Figure 3. Nrp1 acts as downstream effector of Notch.

(a–d) Representative images of the Isolectin-B4-stained retinal vasculature, of P5 Nrp1^fl/fl and Nrp1^fl/fl; Cdh5-CreERT2 mice, injected with 100 μg tamoxifen at P1/P2 and treated with 100 mg kg⁻¹ DAPT at P4; scale bar, 100 μm. (a) Retinal vessels of Nrp1^fl/fl mice; (b) DAPT treatment of Nrp1^fl/fl; (c) Nrp1^fl/fl; Cdh5-CreERT2; (d) DAPT-treated Nrp1^fl/fl; Cdh5-CreERT2 mice. n=number of retinas examined; n=4 (a), n=4 (b), n=6 (c) and n=6 (d). (e–g) P5 retinal vasculature of wt blastocysts expressing DsRED injected with Nrp1^lacZ/+ ES cells, treated with DAPT (100 mg kg⁻¹) at P4. (e) Representative overview of the sprouting front; scale bar, 420 μm. (f) Segmented images; wt nuclei (white) and Nrp1^lacZ/+ nuclei (green); scale bar: 20 μm. (g) Quantification of Nrp1^lacZ/+ cells at the tip, normalized to overall contribution of cells to the endothelium; n=6 (number of retinas analysed); P<0.0001. (h–k) P5 retinas of Nrp1^fl/fl; mTmG; Cdh5-CreERT2 mice injected with 30 μg tamoxifen at P1, and treated with 100 mg kg⁻¹ DAPT at P4. Unrecombined wt cells are labelled with Isolectin-B4 only; recombined Nrp1-deficient cells express GFP. (h) Representative overview of MOCK control. (i) Representative overview of DAPT-treated animals; scale bar, 100 μm, magnification is shown in j; scale bar, 20 μm. (k) Quantification of recombined Nrp1-deficient cells at the tip, normalized to overall contribution of cells to the endothelium. Statistical significance was determined by comparing the proportion of Nrp1-deficient (green) cells at the tip with the total proportion of Nrp1-deficient (green) cells; n=3 (number of retinas analysed); P<0.0013. (g,k) Values represent mean±s.e.m. Statistical significance was assessed using a Student's unpaired t-test.