Fig. 4 | Nature Communications

Fig. 4

From: High mitogenic stimulation arrests angiogenesis

Fig. 4

Tip cells with endogenous high VEGF activity proliferate less than stalk cells. a Confocal micrographs showing that the Esm1-HA-H2B-Cerulean-2A-iCreERT2 allele is expressed by a subset of endothelial tip cells in growing retinal vessels at P6. Only 31% of these cells are cycling (KI67+), contrasting with the adjacent endothelial stalk cells (96% cycling, see Supplementary Fig. 4b and f). b Inhibition of Dll4/Notch signalling upregulates the expression of Esm1-HA-H2B-Cerulean in ECs (Erg+) at the angiogenic front. Chart shows quantification of several microscopic fields from control (n = 3) and mutant (n = 3) mice. c In mice containing the Esm1-HA-H2B-Cerulean-2A-CreERT2 and Rosa26-LSL-TdTomato alleles, the very low-recombination frequency allows individual endothelial tip cells to be genetically pulse–chased and their progeny followed over a few days. Blockade of Dll4/Notch signalling with Anti-Dll4 decreased the proliferation or clonal expansion of Esm1+ tip cells. The line above the bar indicates the most frequent clone size. Number of clones quantified is indicated. Please see also Supplementary Fig. 7b. d High-resolution Dual ifgMosaic labelling (iMb2-Mosaic×iChr2-Mosaic) and clonal analysis of single Cdh5-CreERT2+ stalk cells (see also ref. 13). Recombination was induced with tamoxifen at P3 and vessels were analysed at P6. The histogram shows the frequency of identified dual-labelled cell clones according to their size. Total number of clones quantified is indicated. The line above the bar indicates the most frequent stalk-cell dual-labelled clone size (four cells). Please see also Supplementary Fig. 7c. eg Confocal micrographs showing p21 protein in sprouting tip cells and very few stalk cells during normal retinal angiogenesis. ECs with high-VEGFR2 signalling (MbTomato+) strongly upregulate p21. Charts show quantification of several large microscopic fields from 4 (f) or 2 (g) full retinas per group. hj Dll4/Notch signalling blockade for 24 h induces a strong increase in the frequency of p21-expressing ECs (n = 3 mice per group). Scale bars in all panels, 50 μm. Error bars indicate std. dev.; ***p < 0.0005; **p < 0.005. Two-tailed unpaired t test. Source data are provided as a Source Data file

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