Fig. 4

VEGFR is required for endothelial cell activation of pERK1/2 and vasculogenesis in the developing testis. Section and wholemount immunofluorescent imaging of E12.5 testes cultured for 24h with DMSO or 100, 500 or 2500 nM of VEGFRi. A IF in sections of cultured testes stained with DAPI (blue), pERK1/2 (red) and CD31 (endothelial cells and germ cells; cyan). B Three-dimensional stacked Z-series images of cultured testes showing Oct4-eGFP (germ cells; green), CD31 (endothelial cells and germ cells; blue) and pERK1/2 (red). The DMSO and VEGFRi images should be viewed in conjunction with Movies S9-12 which reveal the 3D organisation of the cells in the tissue. Notably, in the VEGFRi treatment this reveals a thin surface epithelium positive for pERK1/2, but negative for CD31 in the VEGFRi treated samples. Scale bar represents 100 μm. Arrows indicate endothelial cells and asterisks indicate pERK1/2 positive Sertoli cells. Biological replicates; n = 3–4 per treatment