Fig. 1

MiR-132 knockdown and mutation impair brain vascular integrity of larval zebrafish. (A, B) Representative images showing that miR-132 knockdown with morpholino oligonucleotides (MOs) caused intracranial hemorrhage (arrowheads) in wildtype larvae (A) and blood cell leakage (arrowheads) in the brain of Tg(Flk1:eGFP;Gata1:DsRed) larvae (B). The images were taken from larvae at 3 days post fertilization (dpf). (C) Summary of the intracranial hemorrhage effect of miR-132 knockdown. The experiments were repeated 4-8 times, and at each time > 89 embryos were examined for each group. (D, E) Representative images and summary showing that miR-132 knockdown caused more DAPI-positive brain parenchymal nuclei (blue) in 3-dpf Tg(Flk1:eGFP) larvae, in which a mixture of DAPI and Alex-568 dextran were injected into the blood circulation system. In D, the area outlined by the rectangles in the top are enlarged in the bottom, and the number of DAPI-positive nuclei in the area outlined by the dashed lines were counted. 13 control embryos and 18 morphants were analyzed in E. (F) Spatial distribution of leakage sites (red dots) in all 9 miR-132 morphants examined by in vivo time-lapse confocal imaging. (G-I) Transmission electron microscopy (TEM) images showing the ultrastructural changes of brain vessels in miR-132 morphants. The areas outlined by the red dashed rectangles in G were enlarged in (H, “Ctrl MO”) and (I, “miR-132 MO”). The green arrowheads in G point to the abnormal sites of the vessel luminal side, and the red arrowheads in I point to the abnormal junctions between vascular endothelial cells (ECs). (J) Rescue effect of miR-132RNA overexpression on intracranial hemorrhage defects in miR-132 morphants. The experiments were repeated four times, and at each time > 75 embryos were examined for each group. (K) Summary of the intracranial hemorrhage effect of miR-132 mutations generated by co-injection of zebrafish codon-optimized Cas9 mRNA and miR-132 gRNA. The experiments were repeated 3-5 times, and for each time > 21 embryos were examined for each group. Scale bar, 400 μm (top) and 100 μm (bottom) (A), 100 μm (B), 100 μm (top) and 10 μm (bottom) (D), 100 μm (F), 1 μm (G), 200 nm (H and I). Error bars, SEM. n.s., no significant; ^***P < 0.001 (one-way ANOVA with post hoc Bonferroni's multiple comparison test for C and K; unpaired two-tailed Student's t-test for E; one-way ANOVA with post hoc Tukey's multiple comparison test for J).