No vascular patterning in Prx1-Sox9 limbs. Knockout of Sox9 expression in the limb mesenchyme before the formation of condensations prevented mesenchymal condensation and skeleton formation. Examination of E10.5-E12.5 control and Prx1-Sox9 limb vasculature revealed that, in the absence of skeleton formation, the vasculature failed to pattern normally. (A-F) Whole limbs of control (A,C,E) and Prx1-Sox9 (B,D,F) embryos immunostained with anti-CD31 antibody (green) for vascular endothelial cells. (A,B) E10.5; (C,D) E11.5; (E,F) E12.5. Yellow arrows indicate axial arteries; yellow arrowheads indicate metacarpal vascular centers in control limbs and a single center in Prx1-Sox9 limbs; red arrowhead indicates avascular areas in control limbs. (G-L) Immunostaining of control (G,I,K) and Prx1-Sox9 (H,J,L) sections with anti-CD31 for vascular endothelial cells (green) and anti-collagen II for chondrocytes (red). Longitudinal sections (G,H), and transverse sections of autopod (I,J) and zeugopod (K,L). Scale bars: 100 μm in A-F; 200 μm in G-L.

Expression of Vegf in condensed mesenchymal cells. Using mice with an IRES-LacZ reporter cassette inserted into the 3′UTR of Vegf gene (Vegf-lacZ), we detected a dynamic expression of Vegf in the limb. (A-D) X-gal staining of E10.5 (A) and E11.5 (B) whole limbs, and E12.5 (C) and E13.5 (D) longitudinal sections. Black arrows indicate area of digit; black arrowheads indicate areas of radius and ulna. (E,F) X-gal staining of E13.5 transverse sections of the zeugopod, showing Vegf-lacZ expression in areas of forming muscles (red arrows, E), and of the autopod, showing Vegf-lacZ expression in areas of forming tendons (blue arrows, F; u, ulna; r, radius; a, artery; c, condensation). (G) Double immunofluorescence staining showing Vegf expression in areas several rows of cells away from endothelial cells, using anti-VEGF (red) and anti-CD34 (green) antibodies. Scale bars: 1 mm in A-D; 200 μm in E,F; 100 μm in G.

Overexpression of Vegf in condensed mesenchyme increases limb vascularization. (A,B) Immunofluorescence staining with anti-CD31 (green), a marker for vascular endothelial cells, shows vasculature of E12.5 control (A) and Vegf overexpressed (B) whole limbs. (C,D) Enlargements of the blue boxes in A,B that demarcate metacarpal vascular centers; (E,F) enlargements of the red boxes in A,B that demarcate interdigital areas. (G,H) Immunostaining of control (G) and Prx1-Sox9 (H) longitudinal sections with anti-CD31 for vascular endothelial cells (green) and anti-collagen II for chondrocytes (red). Scale bars: 100 μm.

Lack of Vegf in limb mesenchyme results in an absence of vascular morphogenesis. (A-F) Immunofluorescence with anti-CD31 for vascular endothelial cells (green) demonstrates the vasculature of control (A,C,E) and Prx1-Vegf (B,D,F) whole limbs at E10.5 (A,B), E11.5 (C,D) and E12.5 (E,F). (G-J) Longitudinal (G,H) and transverse (I,J) sections of E12.5 control (G,I) and Prx1-Vegf (H,J) limbs stained with anti-CD31 for vascular endothelial cells (G-J; green) and anti-collagen II for chondrocytes (I,J; red). Yellow arrows indicate axial arteries; yellow arrowheads indicate metacarpal vascular centers. Scale bars: 100 μm in A-F and I-J; 200 μm in G,H.

Expression patterns of VEGF receptors in the limb. (A) Detection of VEGF receptors in E12.5 longitudinal sections: in situ hybridization of Flt1 and immunofluorescence of Flk1 using RAFL-1 antibody and of Nrp1 using anti-NRP1 antibody show their expression in endothelial cells. Immunofluorescence of Nrp2 (red) and condensed mesenchyme (green) using anti-β-galactosidase and biotin labeled peanut agglutinin, respectively. Scale bars: 100 μm in Flt1, Flk1, Nrp2; 50 μm in Nrp1. (B) Immunofluorescence staining with anti-CD31 (green) as a marker for vascular endothelial cells reveals that an ablation of VEGF receptors in mesenchyme does not affect vascular patterning: E12.5 longitudinal sections of control and Prx1-Flt1, Prx1-Flk1 and Prx1-Nrp1, Nrp2 limbs. Scale bar: 100 μm.