elife03720.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN"  "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03720</article-id><article-id pub-id-type="doi">10.7554/eLife.03720</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-15537"><name><surname>Gelfand</surname><given-names>Maria V</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-15538"><name><surname>Hagan</surname><given-names>Nellwyn</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-15539"><name><surname>Tata</surname><given-names>Aleksandra</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15540"><name><surname>Oh</surname><given-names>Won-Jong</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15541"><name><surname>Lacoste</surname><given-names>Baptiste</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15542"><name><surname>Kang</surname><given-names>Kyu-Tae</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="aff" rid="aff7"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-18980"><name><surname>Kopycinska</surname><given-names>Justyna</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15543"><name><surname>Bischoff</surname><given-names>Joyce</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-14719"><name><surname>Wang</surname><given-names>Jia-Huai</given-names></name><xref ref-type="aff" rid="aff5"/><xref ref-type="aff" rid="aff6"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-9212"><name><surname>Gu</surname><given-names>Chenghua</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-5"/><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Neurobiology</institution>, <institution>Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution>Korea Brain Research Institute</institution>, <addr-line><named-content content-type="city">Daegu</named-content></addr-line>, <country>Republic of Korea</country></aff><aff id="aff3"><institution content-type="dept">Vascular Biology Program</institution>, <institution>Boston Children's Hospital, Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution content-type="dept">Department of Surgery</institution>, <institution>Boston Children's Hospital, Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><institution content-type="dept">Department of Medical Oncology</institution>, <institution>Dana-Farber Cancer Institute, Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff6"><institution content-type="dept">Department of Cancer Biology</institution>, <institution>Dana-Farber Cancer Institute, Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff7"><institution content-type="dept">College of Pharmacy</institution>, <institution>Duksung Women's University</institution>, <addr-line><named-content content-type="city">Seoul</named-content></addr-line>, <country>Republic of Korea</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Nathans</surname><given-names>Jeremy</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, Johns Hopkins University School of Medicine</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>Chenghua_Gu@hms.harvard.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>22</day><month>09</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03720</elocation-id><history><date date-type="received"><day>18</day><month>06</month><year>2014</year></date><date date-type="accepted"><day>20</day><month>09</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Gelfand et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Gelfand et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03720.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03720.001</object-id><p>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 <italic>Nrp1</italic> locus that selectively abolishes VEGF-NRP1 binding (<italic>Nrp1</italic><sup><italic>VEGF−</italic></sup>). <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that <italic>Nrp1</italic>-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.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.001">http://dx.doi.org/10.7554/eLife.03720.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03720.002</object-id><title>eLife digest</title><p>Blood flows through blood vessels to carry oxygen and nutrients towards, and waste away from, the cells of the body. New blood vessels are formed not only during development but also throughout life as part of normal tissue growth and repair. However, blood vessels may also form as a consequence of diseases, such as cancer. For example, tumors often stimulate the growth of new blood vessels to ensure a good supply of blood carrying nutrients and oxygen. As such, some anti-cancer therapies try to stop blood vessels from developing in an attempt to slow down or prevent tumor growth.</p><p>New blood vessels often form by branching off from existing vessels. One molecule that stimulates this branching process is called vascular endothelial growth factor (or VEGF for short). Three ‘receptor’ proteins found on the outside of cells can bind to the VEGF molecule and then trigger a response inside the cell that guides the development of new blood vessels. VEGF and its receptor proteins—including one called NRP1—are being investigated as a possible target for drugs that could treat cancer and other diseases affecting blood vessels. However, the exact mechanisms that control the formation of new blood vessels are not fully understood, which makes it difficult to develop these treatments.</p><p>Now Gelfand et al. have created mice whose NRP1 receptors cannot bind VEGF. These mice unexpectedly survive to adulthood and develop normal blood vessels. This outcome is in contrast to mice that lack NRP1, which normally die as embryos and have severe defects with their nerves and blood vessels. Gelfand et al. instead found that mice that only lack NRP1 in the cells of their blood vessels had less of another receptor protein called VEGFR2 on the surface of these cells. This result suggests that NRP1 controls blood vessel development, not by binding to VEGF but by affecting how much of the VEGFR2 receptor is available to interact with VEGF.</p><p>These findings challenge the long-held view of how NRP1 functions and lead Gelfand et al. to suggest a new mechanism: NRP1 interacts with VEGFR2, rather than with VEGF, to control the formation of new blood vessels. Future work will aim to uncover how these interactions regulate the normal development of blood vessels, and if other molecules that bind to NRP1 are involved in this process. Furthermore, these findings may help to guide the on-going efforts to develop drugs that target NRP1 into treatments that are effective against diseases that involve problems with blood vessels—including diabetes, immune disorders, and cancer.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.002">http://dx.doi.org/10.7554/eLife.03720.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>Neuropilin-1</kwd><kwd>developmental angiogenesis</kwd><kwd>VEGFR2</kwd><kwd>VEGF</kwd><kwd>mouse genetics</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>R01 NS064583</award-id><principal-award-recipient><name><surname>Gu</surname><given-names>Chenghua</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100006691</institution-id><institution>Harvard Medical School</institution></institution-wrap></funding-source><award-id>Alice and Joseph Brooks Fund Postdoctoral Fellowship</award-id><principal-award-recipient><name><surname>Tata</surname><given-names>Aleksandra</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100006691</institution-id><institution>Harvard Medical School</institution></institution-wrap></funding-source><award-id>Harvard Mahoney Neuroscience Institute Fund Postdoctoral Fellowship</award-id><principal-award-recipient><name><surname>Lacoste</surname><given-names>Baptiste</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000879</institution-id><institution>Alfred P. Sloan Foundation</institution></institution-wrap></funding-source><award-id>Sloan Research Fellowships</award-id><principal-award-recipient><name><surname>Gu</surname><given-names>Chenghua</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100006781</institution-id><institution>Giovanni Armenise-Harvard Foundation</institution></institution-wrap></funding-source><award-id>Armenise Junior Faculty Award</award-id><principal-award-recipient><name><surname>Gu</surname><given-names>Chenghua</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100006691</institution-id><institution>Harvard Medical School</institution></institution-wrap></funding-source><award-id>Genise Goldenson Research Fund</award-id><principal-award-recipient><name><surname>Gu</surname><given-names>Chenghua</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>R01 HL096384</award-id><principal-award-recipient><name><surname>Kang</surname><given-names>Kyu-Tae</given-names></name><name><surname>Bischoff</surname><given-names>Joyce</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>T32 NS007484-12</award-id><principal-award-recipient><name><surname>Hagan</surname><given-names>Nellwyn</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Interactions between Neuropilin-1 and VEGFR2, rather than VEGF-Neuropilin-1 binding, underlie Neuropilin-1's critical function in VEGF-mediated vascular development.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Blood vessels provide oxygen and nutrients to cells throughout the body and are essential for tissue homeostasis and repair as well as tumor growth. The molecular mechanisms underlying angiogenesis have become increasingly clear, and VEGF is an essential player in this process (<xref ref-type="bibr" rid="bib2">Carmeliet et al., 1996</xref>, <xref ref-type="bibr" rid="bib3">1999</xref>; <xref ref-type="bibr" rid="bib8">Ferrara et al., 1996</xref>, <xref ref-type="bibr" rid="bib9">2003</xref>; <xref ref-type="bibr" rid="bib18">Iruela-Arispe and Dvorak, 1997</xref>; <xref ref-type="bibr" rid="bib26">Miquerol et al., 1999</xref>; <xref ref-type="bibr" rid="bib30">Ruhrberg et al., 2002</xref>; <xref ref-type="bibr" rid="bib33">Stalmans et al., 2002</xref>; <xref ref-type="bibr" rid="bib29">Rossant and Hirashima, 2003</xref>; <xref ref-type="bibr" rid="bib25">Maes et al., 2004</xref>; <xref ref-type="bibr" rid="bib5">Coultas et al., 2005</xref>; <xref ref-type="bibr" rid="bib27">Olsson et al., 2006</xref>; <xref ref-type="bibr" rid="bib4">Chung and Ferrara, 2011</xref>). VEGF operates by interacting with three receptors, VEGFR1, VEGFR2 (KDR/Flk1), and NRP1 (<xref ref-type="bibr" rid="bib9">Ferrara et al., 2003</xref>; <xref ref-type="bibr" rid="bib4">Chung and Ferrara, 2011</xref>). Although these three receptors are expressed in spatially and temporally overlapping patterns, they are thought to play different roles in VEGF signaling. The main receptor for VEGF, VEGFR2, is a receptor tyrosine kinase whose activity is crucial for VEGF signaling (<xref ref-type="bibr" rid="bib27">Olsson et al., 2006</xref>). Upon binding VEGF, VEGFR2 phosphorylates intracellular targets leading to a multitude of cellular responses including proliferation, migration, and transcriptional modification via signaling pathways such as PI3K, Src, and PLCϒ (<xref ref-type="bibr" rid="bib27">Olsson et al., 2006</xref>). In contrast, NRP1 is a multifaceted transmembrane receptor that not only binds VEGF and forms a complex with VEGFR2 but also binds a structurally and functionally unrelated family of traditional axon guidance cues, the secreted class 3 semaphorins (SEMA3) (<xref ref-type="bibr" rid="bib15">He and Tessier-Lavigne, 1997</xref>; <xref ref-type="bibr" rid="bib23">Kolodkin et al., 1997</xref>; <xref ref-type="bibr" rid="bib32">Soker et al., 1998</xref>). Consistent with these binding partners, <italic>Nrp1</italic><sup><italic>−/−</italic></sup> mice are embryonically lethal with both neural and vascular defects (<xref ref-type="bibr" rid="bib22">Kitsukawa et al., 1997</xref>; <xref ref-type="bibr" rid="bib20">Kawasaki et al., 1999</xref>), indicating that NRP1 protein is instrumental for developmental angiogenesis. However, how NRP1 functions in conjunction with multiple ligands and receptors to guide vascular development remains elusive.