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First published online 11 June 2008
doi: 10.1242/dev.014902

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Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos

¹ INSERM U833, F-75005, Paris, France.
² Collège de France, 11 Place Marcelin Berthelot, 75005 Paris, France.
³ School of Life Science, Xiamen University 361005 Xiamen, Fujian, China.
⁴ Department of Tumor Biology and Angiogenesis, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.

View larger version (80K): [in this window] [in a new window]   Fig. 1. Cardiovascular phenotype of Nrp1-/- embryos. Dark-field images of freshly dissected yolk sacs from E9.5 embryos with the indicated genotypes (A,E) shows defects in yolk-sac vascular remodeling that are further highlighted by Pecam1 staining of the wild-type (B) and mutant (F) yolk sacs. Angiograms at E8.5, just after the onset of plasma flow (C,G) show that vessels are lumenized in both wild-type (C) and knockout (G) embryos, but that Nrp-1-/- already show altered yolk sac vessel geometry (G). Pecam1 staining of yolk sacs (D,H) were used to quantitate the phenotype based on the number of branchpoints per mm2 (I), average vessel segment length (J) and area of avascular spaces (K) at different stages of development for wild-type and knockout embryos. *P<0.05, **P<0.01, ***P<0.005; Student's two-tailed t-test. A, aorta; H, heart; YS, yolk sac. Scale bars: 1 mm in A,E; 500 µm in B,C,F,G; 100 µm in D,H.

View larger version (111K): [in this window] [in a new window]   Fig. 2. Investigating causes of defective flow in Nrp1-/- embryos. (A) In situ hybridization with an Nrp1 antisense riboprobe on a section from a wild-type embryo at 10 somites shows expression in the atrium (marked A) and not the ventricle (marked V), as well as expression in the dorsal aortae (arrows) and yolk-sac vessels (arrowheads). (B,D) Serial sections of E8.5 wild-type (B) and mutant (D) embryos stained for Pecam1 show that dorsal aortae (DA) are present and lumenized in both cases, though often collapsed in the mutant (D). (C) By E9.5, collagen IV staining highlights the dorsal aortae (DA), gut (G) and heart (H) of wild-type embryos. (E) In mutant embryos, one or both dorsal aortae are often missing (arrow). (F,G) Intersomitic vessel sprouting is visible in E9.5 wild-type embryos stained for Pecam1 (F) but is delayed in mutant embryos (G) and also shows the absent dorsal aorta (arrow). (H,I) The cephalic region of E9.5 wild-type embryos shows an extensive vasculature (H) that is also present in the mutant embryos (I); however, the vasculature of the mutants is more sparse and many vessels do not appear to be lumenized. The internal carotid artery (ICA) has started forming in the wild-type (H) but is lacking in the mutant (I). NT, neural tube. Scale bars: 50 µm in A,B,D; 100 µm in F-I; 200 µm in C,E.

View larger version (67K): [in this window] [in a new window]   Fig. 3. Separating the role of blood flow from Nrp1 function. (A) Pecam1 staining of yolk sacs from cultured heterozygote and mutant embryos in both flow and no-flow conditions, as indicated. In heterozygote embryos with flow, a large remodeled artery (Art) is visible. Heterozygote embryos without flow fail to remodel capillaries into large vessels. Insets show collagen IV staining on sections from E9.5 yolk sacs that also highlight the large vessel diameters in Nrp1-/- yolk sacs compared with wild type. (B-D) Nrp1-/- vessels, with or without flow, show decreased branching (B), increased vessel segment length (C) and increased avascular spaces (D). **P<0.01; ***P<0.005; Student's two-tailed t-test. Scale bar: 100 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 4. Blocking Nrp1 function reproduces the Nrp1-/- yolk sac vessel phenotype. Wild-type embryos from a CD1 background were treated with various proteins by intracardiac injection followed by whole-embryo culture. (A-D) Subsequent to culture, the vasculature was fixed and stained for Pecam1. (E-G) The images were quantified for vascular morphology. Untreated embryos showed remodeling, typical of E9.5 embryos (A). VEGF120 injection caused an increase in overall vascular coverage (B). Injection of antibodies against Nrp1, which are reported to block Sema3A binding (C) and VEGF binding (D) to Nrp1 (Pan et al., 2007b), were both effective at decreasing the number of branchpoints and increasing vessel segment length as seen in the Nrp-1-/- embryos (E,F). Injection of both antibodies did not show a synergistic effect on the number of branchpoints. Only antibodies against the VEGF-binding domain of Nrp1 were capable of increasing average area of avascular space, as seen in Nrp1-/- embryos (G). Injection of Sem3A and Nrp1 had no statistically significant effect on capillary geometry (E-G). *P<0.05, **P<0.01, ***P<0.005; Student's two-tailed t-test. Scale bars: 100 µm.

View larger version (74K): [in this window] [in a new window]   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.

View larger version (81K): [in this window] [in a new window]   Fig. 6. Arterial venous gene expression in Nrp1-/- embryos. (A-F) Serial sections were stained by in situ hybridization for Dll4 (A,B), Cx40 (C,D) and Efnb2 (E,F) in both wild-type (A,C,E) and Nrp1 knockout embryos (B,D,F) at 20 somites. Images show the common atrial chamber (CAC), the vitelline artery (VA) and the dorsal aortae (DA) at either the level of the heart (A-D) or at the caudal end of the embryo (E,F). (B) Red arrow indicates a missing dorsal aorta in the Nrp1-/- embryo; the remaining dorsal aorta still expresses Dll4. (D) Cx40 expression is absent in Nrp1-/- embryos. (F) Normal expression of Efbn2 is present in dorsal aorta and somites of Nrp1-/- embryos, but lacking in the vitelline artery. (G) Levels of gene expression were measured from isolated endothelial cells, either pooled wild-type/heterozygous (denoted wild-type) or knockout, both with and without blood flow for 11 cardiovascular genes: four pan-endothelial genes, four arterial genes and three venous genes. Gene expression was normalized to three housekeeping genes: -tubulin, β-actin and Gapdh. Expression of Cx40 and Efbn2 in Nrp1-/- is downregulated compared with wild type, regardless of flow environment. *P<0.05, **P<0.01, ***P<0.005; Student's two-tailed t-test. Scale bars: 100 µm.

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