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First published online 26 January 2005
doi: 10.1242/dev.01675

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Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback

¹ Division of Biology 216-76, California Institute of Technology, 1201 E. California Boulevard, Pasadena, CA 91125, USA
² Howard Hughes Medical Institute, California Institute of Technology, 1201 E. California Boulevard, Pasadena, CA 91125, USA
³ Department of Molecular Oncology, Genentech, South San Francisco, CA94080, USA
⁴ Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MA21205, USA

View larger version (68K): [in a new window]   Fig. 1. Activity of Wnt1-Cre or Isl1-Cre in peripheral nerves. The spinal cord and dorsal root ganglia (DRG) of E14.5 Rosa26 Cre-dependent lacZ reporter (R26R) line with Wnt1-Cre (A,C,E,G,I) or Isl1-Cre transgene (B,D,F,H,J) are shown. Triple immunofluorescence confocal microscopy with antibodies to ß-galactosidase (ß-gal) (red), the glial marker BFABP (blue) and the neuronal marker HuD (green) is shown. Open arrowheads indicate DRG and open arrows indicate motoneurons in the spinal cord. White arrows indicate Cre activation in motoneurons. Insets are higher-magnification details of the DRG. Scale bar: 100 µm. (K) Schematic illustrating peripheral nerve-specific Cre activation (blue).

View larger version (93K): [in a new window]   Fig. 2. VEGFA is required for arterial differentiation in limb skin. (A-F,H-M) triple immunofluorescence confocal microscopy using antibodies to either NRP1 (A-F, red, arrows and arrowhead) or CX40 (H-M, red, arrows and arrowhead), together with antibodies to PECAM1 (A-C,H-J, blue) and neurofilament (2H3) (A-C,H-J, green, open arrowheads). Arrowheads indicate large-diameter vessels. (G,N) Quantitative analysis of arterial marker expression in small-diameter vessels. Asterisks indicate statistically significant differences (P<0.05) in Vegfflox/flox or Wnt1-Cre; Vegfflox/flox compared with Wnt1-Cre; Isl1-Cre; Vegfflox/flox (Student's t-test). (O-T) Whole-mount double labeling with TUNEL (O-T, red, arrows) and PECAM1 (O-Q, blue) reveals that no significant increase in apoptosis in the absence of nerve-VEGFA, confirmed by quantitative analysis (U). Scale bars: 100µm.

View larger version (95K): [in a new window]   Fig. 3. VEGFA is required for normal DRG vascularization. (A-F) Double immunofluoresence confocal microscopy of E15.5 trunk sections performed using antibodies to PECAM1 (A-D, blue) and HuD (A,B,E,F, green). (G) Quantification of length of PECAM1+ vascular branching in DRG. The internal vascularization of the DRG (C, arrow) is disrupted in the nerve-Vegfa conditional mutants (D), while vessels peripheral to the DRG appeared unaffected (C,D, arrowheads). Asterisk indicates a statistically significant difference (P<0.05) in Vegfflox/flox compared with Wnt1-Cre; Isl1-Cre; Vegfflox/flox (Student's t-test). Scale bar: 100 µm.

View larger version (109K): [in a new window]   Fig. 4. Nerve-blood vessel alignment is unaffected in the absence of nerve-derived VEGFA. (A-H) Whole-mount triple immunofluorescence confocal microscopy using antibodies to BFABP (A-H, red), PECAM1 (A-H, blue) and neurofilament (2H3) (A-D, green) is shown. Close-up images (C,D,G,H) show the boxed regions in A,B,E,F. Arrows indicate blood vessels, arrowheads the aligned nerves and open arrowheads Schwann cells. Scale bars: 100 µm. (I) The alignment of nerves with blood vessels was quantified as the percentage of nerve length aligned with vessels.

View larger version (89K): [in a new window]   Fig. 5. Reduction of VEGFA expression by Cre-mediated recombination. (A-H) Triple immunofluorescence confocal microscopy using antibodies to VEGF (A,B,E,F, red), HuD (C-F, green) and BFABP (G,H, blue). Arrows indicate DRG; arrowheads indicate spinal cord. (I) Quantification of anti-VEGFA immunoreactivity in DRG in embryos of various genotypes. There was no consistent alteration in either HuD+ neuronal (C,D) or BFABP+ glial (G,H) development in multiple specimens. Scale bars: 100 µm. (J) The efficient deletion of the floxed Vegf allele was determined by PCR analysis of DRG. The PCR product of the recombined allele is smaller than that of the non-recombined allele.

View larger version (85K): [in a new window]   Fig. 6. NRP1 is required for arteriogenesis in limb skin. Analysis of E15.5 limbs from Tie2-Cre; Nrp1flox/- mutants or control littermates (Nrp1flox/+) is shown. (A-D,F-I) Triple immunofluorescence confocal microscopy using antibodies to either CX40 (A-D, red) or SMA (F-I, red), together with anti-PECAM1 (A,B,F,G, blue) and neurofilament (2H3) (A,B,F,G, green). (E) Quantification of CX40 expression, and (J) of SMA+ cell coverage in small-diameter vessels. Asterisks indicate statistically significant differences (P<0.05) in Tie2-Cre; Nrp1flox/- compared with Nrp1flox/+ (Student's t-test). Nerve-blood vessel alignment in the Tie2-Cre; Nrp1flox/- mutants appears normal (C versus D, H versus I, arrows and open arrowheads). Scale bars: 100 µm.

View larger version (65K): [in a new window]   Fig. 7. NRP1 expression and arterial induction by VEGF164 in vitro. (A-L) PECAM1+, ephrin B2-ß-gal- embryonic endothelial cells isolated by flow cytometry from Efnb2lacZ/+ knock-in embryos were cultured in 100 pg/ml of VEGF120 or VEGF164 plus 10 ng/ml bFGF for 2 days, and double-labeled with antibodies to either ß-gal (A-F, red) or NRP1 (G-L, red) together with PECAM1 (A-C,G-I, green). Scale bars: 100 µm. (M) Preferential induction of ephrin B2 by VEGF164. Cells as in A-L were cultured in the indicated concentrations of VEGF120 or VEGF164 for 2 days, followed by double-labeling with anti-PECAM and X-gal. Both the percentage of ECs expressing ephrin B2-lacZ (left) and the total number of ECs (right) were determined. The concentrations of VEGF120 were 1.4-fold higher, on a molar basis, then the concentration of VEGF164 used at each serial dilution. Bars represent mean±s.e.m. Asterisks indicate statistically significant differences (P<0.05) between VEGF164 and VEGF120 (Student's t-test).

View larger version (33K): [in a new window]   Fig. 8. Schematic models for nerve-mediated arterial differentiation and vascular branching in the limb skin. (A) Proposed sequence of events in vascularization of limb. A low concentration of VEGFA, or a distinct nerve-derived signal (`factor X') promotes nerve-vessel alignment, followed by VEGFA/NP-1-dependent arteriogenesis in nerve-aligned vessels. Modified from Cleaver and Krieg (Cleaver and Krieg, 1999). (B) VEGF promotes arteriogenesis via an NRP1-mediated positive-feedback loop. All vessels (blue) are initially equivalent. Nerve-derived VEGFA promotes arterial differentiation and NRP1 amplifies the VEGFA effect due to increased sensitivity to VEGF164 in vessels in close proximity to nerves (N, green). A, artery.