</p><p>Previous work has started to systematically dissect NRP1 function in vivo using a combination of structure–function analyses and mouse genetic approaches. In particular, endothelial-specific NRP1 knock-outs (<italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup>) recapitulate the devastating vascular defects observed in <italic>Nrp1</italic><sup><italic>−/−</italic></sup> mice—the vascular network is poorly developed and large endothelial cell aggregates form within the brain (<xref ref-type="bibr" rid="bib13">Gu et al., 2003</xref>). This result strongly demonstrates that NRP1 is a cell autonomously required in endothelial cells for its absolutely essential function in developmental angiogenesis. To pinpoint how SEMA3-NRP1 vs VEGF-NRP1 binding contributes to NRP1's critical role in vascular development, previous work generated a knock-in mouse line, <italic>Nrp1</italic><sup><italic>Sema−</italic></sup>, in which SEMA3-NRP1 interactions were abolished and VEGF-NRP1 binding was maintained (<xref ref-type="bibr" rid="bib13">Gu et al., 2003</xref>). <italic>Nrp1</italic><sup><italic>Sema−</italic></sup> mice mimicked the neural defects observed in the <italic>Nrp1</italic><sup><italic>−/−</italic></sup> but did not exhibit any vascular abnormalities. These data suggest that SEMA3-NRP1 binding does not mediate NRP1's important function in vascular morphogenesis and instead point to the hypothesis that VEGF-NRP1 interactions may be integral for angiogenesis.</p><p>Currently, the dominant view in the field asserts that VEGF-NRP1 binding enhances VEGFR2 activity and downstream signaling. Yet, the functional consequence of VEGF-NRP1 interactions has only been studied indirectly using in vitro methodology and blocking antibodies in vivo (<xref ref-type="bibr" rid="bib28">Pan et al., 2007</xref>; <xref ref-type="bibr" rid="bib16">Herzog et al., 2011</xref>). Specifically, an antibody inhibiting VEGF-NRP1 binding was found to interfere with retinal vascular remodeling as well as tumor angiogenesis (<xref ref-type="bibr" rid="bib28">Pan et al., 2007</xref>) and is currently being developed as a therapeutic strategy to block vessel outgrowth. This study suggests that VEGF-NRP1 binding facilitates pathological angiogenesis. However, in vivo evidence describing a role for VEGF-NRP1 binding in vascular development is currently lacking and the precise function of NRP1 in VEGF-mediated angiogenesis urgently needs to be addressed.</p><p>To delineate the role of VEGF-NRP1 interactions, we identified a single amino acid residue in the b1 domain of NRP1 that is necessary for VEGF-NRP1 binding and generated a mouse harboring this point mutation to abolish VEGF-NRP1 interactions in vivo (<italic>Nrp1</italic><sup><italic>VEGF−</italic></sup>). Surprisingly, although VEGF-NRP1 binding was successfully eliminated, the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants survived into adulthood and did not display any of the severe vascular phenotypes seen in either the <italic>Nrp1</italic><sup><italic>−/−</italic></sup> or the endothelial-specific NRP1 knock-out. Upon closer examination, NRP1-deficient blood vessels in the endothelial-specific NRP1 knock-out exhibited reduced VEGFR2 surface expression, a phenomenon not observed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant. These results challenge the well-accepted view that NRP1 requires VEGF-NRP1 binding to facilitate developmental angiogenesis and points to a provocative new hypothesis that the angiogenic role of NRP1 lies in its capacity as a VEGFR2 co-receptor. Interestingly, retinal angiogenesis and blood flow recovery following hindlimb ischemia were mildly perturbed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant suggesting that the postnatal vascular system is uniquely sensitive to the loss of VEGF-NRP1 binding. Together, this work not only significantly advances our basic scientific understanding of how NRP1 functions in VEGF-mediated angiogenesis, but also provides new insights that may facilitate the development of more effective NRP1-targeted anti-angiogenesis therapies.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Identification of an <italic>Nrp1</italic> mutation that abolishes VEGF-NRP1 binding</title><p>We sought to elucidate the in vivo function of VEGF-NRP1 binding by generating a mouse line that selectively disrupts VEGF binding to NRP1. A previous structure–function analysis revealed that the b1 domain of NRP1 is necessary and sufficient for VEGF binding (<xref ref-type="bibr" rid="bib12">Gu et al., 2002</xref>). However, this b1 region is also required for SEMA3-NRP1 interactions, so a series of <italic>Nrp1</italic> variants containing smaller deletions in the b1 domain were engineered with site-directed mutagenesis to identify a region specific for VEGF-NRP1 binding (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Based upon previous publications, we first targeted two specific sites in the b1 domain: the 7-residue binding site of the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody which competes with VEGF for NRP1 binding (<xref ref-type="bibr" rid="bib19">Jaalouk et al., 2007</xref>) and the 3-residue binding site of the VEGF analog tuftsin (<xref ref-type="bibr" rid="bib34">Vander Kooi et al., 2007</xref>) (<xref ref-type="fig" rid="fig1">Figure 1A–B</xref>). COS-1 cells were transfected with wild-type (WT) or mutant <italic>Nrp1</italic> constructs and assessed for NRP1 expression. PAL-E and tuftsin binding site mutations did not affect NRP1 protein expression at the cell surface as examined by non-permeabilized antibody staining (<xref ref-type="fig" rid="fig1">Figure 1C</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). Ligand binding to NRP1 was assessed using alkaline phosphatase-tagged VEGF (AP-VEGF) and SEMA3A (AP-SEMA3A) in conjunction with alkaline phosphatase histochemistry. All of the PAL-E or tuftsin binding site variants were capable of abolishing VEGF-NRP1 binding, but unfortunately, also eliminated SEMA3-NRP1 binding (<xref ref-type="fig" rid="fig1">Figure 1C</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03720.003</object-id><label>Figure 1.</label><caption><title>Design and assessment of <italic>Nrp1</italic> variants harboring mutations in the VEGF-binding site.</title><p>(<bold>A</bold>) Schematic representation of the NRP1 b1 extracellular domain and crystal structure highlighting three potential mutagenesis sites: the PAL-E binding site (orange circle), tuftsin binding site (blue circle), and electronegative surface (red circle). (<bold>B</bold>) Sequence of the <italic>Nrp1</italic> b1 domain indicating the deletion or mutation sites for the candidate constructs. (<bold>C</bold>) AP-SEMA3A (top row) or AP-VEGF (middle row) binding to COS-1 cells overexpressing the indicated constructs. Deletion of the entire PAL-E binding site (<italic>Nrp1</italic><sup><italic>PAL-EΔ7</italic></sup>) or partial deletion of the PAL-E binding site (<italic>Nrp1</italic><sup><italic>PAL-EΔ6</italic></sup> and <italic>Nrp1</italic><sup><italic>PAL-E Δ5</italic></sup>) eliminated both AP-SEMA3A and AP-VEGF binding. Likewise, mutations in the tuftsin binding site (S346A, E348A, T349A or S346A, E348A) abolished AP-SEMA3A binding and reduced AP-VEGF binding. Although mutations in the NRP1 electronegative surface (E319K, D320K) eliminated AP-VEGF binding and reduced AP-SEMA3A binding, the E319K mutation only slightly reduced AP-SEMA3A binding and maintained AP-VEGF binding. Antibody staining of unpermeabilized cells (lower row) demonstrated normal NRP1 surface expression. Scale bar: 50 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.003">http://dx.doi.org/10.7554/eLife.03720.003</ext-link></p></caption><graphic xlink:href="elife03720f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03720.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Assessment of additional <italic>Nrp1</italic> variants containing mutations in the VEGF-binding site.