Enlarged vascular bed formation in VEGFR-3–deficient embryos.

(A) RT-PCR analysis of gene expression in wild-type (+/+), heterozygous mutant (+/−), and VEGFR-3 homozygous mutant (−/−) embryos at E9.5. PCR primers and animal genotypes are described in “Materials and methods.” Data are from 2 independent littermates. PCR products were electrophoresed on agarose gels. VEGFR-3 message was not detected in the VEGFR-3 homozygous mutant embryos, and message levels for the remaining VEGFRs and VEGFs were unchanged among the 3 genotypes. (B) P-Sp explants derived from E9.5 wild-type (+/+) (Bi), VEGFR-3 heterozygous mutants (+/−) (Bii), VEGFR-3 homozygous mutant (−/−) embryos (Biii), and VEGFR-3 homozygous mutant embryos (−/−) in the presence of VEGFR-3–Fc (100 μg/mL) (Bv) were cultured on OP9 cells. Note that vascular bed formation was enhanced in VEGFR-3 homozygous mutant embryo explants (Biii) compared with in wild-type (Bi) and heterozygous (Bii) littermates. The vascular beds and networks from VEGFR-3–deficient embryo P-Sp expressed VEGFR-2 (Biv). VEGFR-3–Fc inhibited the vascular bed formation from VEGFR-3 homozygous mutant embryo explant (Bv). On the other hand, VEGF-C enhanced the formation of the vascular bed in VEGFR-3–deficient embryo explants (Bvi). (C) Quantitative analysis of the vascular bed area. The vascular areas from P-Sp explants were measured. Each column represents the mean area of the vascular bed. Error bars indicate SEM (n = 4). (D) Addition of VEGFR3–Fc rescued the suppression of hematopoietic cells from VEGFR-3 homozygous mutant P-Sp in a dose-dependent manner. Error bars indicate SEM (n = 3). *P < .001, **P < .01. Scale bar indicates 1 mm (Bi-iii, v, vi), 0.5 mm (Biv).