Fig 1.
Expression patterns of cldn5a and cldn5b.
(A—C) In situ hybridization of cldn5a. Specific expression of cldn5a in KV at 6 ss, marked by a red rectangle (A). Expression of cldn5a in neuroepithelial cells at 18 ss (B) and 24 hpf (C), marked by a red arrow. (D—F) In situ hybridization of cldn5b. No obvious expression of cldn5b at 6 ss (D). Expression of cldn5b in dorsal aorta (DA) and intersegmental vessels (ISV) at 18 ss (E) and 24 hpf (F). DA and ISV are marked by a green arrow and rectangle, respectively. (G—M) Maximum intensity projection images of Cldn5 (red) and sox17:egfp-positive KV cells (green) in 90% epiboly to 13 ss embryos. (N) Single plane image of Cldn5 (red) and sox17:egfp-positive KV cells (green) in 3 ss embryos. Scale bar: 20 μm.
Fig 2.
Laterality of heart was disrupted in cldn5a morphants.
(A—H) Visualization of a heart by in situ hybridization of cmlc1 in 30 hpf embryos. Representative images of control morphants (A), cldn5a morphants (B), DFC cldn5a morphants (C), and yolk cldn5a morphants (D). (E) Stacked bar graph (blue; normal, orange; middle, grey; reversed, control morphants; n = 45, cldn5a morphants; n = 33, DFC control morphants; n = 71, DFC cldn5a morphants; n = 37, yolk control morphants; n = 25, yolk cldn5a morphants; n = 31). Representative images of control morphants with mCherry (F), cldn5a morphants with mCherry (G), cldn5a morphants with mCherry-cldn5a (H). (I) Stacked bar graph (blue; normal, orange; middle, grey; reversed, control morphants with mCherry; n = 132, cldn5a morphants with mCherry; n = 111, cldn5a morphants with mCherry-cldn5a; n = 115). * depicts p < 0.05.
Fig 3.
Expressions of spaw and dand5 were disrupted in DFC cldn5a morphants.
(A—B) Visualization of spaw by in situ hybridization in 18 ss embryos. Representative images of DFC control morphants (A) and DFC cldn5a morphants (B). (C) Stacked bar graph (blue; normal, orange; bilateral, grey; right-sided, DFC control morphants; n = 48, DFC cldn5a morphants; n = 45). (D—E) Visualization of dand5 by in situ hybridization in 6 ss embryos. Representative images of DFC control morphants (D) and DFC cldn5a morphants (E). (F) Stacked bar graph (blue; normal, orange; abnormal, DFC control morphants; n = 26, DFC cldn5a morphants; n = 36). (G—H) Visualization of dand5 by in situ hybridization in 8 ss embryos. Representative images of DFC control morphants (G) and DFC cldn5a morphants (E). (I) Stacked bar graph (blue; normal, orange; abnormal, DFC control morphants; n = 76, DFC cldn5a morphants; n = 82). (J) Relative dand5 mRNA expression in DFC cldn5a morphants versus DFC control morphants at 8 ss. Error bars indicate s.e.m.
Fig 4.
Decreased number of cilia in partially formed KV lumen.
(A—A’, B—B’) Maximum intensity projection images of acetylated tubulin (grey) and sox17:egfp-positive KV cells (green) in 6 ss embryos. Representative images of DFC control morphants (A), and DFC cldn5a morphants (B). (A”, B”) Single plane images of acetylated tubulin (grey) and sox17:egfp-positive KV cells (green) in 6 ss embryos. (C) Statistical column bar graph of cilia number (DFC control morphants; n = 25, DFC cldn5a morphants; n = 30) (D) Statistical column bar graph of cilia length (DFC control morphants; n = 314, DFC cldn5a morphants; n = 128). *** depicts p < 0.001, N.S. (not significant) depicts p > 0.05. Error bars indicate s.e.m. Scale bar: 20 μm.
Fig 5.
Downregulation of cldn5a resulted in defective KV lumen in zebrafish.
(A—F) Maximum intensity projection images of ZO-1 (grey) and sox17:egfp-positive KV cells (green) in 6 ss, 8 ss and 10 ss embryos. Representative images of the DFC control morphants (A—C) and DFC cldn5a morphants (D—F). (G) Statistical box and whisker graph (DFC control morphants at 6 ss; n = 20, DFC control morphants at 8 ss; n = 31, DFC control morphants at 10 ss; n = 15, DFC cldn5a morphants at 6 ss; n = 28, DFC cldn5a morphants at 8 ss; n = 21, DFC cldn5a morphants at 10 ss; n = 18). Scale bar: 20 μm.
Fig 6.
Treatment of forskolin and IBMX failed to recover the KV lumen size in DFC cldn5a morphants.
(A—D) Maximum intensity projection images of ZO-1 in 6 ss embryos. Representative images of DFC control morphants treated with DMSO (A) or forskolin/IBMX (B). Representative images of DFC cldn5a morphants treated with DMSO (C) or forskolin/IBMX (D). (E) Statistical column bar graph (DFC control morphants with DMSO; n = 30, DFC control morphants with forskolin/IBMX; n = 28, DFC cldn5a morphants with DMSO; n = 19, DFC cldn5a morphants with forskolin/IBMX; n = 22). *** depicts p < 0.001, ** depicts p < 0.01, N.S. depicts p > 0.05. Error bars indicate s.e.m. Scale bar: 20 μm.