Figure 3. Nrp1^VEGF- mice selectively abolish VEGF-NRP1 binding in vivo.

(A) Targeting vector design for the generation of Nrp1^VEGF− mice. The WT genomic region contained residue D320 in exon 6 of Nrp1. The targeting vector (TV) introduced the D320K mutation along with an Frt-flanked NeoR cassette to form the targeted allele (TA). After FlpE-mediated excision of the NeoR cassette, the final targeted allele (FTA) had the D320K mutation as well as one remaining Frt site. (B) Section binding assays demonstrated that AP-VEGF binding to the dorsal root entry zone (DREZ) was abolished in the Nrp1^VEGF− mutants (arrows, left panels) while AP-SEMA3A binding to the DREZ appeared similar between Nrp1^VEGF− and control animals (arrows, middle panels). Scale bar: 100 μm. (C) Western blot from E14.5 lung tissue shows that NRP1 protein level was not affected in Nrp1^VEGF− animals. (D and E) Nrp1^VEGF− mutants appear indistinguishable from controls littermates at embryonic (E14.5) and adult stages. (F) Nrp1^VEGF− mutants exhibit normal body weight in adulthood (n = 7, males).

DOI: http://dx.doi.org/10.7554/eLife.03720.007

Figure 3—figure supplement 1. Screening and verification of ES cells for the generation of the Nrp1^VEGF− mutant.

(A) Diagram of the Nrp1 genomic region following successful homologous recombination to insert the targeting vector. The green arrows indicate the primers used in (B), while the blue arrows represent the primers used in (C). (B) PCR screening for the proper insertion of the 3′ homology arm. The 5′ primer was located in the NeoR sequence while the 3′ primer bound to an area outside of the targeting vector. Therefore, WT colonies did not produce a band, while correctly targeted clones produced a band of 1.7 kb. (C) PCR screening for the proper insertion of the 5′ homology arm. The 5′ primer was located outside of the targeting vector area and the 3′ primer was located within the genomic sequence present in the 3′ homology arm. Thus, PCR from a properly targeted clone produced a fragment that was 1.5 kb larger than a negative colony. (D) Sequencing of the D320K region in WT and Nrp1^VEGF− homozygous mutants. The boxed region indicates the altered codon.

DOI: http://dx.doi.org/10.7554/eLife.03720.008

Figure 3—figure supplement 2. The Nrp1^VEGF− mutant mice exhibit normal gross morphology.

(A) Whole-mount images of the heart at P9 show the normal cardiac morphology of the Nrp1^VEGF− mutants. (B and C) Organ weights measured at P9 (B) and adulthood (C) demonstrate that the heart, brain, lung, and kidney undergo appropriate growth in Nrp1^VEGF− animals, n ≥ 5. (D) Western blots from adult heart, brain, lung, and kidney tissue demonstrate that NRP1 protein levels were not affected in Nrp1^VEGF− animals. (E) Viability table depicts the predicted and observed frequencies for each genotype at the indicated developmental stages. The table values represent the percentage of the total number of animals genotyped per age while the total number of animals is shown in parentheses.

DOI: http://dx.doi.org/10.7554/eLife.03720.009