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First published online 11 April 2007
doi: 10.1242/dev.002402

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Selective requirements for NRP1 ligands during neurovascular patterning

Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.

View larger version (36K): [in this window] [in a new window]   Fig. 1. Working models for the role of class 3 semaphorins in mouse vascular development. (A) NRP1 contains several distinct structural domains that cooperate to mediate binding of class 3 semaphorins and VEGF164; the a1 domain is crucial for binding the SEMA domain, the b1 domain for VEGF164 binding. (B) Based on tissue culture models, it has been suggested that SEMA3A modulates VEGF signalling by competing with VEGF164 for binding to NRP1. (C) SEMA3A may signal directly through complexes containing NRP1 and A-type plexins in endothelial cells, as observed in neurons. SEMA3E and possibly other class 3 semaphorins may influence vascular development by signalling through plexin D1-NRP1 complexes (D) and/or through plexin D1 in a mechanism that does not require NRP1 (E). (F) VEGF164 has been implicated as a modifier of neuronal growth and axon guidance based on its ability to compete with SEMA3A for NRP1 binding in a neuronal progenitor cell line in vitro.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Distinct binding specificity of different class 3 semaphorins and VEGF164 for blood vessels and nerves. AP-tagged VEGF164 (A,G), SEMA3A (B,H), SEMA3C (C,I), SEMA3E (D,J), SEMA3F (E,K) and AP alone as a negative control (F,L), were reacted with 11.25 dpc mouse hindbrain tissue to examine their binding preference for growing vessels in the subventricular zone (A-F) or axon tracts on the pial brain surface (G-L; arrowheads); binding to vessels entering the brain from the perineural vascular plexus is indicated with arrows. Each panel is 0.25 mm2.

View larger version (86K): [in this window] [in a new window]   Fig. 3. SEMA3A is not required for vasculogenesis or angiogenesis. Visualisation of the cardiovasculature in stage-matched littermate mouse embryos expressing (A,C,E,G) or lacking (B,D,F,H) SEMA3A in a CD1 background. (A,B) At 9.5 dpc, endomucin-positive vessel networks have extended throughout the embryo and have begun to remodel into the large head vessels. The anterior cardinal vein (arrowhead), dorsal aorta (arrow) and the third and fourth aortic arch arteries are also clearly visible. (C-F) Higher magnification of intersomitic vessels (isv) in the trunk region between the forelimb and hindlimb buds at 9.5 (C,D) and 10.5 (E,F) dpc. Vessel branches extend between intersomitic vessels at 10.5 dpc (wavy arrows), but are rarely seen at 9.5 dpc in this region. (G-J) Double labelling of PECAM-positive vessels and neurofilament-positive nerves at 10.5 dpc reveals axon defasciculation of cranial nerves VII, IX and X (compare I and J), but no vessel defects (compare G and H) in embryos lacking SEMA3A. The heart ventricles (v), atria (a) and fourth and sixth aortic arch arteries are clearly visible in embryos lacking SEMA3A. Scale bar: 250 µm in C,D; 500 µm in A,B,E-J.

View larger version (98K): [in this window] [in a new window]   Fig. 4. Loss of heparin/neuropilin-binding VEGF isoforms, but not loss of SEMA3A, impairs brain vascularisation. Visualisation (A-F) and quantitation (G) of vessel branching in the subventricular zone of 12.5 dpc mouse hindbrains. PECAM-positive vessels are shown in a 0.25 mm2 area of littermate hindbrains with normal (Vegfa+/+) or reduced (Vegfa+/120 and Vegfa120/120) levels of heparin/neuropilin-binding VEGF isoforms (A-C), or in littermate hindbrains expressing (Sema3a+/+) or lacking (Sema3a+/-and Sema3a-/-) SEMA3A (D-F). (G) Quantitation of vessel branching in hindbrains lacking heparin/neuropilin-binding VEGF isoforms or SEMA3A in a CD1 or C57Bl/6 background. Wild types (Vegfa+/+ or Sema3a+/+), dark grey; heterozygous mutants (Vegfa+/120 or Sema3a+/-), light grey; homozygous mutants (Vegfa120/120 or Sema3a-/-), white. LOF, loss of function.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Loss of semaphorin signalling through neuropilins does not impair brain vascularisation. Visualisation (A-D) and quantitation (E) of vessel branching in 12.5 dpc mouse hindbrains. (E) Hindbrains with normal neuropilin function (A) showed a similar amount of vessel branching per 0.25 mm2 as hindbrains lacking semaphorin signalling through NRP1 (B) or NRP2 (C) or lacking both NRP2 and semaphorin signalling through NRP1 (D).

