Morpholinos targeting tnnt, gata1, tif1gamma, or a control morpholino were co-injected with ccm2 guide and Cas9 RNA. (A) Reduction of blood flow in tnnt morphants (A, B, C) resulted in reduced caudal venous plexus (CVP) dilation (A) and intravascular honeycombing (B, C) in 2 days post fertilization (dpf) ccm2 CRISPR Tg(fli1:EGFP) embryos. Arrows indicate intussusceptions. Scale bar: 100 µm. (A) Loss of erythrocytes in gata1 or tif1gamma morphant ccm2 CRISPR embryos also reduced the incidence of CVP dilation. p-Values were calculated using one-way ANOVA. **p<0.01. Error bars indicate SD. (D and E) At 23 hpf, ccm2 CRISPR Tg(klf2a:H2b-EGFP) embryos displayed a mosaic increase of EGFP expression in endothelial cells in the CVP (D), compared with cas9 mRNA control embryos (E). Scale bar: 25 µm. (F) Quantification of the EGFP fluorescence intensity using ImageJ. A total of 20 nuclei were analyzed from ccm2 CRISPR embryos, and 16 nuclei were analyzed from control embryos. Note that 11 nuclei in CRISPR embryo displayed intensity above 3000, while all of the nuclei in control embryo are below 3000. (G) ccm2 CRISPR and tnnt morpholino-injected Tg(klf2a:H2b:EGFP 2 dpf) embryos displayed a mosaic increase of endothelial nuclear EGFP expression in dorsal vein. Scale bar: 50 µm. In A through C, EGFP expression was driven by klf2a promoter in Tg(klf2a:H2b:EGFP) embryo, and endothelial cells were labeled by mcherry in Tg(kdrl:mcherry) transgenic line. Arrows indicated the endothelial nuclei with increased EGFP, and arrowheads indicated the other endothelial nuclei along the ventral wall of dorsal vein.
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