Fig. 3

Retinal angiogenesis is compromised in endothelial Flvcr1a-null mice. A Schematic representation of tamoxifen (TAM) treatment and retina dissection in Flvcr1a;Cdh5-Cre^ERT2 and Flvcr1a control mice. Briefly, pups were injected with TAM into the stomach for 3 consecutive days (P1–P3) to induce Flvcr1a gene deletion in the developing retinal vasculature. Retinas were isolated at P8 to analyze angiogenesis. B Representative PCR products from DNA analysis on Flvcr1a^fl/fl and Flvcr1a^fl/fl;Cdh5-Cre^ERT2 pups after tamoxifen treatment. Deletion of the first exon of Flvcr1a gene mediated by Cre recombinase gave rise to a band referred to as ‘‘null allele.’’ Specific primers were used to amplify Cre recombinase (270 bp), Flvcr1a floxed (280 bp), and Flvcr1a null (320 bp) alleles. C Whole-mount CD31 staining (green) of P8 retinal primary vascular plexus from Flvcr1a^fl/fl and Flvcr1a^fl/fl;Cdh5-Cre^ERT2 mice. Scale bar: 200 µm. D–I Quantification of vascular parameters of Flvcr1a^fl/fl and Flvcr1a^fl/fl;Cdh5-Cre^ERT2 P8 retinas, i.e., D radial expansion, E vascular area, F average nearest neighbor distance (NND), G vessel distribution variability, H vascular total length, I number of branches. J CD31 (gray)/pHH3 (red) double staining of Flvcr1a^fl/fl and Flvcr1a^fl/fl;Cdh5-Cre^ERT2 P8 retinas, and K quantification of pHH3⁺ ECs. Scale bar: 100 µm. L Higher magnification of the angiogenic front in Flvcr1a^fl/fl and Flvcr1a^fl/fl;Cdh5-Cre^ERT2 P8 retinas stained with CD31 antibody to highlight the vasculature. Tip cells are white circled. Scale bar: 100 µm. M Tip cells and N filopodia quantification is shown. O Whole-mount staining of Flvcr1a^fl/fl and Flvcr1a^fl/fl;Cdh5-Cre^ERT2 P8 retinas with CD31 (green) and NG2 (red) antibodies to highlight ECs and pericyte, respectively. Scale bar: 200 µm. P Quantification of pericyte coverage as % ratio of NG2 area and CD31 area. Data are representative of at least 3 independent experiments and are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; For statistical analyses ,an unpaired Student’s t test was used. TAM tamoxifen, P post-natal day, bp base pairs, A.U. arbitrary unit