</title><p>AP-SEMA3A or AP-VEGF was applied to COS-1 cells overexpressing the indicated construct (top and middle row). Non-permeabilized antibody staining was performed with a polyclonal anti-NRP1 antibody to detect NRP1 surface expression (bottom row). Scale bar: 50 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.004">http://dx.doi.org/10.7554/eLife.03720.004</ext-link></p></caption><graphic xlink:href="elife03720fs001"/></fig></fig-group></p><p>We decided to use an unbiased approach and designed our subsequent <italic>Nrp1</italic> variants based upon the crystal structure of the full NRP1 b1 domain. Specifically, we identified a hydrophilic region comprised of several negatively charged residues that provided a promising mutagenesis site for abolishing VEGF-NRP1 binding (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Several of these residues were mutated to amino acids of the opposite charge in order to preserve the hydrophilic nature of the region. As with previous <italic>Nrp1</italic> variants, NRP1 surface expression was unperturbed in transfected COS-1 cells (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). One of these mutations (E282K) did not affect the binding of either AP-SEMA3A or AP-VEGF, while others (E282K and E420K) eradicated binding of both the ligands (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). However, the D320K mutation converting aspartic acid 320 into lysine (<italic>Nrp1</italic><sup><italic>D320K</italic></sup>) successfully abolished VEGF-NRP1 binding while conserving AP-SEMA3A binding as demonstrated through alkaline phosphatase histochemical staining on transfected COS-1 cells (<xref ref-type="fig" rid="fig1">Figure 1C</xref>, <xref ref-type="fig" rid="fig2">Figure 2A,C</xref>). Moreover, the <italic>Nrp1</italic><sup><italic>D320K</italic></sup> mutation also abolished the binding of other VEGF family members including Placenta Growth Factor (PlGF) and Vascular Endothelial Growth Factor B (VEGFB) (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). In a liquid alkaline phosphatase activity assay, <italic>Nrp1</italic><sup><italic>D320K</italic></sup> was co-expressed with <italic>PlexinA4</italic> (<italic>Plex4A</italic>) to more accurately reflect the in vivo situation in which SEMA3A signals through a holoreceptor complex of both NRP1 and PlexinA. AP-SEMA3A binding levels to WT NRP1 and NRP1<sup>D320K</sup> were indistinguishable (<xref ref-type="fig" rid="fig2">Figure 2D</xref>), and the dissociation constant (K<sub>D</sub>) of SEMA3A-NRP1<sup>D320K</sup>/PlexinA4 was unchanged from that of SEMA3A-NRP1/PlexinA4 further verifying that the SEMA3A-NRP1/PlexinA4 interaction was intact (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). Finally, Western blot analysis confirmed that NRP1 protein expression levels were equivalent in COS-1 cells transfected with <italic>WT Nrp1</italic> and <italic>Nrp1</italic><sup><italic>D320K</italic></sup> (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). Taken together, these data demonstrate that the <italic>Nrp1</italic><sup><italic>D320K</italic></sup> mutation is sufficient to eliminate VEGF binding and maintain SEMA3A binding in vitro<italic>.</italic><fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03720.005</object-id><label>Figure 2.</label><caption><title>The Nrp1<sup>D320K</sup> mutation selectively eliminates VEGF-NRP1 binding in vitro.</title><p>(<bold>A</bold>) AP-VEGF binding in COS-1 cells overexpressing the indicated <italic>Nrp1</italic> construct. WT NRP1 bound AP-VEGF strongly, while AP-VEGF binding to NRP1<sup>D320K</sup> was abolished. Scale bar: 100 μm (<bold>B</bold>) Western blot shows that equivalent levels of NRP1 protein in COS-1 cells transfected with the <italic>WT Nrp1</italic> and <italic>Nrp1</italic><sup><italic>D320K</italic></sup>. (<bold>C</bold>) Quantification of the binding assay shows that AP-VEGF-NRP1<sup>D320K</sup> binding was abolished even after normalization for protein content and NRP1 expression. (<bold>D</bold>) Quantification of AP-SEMA3A binding shows comparable AP-SEMA3A binding to WT NRP1 and NRP1<sup>D320K</sup>. (<bold>E</bold>) Measurement of the dissociation constant (K<sub>D</sub>) of AP-SEMA3A demonstrates that AP-SEMA3A bound to the NRP1<sup>D320K</sup>/PlexA4 complex with the same affinity as the NRP1/PlexA4 complex.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.005">http://dx.doi.org/10.7554/eLife.03720.005</ext-link></p></caption><graphic xlink:href="elife03720f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03720.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>VEGFA, VEGFB, and PLFG binding to NRP1 was abolished in the <italic>Nrp1</italic><sup><italic>D320K</italic></sup> mutant.</title><p><italic>Nrp1</italic> constructs were overexpressed in COS-1 cells, and AP-VEGFB or AP-PlGF was applied to cells to observe ligand binding. WT NRP1 bound AP-VEGFB and AP-PlGF strongly, while these ligands did not bind to NRP1<sup>D320K</sup>. Scale bar: 100 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.006">http://dx.doi.org/10.7554/eLife.03720.006</ext-link></p></caption><graphic xlink:href="elife03720fs002"/></fig></fig-group></p></sec><sec id="s2-2"><title>Generation and validation of the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mouse line</title><p>A gene replacement strategy was implemented to generate a mouse line harboring the <italic>Nrp1</italic><sup><italic>D320K</italic></sup> mutation in the endogenous <italic>Nrp1</italic> locus, delineated as <italic>Nrp1</italic><sup><italic>VEGF</italic>−</sup>. Specifically<italic>,</italic> two base pair mutations were introduced into exon 6 of the mouse <italic>Nrp1</italic> gene to produce the D320K mutation in the endogenous Asp320 location (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). After recombineering, embryonic stem cells were screened via PCR and sequenced to confirm that the D320K mutation was appropriately introduced into the <italic>Nrp1</italic> locus (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1A–C</xref>). Once <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mice were obtained, the presence of the D320K mutation was verified by sequencing (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1D</xref>). Importantly, the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> 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 (<xref ref-type="fig" rid="fig3">Figure 3C</xref>, <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2D</xref>). 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 (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) while AP-VEGF binding to the DREZ was abolished in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant (<xref ref-type="fig" rid="fig3">Figure 3B</xref>), confirming that this mutation eliminated VEGF-NRP1 binding in vivo. Moreover, NRP1 immunostaining and AP-SEMA3A binding to the DREZ appeared similar between <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> and control littermates (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). Finally, the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants failed to display the perinatal lethality or cardiac defect observed in the <italic>Nrp1</italic><sup><italic>Sema−</italic></sup> mutants (<xref ref-type="bibr" rid="bib13">Gu et al., 2003</xref>), further confirming functional SEMA3-NRP1 binding in <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mice (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03720.007</object-id><label>Figure 3.</label><caption><title><italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mice selectively abolish VEGF-NRP1 binding in vivo.</title><p>(<bold>A</bold>) Targeting vector design for the generation of <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mice. The WT genomic region contained residue D320 in exon 6 of <italic>Nrp1</italic>. 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. (<bold>B</bold>) Section binding assays demonstrated that AP-VEGF binding to the dorsal root entry zone (DREZ) was abolished in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants (arrows, left panels) while AP-SEMA3A binding to the DREZ appeared similar between <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> and control animals (arrows, middle panels). Scale bar: 100 μm. (<bold>C</bold>) Western blot from E14.5 lung tissue shows that NRP1 protein level was not affected in <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals. (<bold>D</bold> and <bold>E</bold>) <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants appear indistinguishable from controls littermates at embryonic (E14.5) and adult stages. (<bold>F</bold>) <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants exhibit normal body weight in adulthood (n = 7, males).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.007">http://dx.doi.org/10.7554/eLife.03720.007</ext-link></p></caption><graphic xlink:href="elife03720f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03720.008</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Screening and verification of ES cells for the generation of the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant.</title><p>(<bold>A</bold>) Diagram of the <italic>Nrp1</italic> genomic region following successful homologous recombination to insert the targeting vector. The green arrows indicate the primers used in (<bold>B</bold>), while the blue arrows represent the primers used in (<bold>C</bold>). (<bold>B</bold>) 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. (<bold>C</bold>) 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. (<bold>D</bold>) Sequencing of the D320K region in WT and <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> homozygous mutants. The boxed region indicates the altered codon.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.008">http://dx.doi.org/10.7554/eLife.03720.008</ext-link></p></caption><graphic xlink:href="elife03720fs003"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03720.009</object-id><label>Figure 3—figure supplement 2.</label><caption><title>The <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant mice exhibit normal gross morphology.</title><p>(<bold>A</bold>) Whole-mount images of the heart at P9 show the normal cardiac morphology of the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants. (<bold>B</bold> and <bold>C</bold>) Organ weights measured at P9 (<bold>B</bold>) and adulthood (<bold>C</bold>) demonstrate that the heart, brain, lung, and kidney undergo appropriate growth in <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals, n ≥ 5. (<bold>D</bold>) Western blots from adult heart, brain, lung, and kidney tissue demonstrate that NRP1 protein levels were not affected in <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals. (<bold>E</bold>) 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.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.009">http://dx.doi.org/10.7554/eLife.03720.009</ext-link></p></caption><graphic xlink:href="elife03720fs004"/></fig></fig-group></p></sec><sec id="s2-3"><title>VEGF-NRP1 binding is not required for developmental angiogenesis</title><p>Despite the embryonic lethality previously described in <italic>Nrp1</italic><sup>−/−</sup> and <italic>Tie2-Cre,Nrp1</italic><sup><italic>fl/−</italic></sup> animals, <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mice were born at expected Mendelian ratios and maintained their vitality into adulthood (p &gt; 0.05 for observed vs expected, <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2E</xref>). The <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants exhibited normal gross morphology throughout embryonic and postnatal stages (<xref ref-type="fig" rid="fig3">Figure 3D,E</xref>) and failed to develop the cardiac defects previously observed in the <italic>Nrp1</italic><sup>−/−</sup>, <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup>, and <italic>Nrp1</italic><sup><italic>Sema−</italic></sup> mutants (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2A</xref>). Moreover, <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals displayed normal body weight (<xref ref-type="fig" rid="fig3">Figure 3F</xref>), organ growth (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2B,C</xref>), and fertility.