Vascular endothelial growth factor D is dispensable for development of the lymphatic system

Abstract

Vascular endothelial growth factor receptor 3 (Vegfr-3) is a tyrosine kinase that is expressed on the lymphatic endothelium and that signals for the growth of the lymphatic vessels (lymphangiogenesis). Vegf-d, a secreted glycoprotein, is one of two known activating ligands for Vegfr-3, the other being Vegf-c. Vegf-d stimulates lymphangiogenesis in tissues and tumors; however, its role in embryonic development was previously unknown. Here we report the generation and analysis of mutant mice deficient for Vegf-d. Vegf-d-deficient mice were healthy and fertile, had normal body mass, and displayed no pathologic changes consistent with a defect in lymphatic function. The lungs, sites of strong Vegf-d gene expression during embryogenesis in wild-type mice, were normal in Vegf-d-deficient mice with respect to tissue mass and morphology, except that the abundance of the lymphatics adjacent to bronchioles was slightly reduced. Dye uptake experiments indicated that large lymphatics under the skin were present in normal locations and were functional. Smaller dermal lymphatics were similar in number, location, and function to those in wild-type controls. The lack of a profound lymphatic phenotype in Vegf-d-deficient mice suggests that Vegf-d does not play a major role in lymphatic development or that Vegf-c or another, as-yet-unknown activating Vegfr-3 ligand can compensate for Vegf-d during development.

FIG. 1.

Generation of Vegf-d-deficient mice. (A) Homologous recombination of the gene-targeting vector at the Vegf-d locus (wild-type allele; top) was designed to replace the signal sequence for protein secretion and the remaining sequence of the first coding exon with a LacZ-PGK-neo^r cassette. Construction of the gene-targeting vector (middle) is described in Materials and Methods, and the structure of the targeted Vegf-d allele (bottom) is shown. The Vegf-d gene consists of seven coding exons (7), but only the first two are shown here. Arrows indicate the positions of primers used to genotype mice by PCR. The position of the PCR-amplified genomic DNA probe, E1F2R1, used to screen ES cell colonies by Southern blotting is shown. The E1F2R1 probe is 5′ to the region of the Vegf-d gene that underwent homologous recombination and hybridizes to digested fragments of diagnostic sizes in wild-type and targeted ES cells (the sizes of the relevant fragments are shown at the top and bottom). Restriction sites used to construct the gene-targeting vector or digest genomic DNA for screening are also shown: H, HindIII; B, BglII; P, PvuII. (B) Southern blot analysis of genomic DNA extracted from wild-type (W9.5) and targeted (lines 2.2.36 and 2.88) ES cell clones. Genomic DNA from each ES cell line was digested with HindIII (H), BglII (B), and PvuII (P) and analyzed with the E1F2R1 DNA probe. The asterisks in the 2.88 panel indicate hybridization of the E1F2R1 probe to wild-type DNA fragments derived from copurification of PEF DNA with ES cell DNA. The migration of DNA standards (in kilobases) is shown to the left of each panel. (C) Genotypic analysis of mice by PCR. Primers VEGFCM-58 and VEGFCM-36 amplified a 461-bp DNA fragment from a wild-type Vegf-d allele, whereas primers VEGFCM-58 and GenoRC2Alt amplified a 270-bp DNA fragment from a targeted Vegf-d allele. Genotypic analysis of wild-type female (X⁺X⁺) and male (X⁺Y), homozygous Vegf-d-deficient female (X⁻X⁻) and male (X⁻Y), and hemizygous female (X⁺X⁻) Vegf-d mice is shown. The migration of DNA size standards (in base pairs) is shown to the right of the panel.

FIG. 2.