View larger version (112K): [in this window] [in a new window]   Fig. 6. SEMA3A does not affect vascular patterning in the absence of heparin/neuropilin-binding VEGF isoforms. (A-D) The cardiovasculature of 9.5 dpc littermate mouse embryos derived from matings between Sema3a+/- Vegfa+/120 double-heterozygous mice was labelled with antibodies specific for endomucin (blood vessels, green) and smooth muscle actin (heart, red). The paired dorsal aorta and anterior cardinal vein, as well as the pharyngeal arch arteries and heart, were present in all four genotypes. (E-G) Higher magnification of the region containing the dorsal aorta (arrowhead) and anterior cardinal vein (arrow) shows the presence of both these large vessels in littermate wild-type (E), Vegfa120/120 (F) and Sema3a-/- Vegfa120/120 (G) embryos. Note that the strong smooth muscle actin staining of the heart necessitated the use of scanning parameters that did not record staining of the dorsal aorta, even though it was immunoreactive. Scale bars: 500 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 7. Loss of SEMA3A does not rescue the vessel defects caused by the loss of heparin/neuropilin-binding VEGF isoforms. Visualisation (A-F) and quantitation (G) of vessel branching in 12.5 dpc mouse hindbrains derived from Sema3a+/- Vegfa+/120 matings. (A-F) PECAM-positive vessels in 0.25 mm2 areas of hindbrains expressing (Vegfa+/+) or lacking (Vegfa+/120 and Vegfa120/120) heparin/neuropilin-binding VEGF differed with respect to the presence (A-C) or absence (D-F) of SEMA3A. (G) Data from hindbrains expressing (Sema3a+/+) or lacking SEMA3A (Sema3a-/-) were grouped according to their level of VEGF isoform expression. Normal 12.5 dpc hindbrains express more VEGF164 than VEGF120 (Vegfa+/+); mutation of one Vegfa allele increases VEGF120 at the expense of VEGF164 (Vegfa+/120); mutation of both alleles ablates VEGF164 expression (Vegfa120/120) (see Ruhrberg et al., 2002).

View larger version (47K): [in this window] [in a new window]   Fig. 8. SEMA3A patterns axons and VEG164 patterns vessel networks in developing mouse limbs. Double label immunohistochemistry for nerves (A-E) and vessels (F-J) in stage-matched wild-type limbs (Nrp1+/+; A,F) and limbs lacking SEMA3A (B,G), VEGF164 (C,H), SEMA3A and VEGF164 (D,I) or NRP1 (E,J) at 12.5 dpc. (A,F) Vessels and nerves grow in close spatiotemporal proximity on the ventral aspect of the forelimb. The two major nerve branches entering the ventral footplate are normally well separated (asterisk). The microvessel network extends throughout the limb, including the areas under which the cartilage anlagen for the digits will form (one such area is indicated with an arrow). Regions of increased vascular density prefigure the sites where major arterial branches supplying the digits will form (one such area is indicated by an arrowhead in F). Note the axon defasciculation in B,D,E; the reduced microvessel branching (arrowheads) and smaller limb size in H-J; and a slight delay in axon extension, but normal axon patterning, in C. Scale bar: 500 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 9. Loss of VEGF164 but not SEMA3A impairs microvessel branching in the mouse limb. Forelimb vasculature at 12.5 dpc in stage-matched wild-type limbs (Sema3a+/+; A,F), limbs lacking SEMA3A (B,G), VEGF164 (C,H), SEMA3A and VEGF164 (D,I) or NRP1 (E,J). Microvessel networks appear normal in the absence of SEMA3A, whereas loss of VEGF164 or NRP1 impairs vessel branching. Vascular defects in limbs lacking both SEMA3A and VEGF164 are similar to those in limbs lacking only VEGF164. Scale bars: 100 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 10. Loss of SEMA3A but not VEG164 affects sensory nerve branching in the mouse limb. (A-F) Visualisation of limb nerves at 13.5 dpc in wild-type forelimbs (Vegfa+/+; A,B) and forelimbs lacking VEGF164 (Vegfa120/120; C,D) or NRP1 (Nrp1-/-; E,F). Ventral (A,C,E) and dorsal (B,D,F) aspects of the forelimb are shown. Note that the delay in axon extension observed at 12.5 dpc (Fig. 8) is no longer apparent at 13.5 dpc. (G) Quantitation of sensory nerve branching in the dorsal footplate (i.e. above the grey line in B,D,F) of wild-type limbs (combined data for Vegfa+/+, Sema3a+/+ and Nrp1+/+; n=17) and limbs lacking VEGF164 (n=14), SEMA3A (n=5) or NRP1 (n=6).