</p><p>To thoroughly examine vascular integrity during development, isolectin staining was employed to visualize blood vessels in embryonic and perinatal brain sections and vessel ingression, morphology, and branching were assessed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant. Surprisingly, <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals did not exhibit any of the vascular abnormalities observed in the endothelial-specific NRP1 knock-out. As shown in <xref ref-type="fig" rid="fig4">Figure 4A</xref> and quantified in <xref ref-type="fig" rid="fig4">Figure 4B–C</xref>, cortical vessel ingression was nearly absent in <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/fl</italic></sup> animals at E11.5 while ingression was unaffected in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants. In addition, <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/fl</italic></sup> animals had abnormally large vascular aggregates distributed throughout the striatum at E14.5 while vessels were evenly dispersed without aggregates in both control and <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals (<xref ref-type="fig" rid="fig4">Figure 4D–F</xref>). Finally, <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/fl</italic></sup> animals had a significant decrease in vessel branching in the cortex at E14.5 while <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals exhibited normal vessel branching (<xref ref-type="fig" rid="fig4">Figure 4G–I</xref>). Moreover, unlike the endothelial-specific NRP1 knock-out, the long term viability of the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants allowed us to assess cortical vessel branching and coverage at P7 which was indistinguishable from control littermates (<xref ref-type="fig" rid="fig4">Figure 4G–I</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). Therefore, VEGF-NRP1 binding is not required for developmental angiogenesis.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03720.010</object-id><label>Figure 4.</label><caption><title>VEGF-NRP1 binding is not required for developmental angiogenesis.</title><p>(<bold>A</bold>) Vessel staining with isolectin (green) revealed that <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/fl</italic></sup> mutants had delayed vessel ingression into the cerebral cortex at E11.5 while the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants exhibited normal ingression. DAPI was used to stain the nuclei (blue). (<bold>B</bold> and <bold>C</bold>) Quantification of cortical vessel ingression shown in <bold>A</bold>, n = 3. (<bold>D</bold>) <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/fl</italic></sup> mutants exhibited large vessel clumps in the brain (particularly in the striatum) at E14.5, a phenotype not observed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants. (<bold>E</bold> and <bold>F</bold>) Quantification of vessel size in E14.5 striatum shown in <bold>D</bold>, n = 3. (<bold>G</bold>) <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/fl</italic></sup> mutants had reduced vessel branching in the cerebral cortex while the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants displayed normal vessel branching at E14.5. (<bold>H</bold> and <bold>I</bold>) Quantification of vessel branching in E14.5 cortex shown in <bold>G</bold>, n = 4. Scale bar: 200 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.010">http://dx.doi.org/10.7554/eLife.03720.010</ext-link></p></caption><graphic xlink:href="elife03720f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03720.011</object-id><label>Figure 4—figure supplement 1.</label><caption><title>The <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant mice display normal vessel branching and coverage at postnatal stages.</title><p>(<bold>A</bold>) Vessel staining with isolectin (green) demonstrates that the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants have normal vessel coverage and branching in the cerebral cortex at P7. (<bold>B</bold> and <bold>C</bold>) Quantification of vessel coverage and branching in P7 cortex shown in <bold>A</bold>, n = 3. Scale bar: 200 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.011">http://dx.doi.org/10.7554/eLife.03720.011</ext-link></p></caption><graphic xlink:href="elife03720fs005"/></fig></fig-group></p></sec><sec id="s2-4"><title>NRP1 functions to modulate VEGFR2 levels independent of VEGF-NRP1 binding</title><p>The normal developmental angiogenesis observed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants clearly demonstrates that VEGF-NRP1 binding is not responsible for the vascular defects observed in <italic>Nrp1</italic><sup><italic>−/−</italic></sup> or endothelial-specific NRP1 knock-outs. In this regard, NRP1 must function through an alternative mechanism to regulate vascular development during embryogenesis. The intracellular domain of NRP1 does not have any obvious enzymatic activity and is not responsible for the signal transduction mediating angiogenesis (<xref ref-type="bibr" rid="bib7">Fantin et al., 2011</xref>; <xref ref-type="bibr" rid="bib24">Lanahan et al., 2013</xref>). Therefore, two apparent alternatives remain. One possibility is that a yet unidentified ligand outside the VEGF or SEMA3 family binds to NRP1 and instructs developmental angiogenesis. Alternatively, NRP1 may control vascular development by directly regulating its co-receptor, VEGFR2.</p><p>To test this second possibility, VEGFR2 expression was evaluated in the <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> mutants and control littermates via Western blot on E14.5 lung tissue. This biochemical assay revealed that total VEGFR2 protein levels were significantly reduced in the <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> mutants compared to their control littermates (<xref ref-type="fig" rid="fig5">Figure 5A–B</xref>). To determine the cell surface expression of VEGFR2 in vivo, we used fluorescence-activated cell sorting (FACS) to specifically quantify VEGFR2 expression at the cell surface of non-permeabilized endothelial cells derived from the acutely dissociated lungs of <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> and control embryos. Remarkably, <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> mutants displayed a significant decrease in the fluorescence intensity of VEGFR2 labeling as compared to control littermates (<xref ref-type="fig" rid="fig5">Figure 5E–F</xref>), suggesting that NRP1 functions to regulate VEGFR2 surface expression in endothelial cells. In contrast, both Western blot and FACS analysis determined that VEGFR2 protein levels were unperturbed in <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals (<xref ref-type="fig" rid="fig5">Figure 5C–D,G–F</xref>). In addition, co-immunoprecipitation on P7 lung tissue revealed that NRP1 and VEGFR2 are physically associated in both control and <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1B</xref>), validating that NRP1-VEGFR2 receptor complex formation does not require VEGF-NRP1 binding in vivo. This result mimics our co-immunoprecipitation experiments on HEK293T cells transfected with either <italic>WT Nrp1</italic> or <italic>Nrp1</italic><sup><italic>D320K</italic></sup> constructs (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1A</xref>). Together, these findings indicate that NRP1 plays a role in regulating the cell surface expression of VEGFR2 in endothelial cells and that VEGF-NRP1 binding is not necessary for this function in vivo (<xref ref-type="fig" rid="fig5">Figure 5G</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03720.012</object-id><label>Figure 5.</label><caption><title>NRP1 regulates VEGFR2 expression at the cell surface independent of VEGF-NRP1 binding.</title><p>(<bold>A</bold>) Western blot from E14.5 lung tissue treated with 50 ng/ml VEGF for 15 min revealed that VEGFR2 was reduced in <italic>Tie2-CreNrp1</italic><sup><italic>fl/−</italic></sup> mutants while VE-cadherin expression remained at control levels. Western blot for NRP1 demonstrates that the <italic>Tie2-Cre</italic> allele successfully knocked down NRP1 expression. (<bold>B</bold>) Quantification of VEGFR2 expression shown in <bold>A</bold>, n = 4. (<bold>C</bold>) Western blot from E14.5 lung tissue treated with 50 ng/ml VEGF for 15 min demonstrates that VEGFR2, NRP1, and VE-cadherin expression were unperturbed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants. (<bold>D</bold>) Quantification of VEGFR2 expression shown in <bold>C</bold>, n = 5. (<bold>E</bold>) FACS analysis plots illustrate a reduction in VEGFR2 surface expression in endothelial cells isolated from <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> mice. (<bold>F</bold>) Quantification of the VEGFR2 fluorescence intensity from the FACS analysis shown in <bold>E</bold>, n = 5. (<bold>G</bold>) FACS analysis plots demonstrate that VEGFR2 surface expression in endothelial cells isolated from <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mice remained at control levels. (<bold>H</bold>) Quantification of the VEGFR2 fluorescence intensity from the FACS analysis shown in <bold>G</bold>, n ≥ 7. (<bold>I</bold>) Schematic of VEGFR2 and NRP1 at the cell surface illustrates VEGF ligand binding to both VEGFR2 and NRP1. In the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants, VEGF-NRP1 binding is abolished, VEGFR2 has normal cell surface localization, and vascular development proceeds appropriately. However, in <italic>Nrp1</italic><sup><italic>−/−</italic></sup> mutants, VEGFR2 cell surface localization is reduced and vascular development is impaired.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.012">http://dx.doi.org/10.7554/eLife.03720.012</ext-link></p></caption><graphic xlink:href="elife03720f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03720.013</object-id><label>Figure 5—figure supplement 1.</label><caption><title>VEGF-NRP1 binding is not required for NRP1-VEGFR2 complex formation in vitro and in vivo<italic>.</italic></title><p>(<bold>A</bold>) HEK293T cells transfected with <italic>Vegfr2</italic> and either <italic>WT Nrp1</italic> or <italic>Nrp1</italic><sup><italic>D230K</italic></sup> exhibited normal NRP1-VEGFR2 complex formation. (<bold>B</bold>) Lung lysates generated from the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants also displayed normal NRP1-VEGFR2 complex formation comparable to littermate controls<italic>.</italic></p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.013">http://dx.doi.org/10.7554/eLife.03720.013</ext-link></p></caption><graphic xlink:href="elife03720fs006"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03720.014</object-id><label>Figure 5—figure supplement 2.