Extended Data Fig. 4: Validation of iFlpMosaics for ratiometric quantitative gene function analysis in cell migration and proliferation.
a, Representative confocal micrographs of a P6 retina from a Rbpj-floxed mouse carrying the iFlpMTomato-Cre/MYFP and Apln-FlpO alleles. Apln is an X-chromosomal gene (mosaically expressed in females) expressed at the angiogenic front of growing vessels, recombining in angiogenic front ECs that later form the entire vasculature. Apln also recombines in non-endothelial (ERG-negative) cells in the retina. The right micrograph shows ERG-signal segmentation (EC nuclei), with nuclei of MTomato+ (RbpjKO) cells in red and nuclei of MYFP+ (RbpjWT) cells in green. Grey nuclei are in non-recombined ERG+ cells. Dashed white lines demark the leading edge (LE), angiogenic front (AF), and mature area (MA). A, arteries; V, veins. The chart shows the log2 ratios of MTomato+ to MYFP+ ECs in each retinal region, showing the enrichment of RbpjKO cells in the LE and AF, which suggests that loss of this gene induces cell migration to the front of the plexus. b, c Representative confocal micrographs of a P6 retina from Rbpj-floxed mouse carrying the iFlpMTomato-H2BGFP-Cre/MYFP-H2BCherry and Apln-FlpO alleles. This allows the nuclear labeling of RbpjKO (H2B-GFP+) and RbpjWT (H2B-Cherry+) ECs and blood cells, which is more convenient for cell object segmentation and quantification of nuclear-specific proteins, such as the proliferation and cell-cycle arrest markers Ki67 and p21. Nuclear segmentation outlines: red, Cherry+/Ki67- or p21-; pink, Cherry+/Ki67+ or p21+; green, GFP+/Ki67- or p21-; cyan, GFP+/Ki67+ or p21+. Data are presented as mean values +/− SD. For statistics see Source Data file 1. Scale bars, 150μm.
