Extended Data Fig. 3: Generation and functional characterization of EC-specific g6pd and pgd zebrafish mutants.
a, Generation of CRISPR vectors for EC-specific gene targeting. Schematic representation of the two gRNA target sequences (gRNA1 and gRNA2) used to generate endothelial-specific KO for g6pd and pgd. gRNA sequence 1 for both genes is the one used to generate zebrafish mutants. The CRISPR targeted sequence is underlined. The red letters indicate the PAM region for each gene. b, Schematic representation of the workflow for EC-specific CRISPR-based KO technology. This illustration is a modification of BioRender templates. c, Schematic of FACS sorting from from Tg(kdrl:mCherry)uto2 zebrafish embryos to isolate EC (mCherry-positive). Relative mCherry mRNA expression indicated as arbitrary units (A.U.) from EC (mCherry-positive) and non-EC (mCherry-negative). n=3 from two independent experiments for each condition. Data are shown as mean ± SEM. Statistics were done using unpaired Student’s t-test. d, T7E1 assay for checking genomic mutations induced by g6pd or pgd injected plasmids. As previously described, cmlc2-eGFP negative and positive embryos were selected and genomic DNA was extracted from FACS-sorted zebrafish embryos at 3dpf and subjected for T7E1. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. e, Quantification of DA diameter in g6pd and pgd EC-specific KO mutants. DA diameter has been measured in Tg(kdrl:eGFP)s843 embryos, n≥8 from 3 independent experiments. Statistics were done using unpaired Student’s t-test. Data are shown as mean ± SEM. f Confocal images of z-projection of the trunk region (somite 8-14) of Tg(fli-n:GFP)y7 zebrafish embryos at 4dpf after the injection with the Tol2 mRNA plasmid U6:pgdgRNA1; fli1a:Cas9 compared to control plasmid-injected embryos. Scatter plots show the quantification of the number of EC in the DA after the injection with the Tol2 mRNA plasmid U6:pgdgRNA1; fli1a:Cas9 (n=7) compared to control plasmid-injected embryos (n=6) from one independent experiments. Data are shown as mean ± SEM. g, Quantification of the number of EC in the DA in g6pd and pgd EC-specific KO mutants. The number of EC has been measured in Tg(kdrl:eGFP)s843 embryos, n≥6 from three independent experiments. Data are shown as mean ± SEM. Statistics were done using unpaired Student’s t-test.
