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First published online 4 March 2009
doi: 10.1242/dev.034199

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The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf

¹ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
² Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
³ Department of Orthopaedics, Kyoto University, Kyoto 606-8507, Japan.
⁴ Genentech, 1 DNA Way, S. San Francisco, CA 94080, USA.

View larger version (80K): [in this window] [in a new window]   Fig. 1. Vascular and skeletal developments are coordinated. (A-D) Immunofluorescence staining of wild-type embryos with anti-CD31 (green) shows vascular distribution of E10.5 (A), E11.5 (B) and E12.5 (C) whole limbs and interdigital vasculature near the avascular area of future metacarpal (D). Yellow arrows indicate axial arteries; yellow arrowheads indicate vascular-rich stems that divide future metacarpals; white line indicates area of autopod; broken white line indicates area of radius and ulna. (E-J) Immunofluorescence staining with anti-CD31 (vascular endothelial cells, green) and anti-collagen II (chondrocytes, red) antibodies illustrates vascular patterning and chondrocyte differentiation, respectively. (E,H) Vascular patterning in transverse (E) and longitudinal (H) sections of the limb bud at E10.5. (F,I) E11.5 transverse sections of autopod (F) and zeugopod (I) areas, as indicated by unbroken and broken lines in B. Circled areas in F contain mesenchymal cells that undergo differentiation into chondrocytes. (G,J) Transverse (G) and longitudinal (J) sections of the autopod at E12.5. Scale bars: 100 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 2. 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.

View larger version (69K): [in this window] [in a new window]   Fig. 3. 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.

View larger version (64K): [in this window] [in a new window]   Fig. 4. 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.

View larger version (75K): [in this window] [in a new window]   Fig. 5. 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.

View larger version (72K): [in this window] [in a new window]   Fig. 6. 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.

View larger version (51K): [in this window] [in a new window]   Fig. 7. SOX9 is involved in Vegf expression in limb mesenchyme. (A,B) Detection of Vegf expression in E12.5 control (A) and Prx1-Sox9 whole limbs (B) using X-gal staining. (C,D)qRT-PCR on AdβGal (control) and AdCre infected micromass cultures show relative expression levels of Sox9 (C) and Vegf (D) mRNA transcripts. (E) Immunofluorescence staining of Vegf in the autopod of E12.5 control and Sox9-misexpressing limbs, using anti-VEGF antibody. White arrowheads indicate areas of Vegf expression in the digits. (F) Vascular patterning of E12.5 control and Sox9-misexpressing limbs as visualized by whole-mount immunofluorescence staining with anti-CD31 antibody. Scale bars: 100 µm.

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