</label><caption><title>VEGF-induced VEGFR2 phosphorylation is reduced in both the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> and <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> mutants.</title><p>(<bold>A</bold>) Western blot from E14.5 lung tissue shows that VEGFR2 phosphorylation upon VEGF treatment was diminished in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant. (<bold>B</bold>) Quantification of VEGFR2 phosphorylation shown in <bold>A</bold>, n = 7. (<bold>C</bold>) Western blot from E14.5 lung tissue demonstrates that VEGFR2 phosphorylation is significantly reduced in the <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> mutants. (<bold>D</bold>) Quantification of VEGFR2 phosphorylation shown in <bold>B</bold>, n = 5.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.014">http://dx.doi.org/10.7554/eLife.03720.014</ext-link></p></caption><graphic xlink:href="elife03720fs007"/></fig></fig-group></p><p>To examine VEGF signaling in the <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> and <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants, VEGFR2 phosphorylation was examined via Western blot on embryonic lung tissue isolated at E14.5. Specifically, <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/−</italic></sup> mutants had a severe reduction in VEGFR2 phosphorylation at the tyrosine residue 1175 (Y1175) upon VEGF treatment (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2A,B</xref>). Interestingly, <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants also exhibited a mild reduction in VEGFR2 phosphorylation while total VEGFR2 protein levels were well maintained (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2C,D</xref>). Although the level of pVEGFR2 in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant was sufficiently high to support vascular development during embryogenesis, the modest reduction in pVEGFR2 may manifest in issues with angiogenesis, vascular maintenance, and regeneration in the postnatal animal.</p></sec><sec id="s2-5"><title>VEGF-NRP1 binding is required for postnatal angiogenesis</title><p>To directly test the role for VEGF-NRP1 binding in postnatal angiogenesis, whole-mount staining was performed with isolectin and an antibody against α-smooth muscle actin (α-SMA) to visualize the retinal blood vessels and arteries, respectively. At P9, the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants exhibited a reduction in the vascular extension and artery number but did not have any abnormalities in vessel coverage as compared with control littermates (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). In the adult, the vascular extension and vessel coverage in the retina were indistinguishable from controls (<xref ref-type="fig" rid="fig6">Figure 6B</xref>) indicating that the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants experience a delay in the formation of the primary vascular plexus. However, the number of retinal arteries remained lower in <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> adults. These results demonstrate that VEGF-NRP1 interactions are required to some degree for postnatal angiogenesis and artery differentiation in the retina.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03720.015</object-id><label>Figure 6.</label><caption><title>Retinal angiogenesis is perturbed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant.</title><p>(<bold>A</bold>) Isolectin and α-SMA staining on P9 retinal flat-mounts revealed a significant reduction in vascular extension and artery number in <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants. However, vessel coverage in the retina was unperturbed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants, n = 6. (<bold>B</bold>) In the adult, isolectin and α-SMA staining showed that the number of retinal arteries remained lower in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants than littermate controls while vascular extension and vessel coverage in the retina were normal, n = 4. Scale bar: 200 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.015">http://dx.doi.org/10.7554/eLife.03720.015</ext-link></p></caption><graphic xlink:href="elife03720f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03720.016</object-id><label>Figure 6—figure supplement 1.</label><caption><title>The <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants have delayed blood flow recovery following femoral artery ligation.</title><p>(<bold>A</bold>) Laser doppler imaging demonstrates severe hindlimb ischemia directly after femoral artery ligation in both control and <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals (arrows). Five days after surgery, blood flow recovery in the injured hindlimb was significantly greater in control vs <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals (arrowheads). (<bold>B</bold>) Quantification of blood flow recovery following femoral artery ligation, n = 7.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.016">http://dx.doi.org/10.7554/eLife.03720.016</ext-link></p></caption><graphic xlink:href="elife03720fs008"/></fig></fig-group></p><p>In addition, <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals were also assessed for injury-induced arteriogenesis following femoral artery ligation. In this assay, the femoral artery was surgically severed in both <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> and control mice, and blood flow recovery was monitored via deep penetrating laser Doppler imaging. Femoral artery ligation produced a comparable level of hindlimb ischemia in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants and controls (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). However, the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants exhibited a significant delay in hindlimb re-perfusion. Building upon these results, future work will utilize the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> knock-in line to determine if VEGF-NRP1 signaling functions in pathological or physiological angiogenesis in the adult.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>In this study, we identified a single amino acid within the extracellular b1 domain of NRP1 that is required for VEGF-NRP1 binding, but non-essential for SEMA3-NRP1 interactions. A point mutation in this D320 residue was incorporated into the endogenous <italic>Nrp1</italic> locus to generate the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant, a novel mouse line that selectively abolishes VEGF-NRP1 binding in vivo. Recently a cDNA knock-in NRP1 mutant, <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup>, was also developed to examine the role of VEGF-NRP1 binding (<xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref>). However, mice generated with genetically modified cDNA notoriously lack the essential intronic regions that regulate the temporal and spatial expression of the gene. Consequently, the aberrant and severe down-regulation of NRP1 protein expression in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph prevents any definitive conclusions from being garnered about the biological cause of phenotypes present in this mouse. In this regard, abnormalities in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph could originate from two potential sources: the severe reduction in NRP1 levels or the abolishment of VEGF-NRP1 binding. Unlike the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> line, our <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant contains a two base pair replacement in the endogenous <italic>Nrp1</italic> locus and preserves the genetic structure of the <italic>Nrp1</italic> gene. Consequently, <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mice maintain appropriate levels of NRP1 protein expression and allow the first unobscured in vivo assessment of VEGF-NRP1 binding in developmental angiogenesis. Our <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> line provides a powerful new genetic tool for selectively interrogating the function of VEGF-NRP1 binding in broad areas of basic research and translational study.</p><p>Remarkably, our <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant did not recapitulate the early embryonic lethality or developmental angiogenesis phenotypes of the <italic>Nrp1</italic><sup><italic>−/−</italic></sup> and endothelial-specific NRP1 knock-out (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Moreover, the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant did not exhibit any of the cardiac failure, perinatal lethality, or growth defects observed in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph indicating that these phenotypes are attributed to the severe reduction in NRP1 protein in <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> mutants rather than the lack of VEGF-NRP1 binding. However, the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant did exhibit a delay in vascular extension and a reduction in the number of arteries in the postnatal retina. This retinal phenotype is significantly less severe than those observed in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph (<xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref>) or in animals treated with antibodies inhibiting VEGF-NRP1 binding (<xref ref-type="bibr" rid="bib28">Pan et al., 2007</xref>). Together, these results reveal that the retina relies on both VEGF-NRP1 dependent and independent mechanisms to establish the retinal vasculature.</p><p>Our surprising results challenge the well-accepted view that NRP1 depends on VEGF-NRP1 binding to facilitate angiogenesis and points to a provocative new hypothesis that NRP1 functions independently of VEGF-NRP1 binding perhaps via its interaction with an unidentified ligand or in its capacity as a co-receptor for VEGFR2. Our study demonstrates that the NRP1-deficient endothelial cells have reduced VEGFR2 expression at the cell surface, a phenomenon that was not observed in the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutants. This result provides the first in vivo evidence that NRP1 controls VEGFR2 levels at the cell membrane and offers the first in vivo phenotypic characterization linking NRP1 regulated VEGFR2 surface expression to vascular development.