Northern and Western blot analyses of tissues and cell lines derived from Vegf-d-deficient mice. (A) Analysis of Vegf-d expression in E15.5 lungs (left panel) and PEF conditioned media (right panel) derived from wild-type (+/+) and Vegf-d-deficient (−/−) mice (line 2.2.36). Total RNA was analyzed by Northern blotting with the VDEx7 cDNA probe (the same results were obtained with the VD-VHD probe; data not shown). The signal obtained with a cDNA probe for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene is shown as a loading control for E15.5 lung RNA, and methylene blue staining of the 28S rRNA transferred to membranes is indicated as a loading control for PEF RNA. Sizes of Vegf-d transcripts (in kilobases) are indicated to the left of the panels. (B) Western blot analysis of conditioned media of PEFs derived from wild-type (+/+) and Vegf-d-deficient (−/−) embryos (line 2.2.36). Conditioned media were incubated with antiserum A2, which binds to the VHD of Vegf-d, or control antiserum raised against the mouse DokR protein (26). The immunoprecipitate was subjected to SDS-PAGE (reducing condi-tions), transferred to membranes, and blotted with affinity-purified, biotinylated serum raised against the VHD of mouse Vegf-d. The ∼21-kDa mature form, consisting of the VHD, and the ∼31-kDa derivative, consisting of the N-terminal propeptide and the VHD, were detected in conditioned media of wild-type PEFs but not in media of PEFs from Vegf-d-deficient mice. The specificity of A2 and biotinylated VEGF-D antisera for Vegf-d is indicated by the lack of the ∼31- and ∼21-kDa proteins when DokR antiserum was used in the immunoprecipitation reaction. The band of ∼55 kDa detected in all lanes represents the immunoglobulin heavy chain. The migration of molecular mass markers (in kilodaltons) is shown to the right of the panel. (C) Northern blot analysis of mouse tissues for Vegf-c mRNA. Ten micrograms of total RNA from tissues of adult wild-type (+/+) and Vegf-d-deficient (−/−) mice (line 2.2.36) was hybridized with the Vegf-c probe. The position of the 2.4-kb Vegf-c mRNA is marked to the left of the upper panel, and methylene blue staining of 28S rRNA on the filters is shown in the lower panel as a loading control.

FIG. 3.

Immunostaining of lungs for Vegfr-3. (A and C) Immunostaining of vessels (arrows) in the lungs of wild-type and Vegf-d-deficient mice, respectively, with Vegfr-3 monoclonal antibody. (B and D) Serial sections from panels A and C, respectively, in which the Vegfr-3 antibody was omitted from the immunostaining protocol. (E and F) Higher-power images of lung tissues obtained from wild-type and Vegf-d-deficient mice, respectively, and immunostained for Vegfr-3 show bronchioles (asterisks), Vegfr-3-positive lymphatic vessels (arrows), and blood vessels (arrowheads).

FIG. 4.

Visualization of lymphatic vessels and lymph nodes by dye injection. Patent Blue 5 dye was intradermally injected into the ventral skin of wild-type (+/+) and Vegf-d-deficient (−/−) mice. The dye was taken up into a large lymphatic vessel (arrow) but not an accompanying blood vessel in the ventral skin of both wild-type (A) and Vegf-d-deficient (B) mice. The dye was transported to a superficial inguinal lymph node (arrow) of wild-type (C) and Vegf-d-deficient (D) mice as well as the draining axillary lymph node (arrow) of wild-type (E) and Vegf-d-deficient (F) mice. Uptake of the dye into the paraaortic lymph node (arrows) was also observed in wild-type (G) and Vegf-d-deficient (H) mice.

FIG. 5.

Rhodamine-dextran uptake into the superficial lymphatic network of the tails of wild-type and Vegf-d-deficient mice. Rhodamine-dextran was injected into the tips of the tails of wild-type (+/+) (A) and Vegf-d-deficient (−/−) (D) mice, and uptake into the superficial lymphatic network was monitored by confocal microscopy. Uptake of the dye into lymphatic drainage ducts (arrows) of wild-type (C) and Vegf-d-deficient (F) mice was observed at higher magnifications. Autofluorescence in the tails of wild-type (B) and Vegf-d-deficient (E) mice prior to injection of the dye is also shown.