Supplementary Figure 3: Role of endothelial CXCR4 in retinal angiogenesis.

(a) Representative overview confocal images showing IB4-labelled Cxcr4^iΔEC/ΔEC and Cxcr4^iΔEC/KO retinas together with the corresponding controls. Circles indicate vessel outgrowth in the control. (b) Combined ERG (EC nuclei; red), IB4 (green) and EdU (blue) staining shows reduced ERG + EdU + EC numbers in Cxcr4^iΔEC/ΔEC retinal vessels compared to littermate control. EdU pulse-labelling was done 2 h prior to analysis. (c) Representative confocal images of CXCR4 (red/white) and IB4 (green) stained Cxcr4^iΔEC/KO and control retinas. Arrowheads mark sprouts at the angiogenic front. (d) ESM1 (white) and IB4 (green) staining of Cxcr4^iΔEC/ΔEC and control retinas. ESM1 expression is not overtly changed. (e) Quantitative analysis of EC proliferation for Cxcr4^iΔEC/ΔEC and control littermates (n = 7 control and 8Cxcr4^iΔEC/ΔEC retinas from two independent tamoxifen injections). Graphs indicate reduction of Cxcr4^iΔEC/ΔEC ERG + EdU + cells per EC area at the angiogenic front relative to control, while the number of ERG + cells was not significantly changed because of the impaired outgrowth. Data represent mean ± s.e.m. P values, two-tailed unpaired t-test. (f) Quantitative analysis of vascular parameters for Cxcr4^iΔEC/KO, Cxcr4^KO/+ and control retinas: retinal vessel progression, EC area, branching points (n = 7 control, 10 Cxcr4^KO/+ and 6 Cxcr4^iΔEC/KO retinas), and sprouts (n = 5 control and 6 Cxcr4^iΔEC/KO retinas). Note absence of significant differences between Cxcr4^KO/+ heterozygotes and controls. Data represent mean ± s.e.m. P values, two-tailed unpaired t-test and one-way ANOVA with Tukey’s multiple comparison post-hoc test. (g) RT-qPCR analysis for Cxcr4 (n = 6 control and 7 Cxcr4^iΔEC/ΔEC mice), Vegfa(n = 6 control and 12 Cxcr4^iΔEC/ΔEC mice) and Esm1 (n = 5 control and 7 Cxcr4^iΔEC/ΔEC mice) in sorted Cxcr4^iΔEC/ΔEC and control retinal ECs. Data represent mean ± s.e.m. P values, two-tailed unpaired t-test. (h) RT-qPCR expression analysis of the Notch pathway components Dll4 (n = 7 control and 9 Cxcr4^iΔEC/ΔEC mice), Jag1 (n = 8 control and 12 Cxcr4^iΔEC/ΔEC mice) Hey1 (n = 4 control and 10 Cxcr4^iΔEC/ΔEC mice) and Hes1 (n = 5 control and 12 Cxcr4^iΔEC/ΔEC mice) in sorted Cxcr4^iΔEC/ΔEC and control retinal ECs. Data represent mean ± s.e.m.P values, two-tailed unpaired t-test.