</p><p>Consistent with our in vivo observations, several lines of in vitro work using multiple cell culture systems demonstrate that NRP1 is essential for the proper presentation, recycling, and degradation of VEGFR2 (<xref ref-type="bibr" rid="bib31">Shintani et al., 2006</xref>; <xref ref-type="bibr" rid="bib17">Holmes and Zachary, 2008</xref>; <xref ref-type="bibr" rid="bib1">Ballmer-Hofer et al., 2011</xref>; <xref ref-type="bibr" rid="bib14">Hamerlik et al., 2012</xref>). The loss of function and gain of function studies in human umbilical vein endothelial cells (HUVECs) found that VEGFR2 protein levels were decreased in the absence of NRP1 while <italic>Vegfr2</italic> mRNA levels were unaffected by Nrp1 siRNA (<xref ref-type="bibr" rid="bib31">Shintani et al., 2006</xref>; <xref ref-type="bibr" rid="bib17">Holmes and Zachary, 2008</xref>). Similarly, <xref ref-type="bibr" rid="bib14">Hamerlik et al. (2012)</xref> examined human glioblastoma multiforme cells and found that shRNA mediated knock-down of NRP1 resulted in dramatically decreased VEGFR2 protein levels accompanied by a lower surface presentation of VEGFR2 and a decrease in cell viability. Moreover, cell surface protein biotinylation and immunofluorescence staining with confocal microscopy confirmed the co-localization of VEGFR2-NRP1 with the early/recycling endosome. Finally, <xref ref-type="bibr" rid="bib1">Ballmer-Hofer et al., (2011)</xref> used stably transfected porcine aortic endothelial cell (PAEC) lines in conjunction with immunostaining to visually follow VEGFR2 trafficking in the presence and absence of NRP1. Their experiments revealed that upon VEGF stimulation, VEGFR2 is internalized in Rab7 vesicles for degradation. However, in the presence of NRP1, VEGFR2 is stabilized in Rab11 vesicles and recycled back to the cell surface. In conjunction with our in vivo results, these data demonstrate that NRP1 guides vascular development through its capacity as a VEGFR2 co-receptor rather binding to VEGF. In this manner, NRP1 regulates angiogenesis by controlling the amount of VEGFR2 expression at the cell surface and consequently the level of VEGFR2-VEGF signaling.</p><p>The modulation of co-receptors may function as a general mechanism for regulating cell signaling and behavior. A prior in vitro study identified a similar relationship between the membrane protein, neural cell adhesion molecule (NCAM) and fibroblast growth factor receptor-1 (FGFR1) (<xref ref-type="bibr" rid="bib10">Francavilla et al., 2009</xref>). This previous work discovered that NCAM induced sustained FGFR1 activation by controlling the intracellular trafficking of the FGFR1 receptor. Specifically, NCAM was capable of re-targeting internalized FGFR1 from the lysosomal degradation pathway to Rab11-postive recycling vesicles and increased FGFR1 expression at the cell surface. In this regard, the co-receptor interaction between NRP1 and VEGFR2 may be representative of a more universal phenomenon in which membrane proteins function to regulate the cell surface expression and subsequent downstream signaling of receptors.</p><p>Ultimately, our findings mark a pivotal step toward understanding the role of NRP1 in developmental angiogenesis and indicate that NRP1-VEGFR2 interactions rather than VEGF-NRP1 binding underlie NRP1's critical function in VEGF-mediated vascular development. Given the substantial resources invested in NRP1-targeted anti-angiogenesis therapies for vascular disease and cancer, the information gleaned from this study will be invaluable in identifying the cellular and molecular mechanisms underlying angiogenesis and ultimately using this information to instruct the development of new therapeutic approaches.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Site-directed mutagenesis and targeting vector construction</title><p>Rat Neuropilin1 cDNA was re-cloned from pMT21 into pCS2+ using the original EcoRI and XhoI sites present in both vectors. Mutations were made using PCR, and the mutated fragment was subcloned back into pCS2-Nrp1 using endogenous restriction sites. The targeting vector (TV) was constructed using a combination of traditional cloning and recombineering along with point mutagenesis. Genomic DNA was obtained from the 129S7-AB2.2 BAC library, clone #bMQ-373E22. The short (3′) arm (1.3 kb) was cloned into the HpaI and EcoRI sites of 4600C-loxP. Two short homology arms (900 bp, total) were created and cloned into the XhoI and NotI sites of 4600C-loxP, with the two arms joined by a SalI site. The homology arms were ligated in a triple ligation to 4600C-loxP as well as to each other. The vector was then linearized with SalI and electroporated into modified electrocompetent DH10B cells containing the previously mentioned BAC in order to facilitate homologous recombination to insert the remainder of the long arm. Recombineering was performed as described by the NCI-Frederick. After a full-length TV was made, the D320K mutation was introduced. The final TV was linerarized and electroporated into ES cells. All primer sequences used for the targeting vector construction are provided in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>.</p></sec><sec id="s4-2"><title>Alkaline-phosphatase-tagged ligand production</title><p>HEK293T cells were transfected with AP-SEMA3A, AP-VEGF A, AP-VEGF B, or AP-PlGF expression constructs using a calcium phosphate transfection method. Media was changed after 6 hr. Cells were cultured for an additional 48 hr in DMEM + 10% FBS. After 48 hr the media were collected, filtered to remove the cell debris, and AP activity was measured. The ligands were frozen at −80°C until use.</p></sec><sec id="s4-3"><title>Binding of AP-tagged protein to cells and unpermeabilized antibody staining</title><p>COS-1 cells were grown in DMEM + 10% fetal bovine serum (FBS) + 1% Penicillin-Streptomycin. Cells were transfected with the indicated expression vectors using Lipofectamine-2000 (Invitrogen, Carlsbad, CA) in 6-well plates. 24 hr later, transfected cells were split into 24-well plates for parallel AP-binding and antibody staining. 24 hr after splitting, binding was performed using AP-tagged ligands (AP-VEGF A, AP-SEMA3A, AP-VEGF B, AP-PlGF). The binding protocol was as follows: cells were washed 1× with HBHA (1× HBSS, 0.5 mg/ml BSA, 0.5% sodium azide, and 20 mM HEPES [pH 7]), then incubated for 75 min with 0.3 ml of 2 nM ligand. Cells were then washed 7× with HBHA on a rotating platform and 110 µl of cell lysis buffer (1% Triton X-100 and 10 mM Tris–HCl [pH 8]) was added to each well. Cells and buffer were scraped into Eppendorf tubes, then vortexed for 5 min to fully lyse them. The lysates were then spun down for 5 min, and the supernatant was heat inactivated at 65°C for 10 min to inactivate endogenous alkaline phosphatases. AP-activity was measured by adding 2× SEAP buffer (50 ml 2 M diethanolamine [pH 9.8], 50 µl 1 M MgCl<sub>2</sub>, 224 mg L-homoarginine, 50 mg BSA, 445 mg p-nitrophenylphosphate) and measuring optical absorbance at 405 nm every 15 s for 1 min. Antibody staining of these cells was done as follows: non-specific binding was blocked with 5% Normal Goat Serum in DMEM for 30 min at 4°C. Cells were then incubated with primary antibody (Rabbit anti-NRP1, gift of Dr David Ginty) for 2 hr at 4°C. They were then washed 6× with cold HBHA, then incubated with a secondary antibody (AP-tagged anti-rabbit) for 1.5 hr at 4°C. Cells were then washed 3× in cold HBHA, then lysed as described above. AP-activity was measured from lysed extracts. Binding of AP-tagged ligands was normalized to protein content of each well and to antibody staining with an anti-NRP1 antibody. Each AP-binding assay was independently repeated three times.</p></sec><sec id="s4-4"><title>Animal care</title><p><italic>Nrp1</italic><sup><italic>VEGF−</italic></sup>, <italic>Tie2</italic>-Cre, <italic>Nrp1</italic><sup><italic>fl</italic></sup>, and <italic>Nrp1</italic><sup><italic>−</italic></sup> (<xref ref-type="bibr" rid="bib13">Gu et al., 2003</xref>) mice were maintained on a C57Bl/6 background. <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mice were genotyped with traditional PCR techniques. The expected WT band is 305 bp, while the targeted allele is 350 bp due to the remaining presence of one FRT site. To sequence the mutation site, PCR was performed to generate a fragment around the mutation site. The primer sequences for genotyping and sequencing are included in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>. <italic>Tie2-Cre</italic>, <italic>Nrp1</italic><sup><italic>fl</italic></sup>, and <italic>Nrp1</italic><sup><italic>−</italic></sup> genotyping was performed as previously published. All animals were treated according to institutional and NIH guidelines approved by IACUC at Harvard Medical School.</p></sec><sec id="s4-5"><title>AP-ligand binding to tissue sections</title><p>Embryos were dissected and frozen immediately in liquid nitrogen, then stored at −80°C until use. Sections were cut at 25 µm with a cryostat, then fixed for 8 min in ice-cold methanol. Sections were then washed 3× in PBS + 4 mM MgCl<sub>2</sub>. Non-specific binding was reduced by blocking the sections with DMEM + 10% FBS for 45 min. After fixation, sections were incubated with 2 nM AP-tagged ligand, diluted with PBS + 4 mM MgCl<sub>2</sub>, and buffered with HEPES, pH 7 for 1.5 hr at room temperature in a humidified chamber. The sections were washed 5× in PBS + 4 mM MgCl<sub>2</sub>, then fixed with a fixative solution (60% acetone, 1% formaldehyde, 20 mM HEPES, pH 7). Sections were washed 3× in PBS and incubated in PBS at 65°C for 2 hr to heat inactive endogenous alkaline phosphatases and then incubated overnight in developing solution (100 mM Tris–HCl pH 9.5, 100 mM NaCl, 5 mM MgCl<sub>2</sub>) with NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine). AP-ligand binding was analyzed in sections from at least three animals across two different litters per genotype.</p></sec><sec id="s4-6"><title>Western blotting</title><p>For immunoblotting, E14.5 lung samples were loaded on 8% polyacrylamide gels and run until the appropriate protein separation was achieved. Samples were electrophoretically transferred onto the PVDF membrane. Non-specific binding was blocked by a 1 hr incubation in 5% non-fat milk in TBST (Tris-buffered saline + 0.1% Tween-20). The membranes were then incubated overnight with the following primary antibodies, as indicated below, at 4°C: anti-NRP1 (#ab81321 Abcam, Cambridge, MA or gift of Dr David Ginty, see <xref ref-type="bibr" rid="bib11">Ginty et al., 1993</xref> for details), anti-VEGFR2 (gift of Procter and Gamble, see <xref ref-type="bibr" rid="bib13">Gu et al., 2003</xref> for details), anti-VE-cadherin (#ab33168 Abcam, Cambridge, MA), anti-p-VEGFR2 (p1175) (#2478 Cell Signaling Technology, Danvers, MA), and anti-α-Tubulin (#T5168 Sigma-Aldrich, Natick, MA). After incubation with primary antibodies, the membranes were washed 3× in TBST then incubated with the appropriate HRP-labeled secondary antibody in TBST or 5% milk in TBST for 1 hr at room temperature. Membranes were then washed 3× with TBST then developed with regular or super ECL (GE Amersham, United Kingdom or Thermo Scientific, Waltham, MA). The intensity of individual bands was quantified using ImageJ.