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Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Gelfand MV, Hagan N, Tata A, Oh WJ, Lacoste B, Kang KT, Kopycinska J, Bischoff J, Wang JH, Gu C - Elife (2014)

Bottom Line: Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis.Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2.Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.

ABSTRACT
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

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Screening and verification of ES cells for the generation of the Nrp1VEGF− 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 Nrp1VEGF− homozygous mutants. The boxed region indicates the altered codon.DOI:http://dx.doi.org/10.7554/eLife.03720.008
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fig3s1: Screening and verification of ES cells for the generation of the Nrp1VEGF− 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 Nrp1VEGF− homozygous mutants. The boxed region indicates the altered codon.DOI:http://dx.doi.org/10.7554/eLife.03720.008

Mentions: A gene replacement strategy was implemented to generate a mouse line harboring the Nrp1D320K mutation in the endogenous Nrp1 locus, delineated as Nrp1VEGF−. Specifically, two base pair mutations were introduced into exon 6 of the mouse Nrp1 gene to produce the D320K mutation in the endogenous Asp320 location (Figure 3A). After recombineering, embryonic stem cells were screened via PCR and sequenced to confirm that the D320K mutation was appropriately introduced into the Nrp1 locus (Figure 3—figure supplement 1A–C). Once Nrp1VEGF− mice were obtained, the presence of the D320K mutation was verified by sequencing (Figure 3—figure supplement 1D). Importantly, the Nrp1VEGF− mutants expressed normal levels of NRP1 protein as assessed by Western blot on embryonic day 14.5 (E14.5) lung and adult heart, brain, lung and kidney (Figure 3C, Figure 3—figure supplement 2D). AP-VEGF and AP-SEMA3A binding was examined at E12.5 in the dorsal root entry zone (DREZ), where NRP1-expressing axons from the dorsal root ganglion enter the spinal cord. Both AP-VEGF and AP-SEMA3A bound to the DREZ in control animals (Figure 3B) while AP-VEGF binding to the DREZ was abolished in the Nrp1VEGF− mutant (Figure 3B), confirming that this mutation eliminated VEGF-NRP1 binding in vivo. Moreover, NRP1 immunostaining and AP-SEMA3A binding to the DREZ appeared similar between Nrp1VEGF− and control littermates (Figure 3B). Finally, the Nrp1VEGF− mutants failed to display the perinatal lethality or cardiac defect observed in the Nrp1Sema− mutants (Gu et al., 2003), further confirming functional SEMA3-NRP1 binding in Nrp1VEGF− mice (Figure 3—figure supplement 1).10.7554/eLife.03720.007Figure 3.Nrp1VEGF- mice selectively abolish VEGF-NRP1 binding in vivo.


Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Gelfand MV, Hagan N, Tata A, Oh WJ, Lacoste B, Kang KT, Kopycinska J, Bischoff J, Wang JH, Gu C - Elife (2014)

Screening and verification of ES cells for the generation of the Nrp1VEGF− 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 Nrp1VEGF− homozygous mutants. The boxed region indicates the altered codon.DOI:http://dx.doi.org/10.7554/eLife.03720.008
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197402&req=5

fig3s1: Screening and verification of ES cells for the generation of the Nrp1VEGF− 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 Nrp1VEGF− homozygous mutants. The boxed region indicates the altered codon.DOI:http://dx.doi.org/10.7554/eLife.03720.008
Mentions: A gene replacement strategy was implemented to generate a mouse line harboring the Nrp1D320K mutation in the endogenous Nrp1 locus, delineated as Nrp1VEGF−. Specifically, two base pair mutations were introduced into exon 6 of the mouse Nrp1 gene to produce the D320K mutation in the endogenous Asp320 location (Figure 3A). After recombineering, embryonic stem cells were screened via PCR and sequenced to confirm that the D320K mutation was appropriately introduced into the Nrp1 locus (Figure 3—figure supplement 1A–C). Once Nrp1VEGF− mice were obtained, the presence of the D320K mutation was verified by sequencing (Figure 3—figure supplement 1D). Importantly, the Nrp1VEGF− mutants expressed normal levels of NRP1 protein as assessed by Western blot on embryonic day 14.5 (E14.5) lung and adult heart, brain, lung and kidney (Figure 3C, Figure 3—figure supplement 2D). AP-VEGF and AP-SEMA3A binding was examined at E12.5 in the dorsal root entry zone (DREZ), where NRP1-expressing axons from the dorsal root ganglion enter the spinal cord. Both AP-VEGF and AP-SEMA3A bound to the DREZ in control animals (Figure 3B) while AP-VEGF binding to the DREZ was abolished in the Nrp1VEGF− mutant (Figure 3B), confirming that this mutation eliminated VEGF-NRP1 binding in vivo. Moreover, NRP1 immunostaining and AP-SEMA3A binding to the DREZ appeared similar between Nrp1VEGF− and control littermates (Figure 3B). Finally, the Nrp1VEGF− mutants failed to display the perinatal lethality or cardiac defect observed in the Nrp1Sema− mutants (Gu et al., 2003), further confirming functional SEMA3-NRP1 binding in Nrp1VEGF− mice (Figure 3—figure supplement 1).10.7554/eLife.03720.007Figure 3.Nrp1VEGF- mice selectively abolish VEGF-NRP1 binding in vivo.

Bottom Line: Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis.Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2.Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.

ABSTRACT
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

Show MeSH
Related in: MedlinePlus