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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.

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