</p></sec><sec id="s4-7"><title>Phenotypic analysis of the <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> mutant</title><p>At the indicated stages, embryos were dissected, fixed with 4% paraformaldehyde, equilibrated in a sucrose gradient, embedded in OCT, and sectioned in the coronal plan at 12 µm with a Leica CM3050S cryostat. Likewise, the brains of postnatal pups (P7) were dissected, fixed, cryo-protected, and sectioned at 20 µm. Tissue sections were washed 3× for 5 min in 0.2% PBT (0.2% Triton X-100 in PBS), incubated with Isolectin GS-IB4 (#I21411 Life Technologies, Grand Island, NY) overnight at 4°C, washed 3× for 5 min in PBS, and coverslipped with using ProLong Gold/DAPI antifade reagent (#P36935 Molecular Probes, Eugene, OR). Sections were imaged by fluorescence microscopy using a Nikon Eclipe 80i microscope equipped with a Nikon DS-2 digital camera. Quantification was performed using ImageJ. Vessel coverage delineates the percent of cortical pixel area covered by isolectin-positive pixels while vessel size quantifies the pixel area of each discrete vascular aggregate identified by isolectin staining.</p></sec><sec id="s4-8"><title>VEGF lung treatment</title><p>E14.5 mouse lungs were dissected in cold PBS and minced finely using a razor blade. The tissue was then incubated with plain EBM (Lonza, Switzerland) or EBM containing 50 ng/ml VEGF for 15 min at 37°C. Lysis buffer (50 mM Tris/HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 2 mM DTT) containing complete proteinase inhibitors (Roche, Switzerland), PhosSTOP (Roche, Switzerland), and sodium orthovanadate was added to the tissue, which was then pulverized with a pestle and incubated for 30 min while rotating at 4°C. Tissue was spun down and protein quantification was performed. The tissue was treated as described in the Western blotting section.</p></sec><sec id="s4-9"><title>Co-immunoprecipitation</title><p>HEK293T cells were transfected with the indicated constructs using Lipofectamine-2000 (Invitrogen, Carlsbad, CA). They were then grown in DMEM + 10% fetal bovine serum + 1% Penicillin-Streptomycin and 48 hr after transfection cells were washed and harvested in ice-cold PBS. Cells were lysed using lysis buffer (50 mM Tris/HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 2 mM DTT) containing complete proteinase inhibitors (Roche, Switzerland). After 30 min of rotation in the cold room and subsequent centrifugation, protein was quantified and 20 µg of protein was frozen down as input controls. 0.5 µg of anti-VEGFR2 antibody (gift of Procter and Gamble, see <xref ref-type="bibr" rid="bib13">Gu et al., 2003</xref> for details) was added to 500 µg of protein and rotated in the cold room for 1 hr. Then, 20 µl of protein A/G beads (Thermo Scientific, Waltham, MA) were added to the protein and rotated overnight in the cold room. Beads were washed 3× with lysis buffer and two times with wash buffer (lysis buffer with 300 mM NaCl). Protein was eluted by the addition of 2× SDS-PAGE sample buffer and boiling for 10 min. Co-immunoprecipitation was also performed on P7 lung lysates isolated from control and <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> animals treated with VEGF as described above.</p></sec><sec id="s4-10"><title>FACS</title><p>Analysis of E14.5 mouse embryos were performed on single cells from dissociated lungs. In brief, microdissection techniques were used to isolate the lung. Lungs were then rinsed in PBS and incubated in 2 mg/ml collagenase and 20 μg/ml DNase I 3× for 15 min at 37°C and gently pipetted. The collagenase was inactivated using 5 ml of ice-cold 10% FBS/PBS, centrifuged at 1000×<italic>g</italic> for 5 min, and suspended in 400 µl of red blood cell (RBC) lysis buffer (Sigma-Aldrich, Natick, MA). Following a 5 min incubation at room temperature, 2 ml of ice-cold 5% FBS/PBS was added and cells were centrifuged at 1000×<italic>g</italic> for 5 min at 4°C. Cells were then blocked in Fc-blocking solution (#553142; BD) for 20 min on ice, centrifuged, incubated with the labeled conjugated primary antibodies–PE-anti-CD31 (PECAM) (#553373 BD Pharmingen, Franklin Lakes, NJ) and APC-anti-Flk1-1 (VEGFR2) (#560070 BD Pharmingen, Franklin Lakes, NJ), for 30 min on ice with agitation every 10 min. After incubation, the cells were spun down, the supernatant was removed, and the cell pellet was resuspend in 1:10K Sytox in PBS/5%FBS. Cells were analyzed on a LSR II Flow Cytometer. Cells incubated with no antibody, APC-anti-Flk1, or PE-anti-CD31 only served as the control population.</p></sec><sec id="s4-11"><title>Phenotypic analysis of the developing retina</title><p>Whole-mount retina immunohistochemistry was performed as previously described in <xref ref-type="bibr" rid="bib21">Kim et al., (2011)</xref>. Briefly, eyes were extracted and fixed in 4% paraformaldehyde for 10 min at room temperature. Retinas were dissected in PBS and post-fixed in 4% paraformaldehyde overnight at 4°C. Retinas were then permeabilized in PBS, 1% BSA, and 0.5% Triton X-100 at 4°C overnight, washed 2× for 5 min in 1% PBT (1% Triton X-100 in PBS), and incubated in Isolectin GS-IB4 (1:200, #I21411 Life Technologies, Grand Island, NY) and anti-αSMA Cy3 (1:100, #C6198 Sigma-Aldrich, Natick, MA) in 1% PBT overnight at 4°C. Retinas were washed 3× for 5 min and flat-mounted using ProLong Gold antifade reagent (#P36934 Molecular Probes, Eugene, OR). Flat-mounted retinas were analyzed by fluorescence microscopy using a Nikon Eclipe 80i microscope equipped with a Nikon DS-2 digital camera and by confocal laser scanning microscopy using an Olympus FV1000 confocal microscope. Quantification was performed using MetaMorph Image Analysis Software and ImageJ. At least four retinal leaves were quantified per animal to determine the vascular extension ratio, both eyes were examined in each animal for artery number, and three representative images were quantified from each animal for vascular coverage (representing the total isolectin-positive pixel area per image).</p></sec><sec id="s4-12"><title>Femoral artery ligation</title><p>Ketamine (80–100 mg/kg) and xylazine (5–10 mg/kg) delivered by IP injection were used to anesthetize 12-week old male <italic>Nrp1</italic><sup><italic>VEGF−</italic></sup> and control littermates. After anesthesia was achieved, the bilateral hindlimbs and lower abdomen were cleared of hair and cleaned with 10% betadine and 70% alcohol. An incision of 3–4 mm was made in the right inguinal area to visualize the femoral artery. Two 6–0 silk sutures were tied in the proximal femoral artery and the deep femoral and epigastric artery branches were cauterized. The femoral artery was then ligated between the two sutures. The skin was sutured with one 4–0 prolene sutures. Immediately before and after surgery, each animal was scanned with a non-invasive laser doppler imaging system (moorLD12-HR Moor Instruments, Wilmington, DE) under 1–3% isofluorane anesthesia. Blood flow recovery in the hindlimbs was further assessed on 3, 5, and 7 days post-surgery and quantified via Moor LDI Software.</p></sec><sec id="s4-13"><title>Statistical analysis</title><p>The standard error of the mean was calculated for each experiment and error bars in the graphs represent the standard error. A paired Student's <italic>t</italic>-test was used to determine the statistical significance of differences between samples, and the genotype distribution was analyzed using a Chi-square test. Statistical analyses were performed with Prism 4 (GraphPad Software) and p values are indicated by * ≤ 0.05, ** ≤ 0.01, and *** ≤ 0.001.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank the members of the Gu laboratory for helpful comments on the manuscript, Lauren Byrnes for technical support, and both the Flow Cytometry Facility in the Systems Biology Department and the Neurobiology Imaging Facility in the Neurobiology Department of Harvard Medical School for consultation and instrument availability that facilitated this work. The Neurobiology Imaging Facility is supported in part by the Neural Imaging Center as part of an NINDS P30 Core Center grant #NS072030. This study was supported by the National Institutes of Health Fundamental Neurobiology Training grant T32 NS007484-12 (N Hagan), the Alice and Joseph Brooks Fund Postdoctoral Fellowship (A Tata), Harvard Mahoney Neuroscience Institute Fund Postdoctoral Fellowship (B Lacoste), National Institutes of Health grant R01 HL096384 (K Kang and J Bischoff), and the following grants to C Gu: Sloan research fellowship, Armenise junior faculty award, the Genise Goldenson fund, and National Institutes of Health grant R01 NS064583.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>MVG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>NH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>AT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>W-JO, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>BL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>K-TK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con7"><p>JK, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con8"><p>JB, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con9"><p>CG, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con10"><p>J-HW, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animals were handled according to approved institutional animal care and use committee (IACUC) protocols at Harvard Medical School (IACUC Study ID: IS00000045).</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03720.017</object-id><label>Supplementary file 1.</label><caption><p>Primers used for generating, genotyping, and sequencing the <italic>Nrp1<sup>VEGF-</sup></italic> knock-in mouse line.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03720.017">http://dx.doi.org/10.7554/eLife.03720.017</ext-link></p></caption><media mime-subtype="pdf" mimetype="application" xlink:href="elife03720s001.pdf"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group 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USA</source><volume>104</volume><fpage>6152</fpage><lpage>6157</lpage></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03720.018</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Nathans</surname><given-names>Jeremy</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, Johns Hopkins University School of Medicine</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Neuropilin-1 controls developmental angiogenesis by regulating VEGFR2 independent of VEGF-Neuropilin-1 binding” for consideration at <italic>eLife.</italic> Your article has been favorably evaluated by Janet Rossant (Senior editor), Jeremy Nathans (Reviewing editor), and 3 reviewers.