Figure 7.
Figure 7. Caveolae are essential for maintaining muscle integrity in the zebrafish. (A) Temporal expression of cavin-1a mRNA in 1.3–72-hpf zebrafish embryos. (B) Expression pattern of cavin-1a in zebrafish embryos using whole-mount mRNA in situ hybridization. Dorsal view of a 12-somite (12S) embryo shows faint labeling for cavin-1a (arrowheads). At 48 hpf, cavin-1a expression is observed exclusively within the zebrafish myotomes, shown here in dorsal (top right) and lateral (bottom left) view; anterior to left in both images. Note the lack of notochord labeling in the dorsal view. Image in bottom right represents magnification of boxed area. (C) WT and tp53zdf1 zebrafish embryos were injected with control or cavin1a MO (CtrMO and cavin1aMO, respectively) and abnormal morphants (classified as mild or severe) imaged at 72 hpf. Arrowheads indicate cardiac edema. (D) Ruthenium red–labeled isolated muscle fibers from control MO and cavin1aMO embryos. Arrows indicate caveolae (left image) or endosomes (middle and right images). Inset represents boxed area. (E) Relative caveolae density of muscle fibers from cavin1aMO embryos was 1.4 ± 2.0% (means ± SD), compared with muscle fibers from control MO embryos. Quantitation performed on four control MO and six cavin1a MO muscle fibers. (F) EBD uptake in 96 hpf Tg(actb2:EGFP-CAAX)pc10 embryos expressing EGFP-CAAX injected with control MO or cavin1a MO and incubated in 3% MC. Inset shows higher magnification of EGFP-CAAX/EBD-positive muscle fibers. (G) 0.0 ± 0.0% of control MO and cavin1aMO embryos were EBD positive after incubation in E3 (means ± SEM; 28 control MO and 47 cavin1a MO embryos from three separate microinjections). 0.8 ± 0.8% of control MO and 17.0 ± 7.0% of cavin1a MO embryos were EBD positive after incubation in 3% MC (means ± SEM; 105 control MO and 111 cavin1a MO embryos from eight separate microinjections). (H) EBD uptake in 96 hpf embryos expressing Cav3-WT-GFP (WT) or Cav3-R26Q-GFP (R26Q) after incubation in 3% MC. Inset shows higher magnification of GFP/EBD-positive muscle fibers. (I) 0.8 ± 0.8% of WT and 2.1 ± 2.1% R26Q embryos were EBD positive after incubation in E3 (means ± SEM; 71 WT and 33 R26Q embryos from five and four clutches, respectively). 7.1 ± 2.6% of WT and 31.5 ± 5.9% R26Q embryos were EBD positive after incubation in 3% MC (130 WT and 126 R26Q embryos from seven and six clutches, respectively). Uptake was performed in two separate founder lines for both WT and R26Q to ensure that phenotypes observed were not caused by Tol2 integration sites. *, P ≤ 0.05; **, P ≤ 0.01. ns, not significant. Bars: (B and C) 200 µm; (D, main images) 1 µm; (D, inset) 200 nm; (F and H, main images and insets) 50 µm.

Caveolae are essential for maintaining muscle integrity in the zebrafish. (A) Temporal expression of cavin-1a mRNA in 1.3–72-hpf zebrafish embryos. (B) Expression pattern of cavin-1a in zebrafish embryos using whole-mount mRNA in situ hybridization. Dorsal view of a 12-somite (12S) embryo shows faint labeling for cavin-1a (arrowheads). At 48 hpf, cavin-1a expression is observed exclusively within the zebrafish myotomes, shown here in dorsal (top right) and lateral (bottom left) view; anterior to left in both images. Note the lack of notochord labeling in the dorsal view. Image in bottom right represents magnification of boxed area. (C) WT and tp53zdf1 zebrafish embryos were injected with control or cavin1a MO (CtrMO and cavin1aMO, respectively) and abnormal morphants (classified as mild or severe) imaged at 72 hpf. Arrowheads indicate cardiac edema. (D) Ruthenium red–labeled isolated muscle fibers from control MO and cavin1aMO embryos. Arrows indicate caveolae (left image) or endosomes (middle and right images). Inset represents boxed area. (E) Relative caveolae density of muscle fibers from cavin1aMO embryos was 1.4 ± 2.0% (means ± SD), compared with muscle fibers from control MO embryos. Quantitation performed on four control MO and six cavin1a MO muscle fibers. (F) EBD uptake in 96 hpf Tg(actb2:EGFP-CAAX)pc10 embryos expressing EGFP-CAAX injected with control MO or cavin1a MO and incubated in 3% MC. Inset shows higher magnification of EGFP-CAAX/EBD-positive muscle fibers. (G) 0.0 ± 0.0% of control MO and cavin1aMO embryos were EBD positive after incubation in E3 (means ± SEM; 28 control MO and 47 cavin1a MO embryos from three separate microinjections). 0.8 ± 0.8% of control MO and 17.0 ± 7.0% of cavin1a MO embryos were EBD positive after incubation in 3% MC (means ± SEM; 105 control MO and 111 cavin1a MO embryos from eight separate microinjections). (H) EBD uptake in 96 hpf embryos expressing Cav3-WT-GFP (WT) or Cav3-R26Q-GFP (R26Q) after incubation in 3% MC. Inset shows higher magnification of GFP/EBD-positive muscle fibers. (I) 0.8 ± 0.8% of WT and 2.1 ± 2.1% R26Q embryos were EBD positive after incubation in E3 (means ± SEM; 71 WT and 33 R26Q embryos from five and four clutches, respectively). 7.1 ± 2.6% of WT and 31.5 ± 5.9% R26Q embryos were EBD positive after incubation in 3% MC (130 WT and 126 R26Q embryos from seven and six clutches, respectively). Uptake was performed in two separate founder lines for both WT and R26Q to ensure that phenotypes observed were not caused by Tol2 integration sites. *, P ≤ 0.05; **, P ≤ 0.01. ns, not significant. Bars: (B and C) 200 µm; (D, main images) 1 µm; (D, inset) 200 nm; (F and H, main images and insets) 50 µm.

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