comparison to WT. Quantitation from n = 40 WT and n = 39 cavin4a−/− embryos from two clutches each, and n = 52 WT and n = 52 cavin4b−/− embryos from four clutches each (two-tailed t test). Colored circles represent individual embryos. ****, P ≤ 0.0001. (F–I) Confocal images of Cav3-EGFP in muscle fibers of live 4-dpf WT (F and H) cavin4a−/− (G) and cavin4b−/− (I) embryos. Cav3-EGFP–positive embryos were generated from homozygote × heterozygote crosses, and genotyping was performed on embryos after imaging. Area highlighted by the asterisk is magnified in bottom panel (shown as inverted images, F'–I'). Scale bars: 10 µm. (J and K) Ratio of T-tubule to sarcolemmal CAV3-EGFP fluorescence intensity in cavin4a−/− (J) and cavin4b−/− muscle fibers (K) in comparison to WT. Quantitation performed on muscle fibers from n = 12 WT and n = 11 cavin4a−/− embryos from two clutches each and from n = 20 WT and n = 19 cavin4b−/− embryos from three clutches each (two-tailed t test). Colored circles represent individual embryos. ****, P ≤ 0.0001. Error bars represent mean ± SD.

Generation and characterization of cavin4a and cavin4b zebrafish mutant lines. (A) Alignment of nucleotide and amino acid sequences for cavin4auq5rp (cavin4a−/−) and cavin4buq6rp (cavin4b−/−) mutant lines in comparison to WT sequences. The CRISPR-generated cavin4a−/− line harbors a single base pair insertion (underlined), resulting in a frameshift (truncating stop codon indicated by asterisk) within the helical region 1 (HR1) domain of Cavin4a (arrow). Cavin4a protein domains include DR1 (residues 1–15), DR2 (residues 124–260), and DR3 (residues 270 to C’ end) shaded gray; helical regions HR1 (residues 16–123) and helical region 2 (HR2; residues 160–270) shaded yellow. The cavin4b−/− line, identified by a TILLING mutagenesis screen, harbors a single point mutation (underlined) resulting in a nonsense mutation within the HR1 domain of Cavin4b (arrow). Cavin4b protein domains: Disordered regions DR1 (residues 1–11), DR2 (residues 120–169) and DR3 (residues 256–319) shaded gray; helical regions HR1 (residues 12–119) and HR2 (residues 170–255) shaded yellow. (B) qRT-PCR of caveolae-associated genes (relative to β-actin) in WT and cavin4a−/− 5-dpf embryos (n = 3 WT clutches and n = 4 cavin4a−/− clutches, performed in triplicate, two-tailed t test). **, P ≤ 0.01 for cavin4a expression; the remaining comparisons were not significant. (C) qRT-PCR of caveolae-associated genes (relative to β-actin) in WT and cavin4b−/− 5-dpf embryos (n = 4 clutches from each, performed in triplicate, two-tailed t test). ****, P ≤ 0.0001 for cavin4b expression; the remaining comparisons were not significant. (D) Embryo length (millimeters) for 72-hpf cavin4a−/− and cavin4b−/− zebrafish embryos in comparison to WT. Quantitation from n = 40 WT and n = 40 cavin4a−/− embryos from two clutches, and n = 67 WT and n = 50 cavin4b−/− embryos from four and three clutches, respectively (two-tailed t test). Colored circles represent individual embryos. ****, P ≤ 0.0001. (E) Mean intensity of birefringence (measured as average gray value of pixels per area) in 5-dpf cavin4a−/− and cavin4b−/− zebrafish embryos in comparison to WT. Quantitation from n = 40 WT and n = 39 cavin4a−/− embryos from two clutches each, and n = 52 WT and n = 52 cavin4b−/− embryos from four clutches each (two-tailed t test). Colored circles represent individual embryos. ****, P ≤ 0.0001. (F–I) Confocal images of Cav3-EGFP in muscle fibers of live 4-dpf WT (F and H) cavin4a−/− (G) and cavin4b−/− (I) embryos. Cav3-EGFP–positive embryos were generated from homozygote × heterozygote crosses, and genotyping was performed on embryos after imaging. Area highlighted by the asterisk is magnified in bottom panel (shown as inverted images, F'–I'). Scale bars: 10 µm. (J and K) Ratio of T-tubule to sarcolemmal CAV3-EGFP fluorescence intensity in cavin4a−/− (J) and cavin4b−/− muscle fibers (K) in comparison to WT. Quantitation performed on muscle fibers from n = 12 WT and n = 11 cavin4a−/− embryos from two clutches each and from n = 20 WT and n = 19 cavin4b−/− embryos from three clutches each (two-tailed t test). Colored circles represent individual embryos. ****, P ≤ 0.0001. Error bars represent mean ± SD.

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