Developmental vessel regression does not depend on endothelial cell death.
A, Overview of a wild-type postnatal day 6 (P6) mouse retina highlighting all regression profiles (blue lines). Regression profiles are vessel segments with collagen IV-positive vessel segments and negative for IsolectinB4. B, Quantification of number of regressing segments at P4, P6, and P8 retinas per vascularized area. C, Representative image of a P6 mouse retina labeled with Col.IV (green), cleaved caspase-3 (red) and IsolectinB4 (blue) showing regression profiles (white arrows) associated with cleaved caspase-3-positive cells (yellow arrows). D, Quantification of total numbers of cleaved caspase-3 events in entire P4, P6, and P8 mouse retinas, normalized for 100 μm2 of vascularized tissue. At P6, only 4.82% ± 0.76 (n = 5 retinas) of regression events are associated with caspase-3-positive labeled endothelial cells. Data given as mean ± SD. E, Confocal images of P6 wild-type retinas after 4h EdU-treatment (EdU, blue), endothelial cell nuclei (Erg, green) and blood vessels (ICAM2, red). F, Quantification of total number of endothelial cells, percentage of ETS related gene (Erg)- and 5-ethynyl-2'-deoxyuridine (EdU)-positive cells to total number of endothelial cells, and number of endothelial cells per vascularized area at specified mouse retina developmental stages. Mean ± SEM; n = 4 mice, 2 litters. Scale bars (A and E: 200 μm; C: 25 μm). The data used to make this figure can be found in S1 Data.
