Figure S3.
A loss of Cavin4b leads to reduced caveolae density and impaired swimming ability in zebrafish muscle. (A) Whole-mount ISH of cavin4a and cavin4b in WT and cavin4b−/− 24-hpf zebrafish embryos, showing loss of cavin4b signal in cavin4b−/− embryos. Images shown anterior to left in both dorsal and lateral view (upper and lower panel, respectively). Scale bars: 200 µm. (B–D) Ultrastructural analysis of 5-dpf WT (B) and cavin4b−/− (C-D) zebrafish embryos. Normal sarcomeric structure and T-system (arrows, B and D) was observed in WT and cavin4b−/− embryos. An abundance of caveolae was observed in WT muscle (arrowheads, B), but relatively few caveolae were observed in cavin4b−/− muscle (arrowhead, D). Scale bars: B and D, 500 nm; C, 1 µm. (E) Relative caveolae density was 43.7 ± 15.0% in 5-dpf cavin4b−/− muscle in comparison to WT (n = 3 each of WT and cavin4b−/− fibers, two-tailed t test). **, P ≤ 0.01. (F) Cav3 immunofluorescence (with DAPI counterstain) in isolated muscle fibers from 4-dpf WT and cavin4b−/− embryos. Muscle fibers are highlighted with a dashed line. Images in right panel represent higher magnification of boxed areas. Scale bars: 30 µm (left panel); 5 µm (right panel). (G) CTCF of Cav3 in 4-dpf WT and cavin4b−/− muscle fibers (n = 86 fibers from 4 WT clutches and n = 65 fibers from 4 cavin4b−/− clutches, two-tailed t test). CTCF values (AUF, arbitrary unit of fluorescence) were calculated by subtracting mean fluorescence of background signal from the integrated density of a whole muscle fiber. Colored circles represent the four independent clutches. (H and I) 7-dpf WT (+/+), cavin4b+/− (+/−), and cavin4b−/− (−/−) zebrafish embryos were placed in a 24-well plate, and total swimming distance (millimeters) over 10 min under light was recorded (one-way ANOVA with multiple-comparison Dunnett's test). H and I represent two independent experiments. Cavin4b+/− embryos were generated from WT × cavin4b−/− crosses. Colored circles represent individual embryos (n = 24 each). *, P ≤ 0.05; **, P ≤ 0.01. (J) Critical swimming speed (Ucrit) measured as body lengths per second (BL/s) in WT and cavin4b−/− adult zebrafish. Colored circles represent individual fish (n = 6 each, two-tailed t test). *, P ≤ 0.05. Error bars represent mean ± SD.

A loss of Cavin4b leads to reduced caveolae density and impaired swimming ability in zebrafish muscle. (A) Whole-mount ISH of cavin4a and cavin4b in WT and cavin4b−/− 24-hpf zebrafish embryos, showing loss of cavin4b signal in cavin4b−/− embryos. Images shown anterior to left in both dorsal and lateral view (upper and lower panel, respectively). Scale bars: 200 µm. (B–D) Ultrastructural analysis of 5-dpf WT (B) and cavin4b−/− (C-D) zebrafish embryos. Normal sarcomeric structure and T-system (arrows, B and D) was observed in WT and cavin4b−/− embryos. An abundance of caveolae was observed in WT muscle (arrowheads, B), but relatively few caveolae were observed in cavin4b−/− muscle (arrowhead, D). Scale bars: B and D, 500 nm; C, 1 µm. (E) Relative caveolae density was 43.7 ± 15.0% in 5-dpf cavin4b−/− muscle in comparison to WT (n = 3 each of WT and cavin4b−/− fibers, two-tailed t test). **, P ≤ 0.01. (F) Cav3 immunofluorescence (with DAPI counterstain) in isolated muscle fibers from 4-dpf WT and cavin4b−/− embryos. Muscle fibers are highlighted with a dashed line. Images in right panel represent higher magnification of boxed areas. Scale bars: 30 µm (left panel); 5 µm (right panel). (G) CTCF of Cav3 in 4-dpf WT and cavin4b−/− muscle fibers (n = 86 fibers from 4 WT clutches and n = 65 fibers from 4 cavin4b−/− clutches, two-tailed t test). CTCF values (AUF, arbitrary unit of fluorescence) were calculated by subtracting mean fluorescence of background signal from the integrated density of a whole muscle fiber. Colored circles represent the four independent clutches. (H and I) 7-dpf WT (+/+), cavin4b+/− (+/−), and cavin4b−/− (−/−) zebrafish embryos were placed in a 24-well plate, and total swimming distance (millimeters) over 10 min under light was recorded (one-way ANOVA with multiple-comparison Dunnett's test). H and I represent two independent experiments. Cavin4b+/− embryos were generated from WT × cavin4b−/− crosses. Colored circles represent individual embryos (n = 24 each). *, P ≤ 0.05; **, P ≤ 0.01. (J) Critical swimming speed (Ucrit) measured as body lengths per second (BL/s) in WT and cavin4b−/− adult zebrafish. Colored circles represent individual fish (n = 6 each, two-tailed t test). *, P ≤ 0.05. Error bars represent mean ± SD.

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