The cytoplasmic domain of neuropilin 1 is dispensable for angiogenesis, but promotes the spatial separation of retinal arteries and veins

Abstract

Neuropilin 1 (NRP1) is a transmembrane glycoprotein that is essential for blood vessel development in vertebrates. Best known for its ability to bind members of the vascular endothelial growth factor (VEGF) and class 3 semaphorin families through its extracellular domain, it also has a highly conserved cytoplasmic domain, which terminates in a SEA motif that binds the PDZ protein synectin/GIPC1/NIP. Previous studies in zebrafish embryos and tissue culture models raised the possibility that the SEA motif of NRP1 is essential for angiogenesis. Here, we describe the generation of mice that express a form of NRP1 that lacks the cytoplasmic domain and, therefore, the SEA motif (Nrp1(cyto)(Δ)(/)(Δ) mice). Our analysis of pre- and perinatal vascular development revealed that vasculogenesis and angiogenesis proceed normally in these mutants, demonstrating that the membrane-anchored extracellular domain is sufficient for vessel growth. By contrast, the NRP1 cytoplasmic domain is required for normal arteriovenous patterning, because arteries and veins crossed each other at an abnormally high frequency in the Nrp1(cyto)(Δ)(/)(Δ) retina, as previously reported for mice with haploinsufficient expression of VEGF in neural progenitors. At crossing sites, the artery was positioned anteriorly to the vein, and both vessels were embedded in a shared collagen sleeve. In human eyes, similar arteriovenous crossings are risk factors for branch retinal vein occlusion (BRVO), an eye disease in which compression of the vein by the artery disrupts retinal blood flow, causing local tissue hypoxia and impairing vision. Nrp1(cyto)(Δ)(/)(Δ) mice may therefore provide a suitable genetic model to study the aetiology of BRVO.

Fig. 1.

Generation of mouse mutants lacking the NRP1 cytoplasmic domain. (A) The targeting construct contains a stop codon after the sequence encoding the transmembrane domain. (B,C) Southern blot of EcoRI-digested genomic DNA from neomycin-resistant ES cell clones (B) and gene-targeted mice after removal of the neomycin cassette (C) with the 3′ probe (orange). (D) PCR analysis of genomic mouse DNA with the oligonucleotides indicated in A. (E,F) cDNA sequencing of E9.5 embryos; sequencing traces in the mutated area and the predicted amino acid sequence are shown above the nucleotide sequence. Wild-type cDNA (E) encodes an extracellular (red; only the C-terminal end is shown), a transmembrane (blue) and a cytoplasmic (green) domain. Mutant cDNA (F) contains a 4 bp insertion (AATT, red), which introduces a stop codon after the transmembrane domain and shifts the reading frame. (G) Immunoblot analysis of protein from wild-type and Nrp1^cytoΔ/Δ primary cardiac endothelial cells with a NRP1 cytoplasmic domain antibody (top panel; αC); the blot was stripped and reprobed with an antibody for the NRP1 extracellular domain (bottom panel; αN).

Fig. 2.

The NRP1 cytoplasmic domain is not essential for embryonic vasculogenesis or angiogenesis. (A-F′) Immunostaining of E9.5 littermates with endomucin (green) reveals microvessels and pharyngeal arch arteries (asterisks in A,D); higher magnification images show head vessels (B,E), ISVs and microvessels at forelimb level (C,F); an antibody for the NRP1 extracellular domain stains blood vessels (endomucin/NRP1-double positive vessels appear yellow) and trigeminal ganglia (red; arrowheads in B′,E′). (G-I) PECAM immunohistochemistry of E12.5 hindbrains expressing (G) or lacking (H) the NRP1 cytoplasmic domain or lacking NRP1 entirely (I). Scale bars: 500 μm in A,D; 200 μm in B-C′,E-F′; 100 μm in G-I. (J) Quantitation of vascular density in the E12.5 hindbrain SVP of Nrp1^cytoΔ/Δ and wild-type littermates; P>0.05; n≥4. Data are mean±s.d.

Fig. 3.

Increased incidence of arteriovenous crossing points in the Nrp1^cytoΔ/Δ retina. (A-B′,E-H) Immunostaining of retinas for IB4 and SMA at P7 (A,B) or for SMA at P21 (G,H); boxed areas are shown at higher magnification in E,F. Scale bars: 1 mm in A,B; 50 μm in G,H. (C,D) Quantitation of vascular extension and SMC coverage, expressed as fold change relative to wild-type littermates; n≥8 in C, n≥4 in D; P>0.05. (I) Quantitation of artery number; n≥8; P>0.05. (J) Quantitation of artery/vein crossings; n≥14; ^***P<0.001. Data are mean±s.d.

Fig. 4.

Anatomy of arteriovenous crossings in Nrp1^cytoΔ/Δ retinas. (A,B) Confocal z-stacks of arteriovenous crossings (solid arrowheads) in Nrp1^cytoΔ/Δ retinas, stained for IB4 (blue) and NRP2 (red in A) at P7 or IB4, NRP1 (red in B) and collagen IV (not shown in B) at P21. The artery (a) is strongly positive for IB4 and NRP1, the vein (v) for IB4 and NRP2; axons underneath the vessel plexus are NRP2-positive. (A′-B′) Anterior (A′,B′) and posterior angle (A′,B′) snapshots of IB4 staining after three-dimensional surface rendering of z-stacks. (C) Selected x and y coordinates of optical cross-sections through the z-stack in B; only collagen IV is shown (green); arrows indicate basement membrane separating artery and vein; arrowheads indicate a contiguous extracellular matrix wrapping artery and vein. Scale bars: 50 μm.