Fig. 5. Replication, sprouting and migration in Nrp1^-/- yolk sacs. (A) Endothelial replication was quantitated using double staining with phospho-histone 3 and Pecam1 at different stages of development. No statistically significant difference was seen at any of the stages examined. (B) The number of Pecam1-positive unconnected segments extending into avascular spaces, which may represent sprouts or regressing vessels, was assessed from images of the yolk sac at E9.5. No significant differences were observed between the different groups. (C-G) Endothelial cell migration was assessed by focal injection of cell tracker CM-DiI into a capillary region of the yolk sac followed by quantification of the maximum migration distance per embryo (C). The initial injection site can be observed in the white light images (red arrows, D,E). Endothelial cells did not migrate very far away from the site of injection in wild-type embryos (F) but were seen migrating longer distances along existing capillaries for knockout embryos (arrowheads, G). ^*P<0.05, Student's two tailed t-test. Scale bar: 1 mm.