</p><p>The manuscript has been reviewed by three expert reviewers, and their assessments together with the Reviewing editor form the basis of this letter. I am also including the three reviews in their original form at the end of this letter, as there are many specific and useful suggestions in them that will not be repeated in the summary here.</p><p>All of the reviewers were impressed with the incisive design and execution of your experiments. The mutant mouse that you constructed is the cleanest experimental approach to date to address the question of whether direct VEGF binding to Neuropilin1 (Nrp1) plays a role in vivo. Our consensus is that this is an important body of work but that a few key questions have been left open and that addressing them would greatly enhance the impact of the study.</p><p>These are:</p><p>Is the Nrp1 VEGF binding mutant mouse really normal? A close look at the retinal vasculature (e.g. at P7, P9, and adulthood) and at health/fertility at later ages would be useful. You might consider stressing the system, for example with the oxygen induced retinopathy model (your colleague Lois Smith at Children's Hospital is the expert) or with a test of tumor angiogenesis, both of which tie into the applied aspects of this work as related to the efficacy of anti-Nrp1 antibodies. Comparing the phenotypes to those reported by <xref ref-type="bibr" rid="bib6">Fantin et al (2014)</xref> would be useful.</p><p>The decrease in VEGFR2 levels (and surface VEGFR2), which is presented as the mechanism by which the Nrp1 KO exerts its effects, could use more experimental backing. Checking the level of VEGFR2 phosphorylation in response to VEGF and an assessment of VEGFR2 trafficking with vs. without Nrp1 vs. with the Nrp1 VEGF binding mutant (in cell culture) would be very helpful.</p><p>Is the VE cadherin level in <xref ref-type="fig" rid="fig5">Figure 5</xref> lower in the absence of Nrp1? If so, does this suggest other roles for Nrp1? Also, the decrease in VEGFR2 by Western blot seems more dramatic than the decrease shown by FACS. Is the lower level of VEGFR2 shown by FACS in the absence of Nrp1 really insufficient for VEGF signaling? Or is something more subtle at play (defective trafficking?)?</p><p>Reviewer #1:</p><p>Clear mechanistic understanding of the VEGF signaling pathway is of paramount importance since this pathway lies in the heart of vascular biology and is being targeted to treat a number of human diseases. The role of Neuropilin-1 is an important piece of the puzzle in VEGF signaling, and the central dogma about Neuropilin-1 is that binding to VEGFA is essential for its function despite lacking direct evidence to support it. Recent attempt to provide direct evidence by assessing a Neuropilin-1 allele that lacks VEGF-A binding capacity fell short to answer this question due to the severe reduction of expression (<xref ref-type="bibr" rid="bib6">Fantin et al 2014</xref>). In this report, Gelfand et al carried out rigorous analyses of a knockin Neuropilin-1 allele that abolished VEGFA binding yet preserved expression levels, and provided convincing evidence to demonstrate that the VEGFA binding capacity is dispensable for embryonic vascular development. They also provided biochemical evidence implicating Neuropilin-1 in the regulation of VEGFR2 levels. This study unequivocally refutes a dogma in the VEGF signaling field and reveals a novel Neuropilin-1 function. It is suitable for publication in <italic>eLife</italic>. The overall quality of the paper can be improved by addressing the following comments.</p><p>1) The degree of surface VEGFR2 reduction is much less than that of total VEGFR2 when Npn1 was deleted in ECs (compare <xref ref-type="fig" rid="fig5">Figure 5D</xref> with 5A), suggesting that the loss of Npn1 effect on surface VEGFR2 might be secondary to a reduction of total VEGFR2. Since the exiting data cannot distinguish between maintaining surface VEGFR2 levels versus regulating total VEGFR2 levels, the mechanism depicted in <xref ref-type="fig" rid="fig5">Figure 5G</xref> is not supported by actual data. The diagram should be modified to reflect a reduction in total VEGFR2, and the related text descriptions throughout the manuscript should be revised accordingly.</p><p>2) Since the reduction of surface VEGFR2 is modest (∼ 30%, <xref ref-type="fig" rid="fig5">Figure 5D</xref>) in the Npn1 null ECs, the functional significance of reducing VEGFR2 levels has not been clearly established. This question can be addressed by including a phospho-VEGFR2 blot or ELISA in <xref ref-type="fig" rid="fig5">Figure 5</xref>.</p><p>3) Data from <xref ref-type="bibr" rid="bib6">Fantin et al. 2014</xref> indicated that either severely reduced NRP1 levels and/or abolishment of NRP1-VEGFA binding plays an important role in postnatal angiogenesis. This is an important issue in the angiogenesis field. The authors have the proper tool and data to address this question. They can contribute to the angiogenesis community by mentioning some aspects of postnatal development in their Npn1VEGFR- mice. For example, they can include a table that documents mouse numbers at different age after birth to demonstrate if there is reduced viability overtime, instead of only giving a statement about a single time point. They can also point out if they have seen overt phenotypes reported by <xref ref-type="bibr" rid="bib6">Fantin et al. 2014</xref>, for example, have they ever seen evidence of cardiac failure in some of their knockin mice?</p><p>4) Some mechanistic data related to how NPN1 regulates VEGFR2 level will significantly elevate this manuscript to a different level. The authors should consider including one or two in vitro analysis similar to the NCAM / FGFR1 study (<xref ref-type="bibr" rid="bib10">Francavilla et al 2009</xref>).</p><p>Reviewer #2:</p><p>Gelfand et al present evidence that a point mutation (D320K) in the extracellular domain of neuropilin 1 (Npn1) prevents its binding to VEGF while leaving relatively intact Nrp1 association with Sema 3A. By gene replacement strategy they also generated a mouse line harboring the D320K mutation in the Npn1 locus.</p><p>The major finding of this paper is that, in absence of VEGF binding to Npn1, embryo vascular development is unaffected. Furthermore, it is reported that Npn1 co-immunoprecipitates with VEGFR2 and stabilizes VEGFR2 levels. The authors therefore hypothesize that Npn1 acts by controlling VEGFR2 levels through a still undefined mechanism.</p><p>1) The authors are not giving a fair representation of the literature. In the Abstract they report: “Npn1 is essential for vascular morphogenesis, but how Npn1 functions to guide vascular development remains elusive”. This statement is unfair considering several previous papers: Lanahan et al Dev Cell 2013; Fantin et al Development 2014 or Raimondi et al J Exp Med 2014. Furthermore, similarly to what is reported here, <xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref> already generated mice expressing Npn1 with a mutation that prevents VEGF binding. Homozygous mutated mice were viable and no severe and lethal cardiovascular phenotypes were observed implying that Npn1 could have VEGF-independent role during vascular development. Although the experimental approaches used in the two papers are different, the final conclusion remains the same.</p><p>2) The reduction in VEGFR2 levels in Npn1 mutants is indeed striking but does not correspond to the findings of many other authors where the uncoupling or strong reduction on Npn1 did not modify VEGFR2 or VEGFR1 expression (see, as an example, Fantin et al. Development 2014). In addition, while in <xref ref-type="fig" rid="fig5">Figure 5 A</xref> the drop in receptor expression is almost complete; in <xref ref-type="fig" rid="fig5">Figure 5 D</xref> it is around 35% only.</p><p>Since Title, Abstract and Discussion are focused on this finding the authors should present more solid data to support their conclusions. Last but not least, if the decrease in VEGFR2 is in the range presented in <xref ref-type="fig" rid="fig5">Figure 5 A</xref> the phenotype of Npn1 KO should be comparable to that of VEGFR2 KO and this is not the case.</p><p>3) The authors do not investigate the possible effects of their Npn1 mutant on neurons. Taking into account that some types of neurons rely on VEGF165 and Npn1 signaling, these observations are needed for a more complete interpretation of the mutant phenotype.</p><p>4) Introduction. Most of previously published data converge in saying that the domain of Npn1 responsible for the binding of Sema 3a is the b1 region and not the a1 (see for instance <xref ref-type="fig" rid="fig2">Figure 2</xref> Nat. Rev. Immunol. 2103 by Kikutani). This should be introduced and discussed in a better way in the text.</p><p>In conclusion, the model presented is of particular value and the data shown demonstrate unequivocally that Npn1 acts independently from binding to VEGF 165. However, more solid data on the mechanism of action of Npn1 are needed to make the paper novel enough for publication in the Journal.</p><p>Reviewer #3:</p><p>In this study the VEGF binding site of Neuropilin has been mutated in mice. This has been executed very well, with careful in vitro validation of the mutation to ensure that the mutation (D320K) only affects VEGF but not Sema3 binding. This approach is more sophisticated and better controlled than a previous study from another group who created a Nrp1Y297A mutation (PMID: 24401374), also aimed at ablating VEGF binding in Nrp1. The NrpY297A study suffered from a poor genetic targeting strategy that resulted in reduced Nrp1 expression (in vivo). Surprisingly, both studies found that VEGF binding to Nrp1 is not required for normal embryonic development. The current study shows this more convincingly, because the Nrp1Y297A study was more difficult to interpret because of the reduced Nrp1 levels caused by the Nrp1Y297A mutation. The novel Nrp1D320K strain described here will therefore be a very useful, well validated research tool.</p><p>Furthermore, the authors found that the Nrp1D320K mutation caused a dramatic reduction of VEGFR expression. This is a very intriguing observation and it would be interesting to know the reason for this and what the functional consequences are. The study does not explore this but the authors conclude (in the Discussion) that “Nrp1 regulates angiogenesis by controlling the amount of VEGFR2 expression at the cell surface and consequently the level of VEGFR2-VEGF signaling.”</p><p>There are several problems with this statement:</p><p>1) It is logically wrong. Since the Npn1VEGF mice have reduced VEGFR2 levels but develop normally, Nrp1 obviously is not regulating angiogenesis via VEGFR2 levels, otherwise the mice would have a Nrp1 KO phenotype.</p><p>2) The authors have not actually shown that VEGFR2 signalling is reduced. This would have to be shown more directly (e.g. VEGFR2 phosphorylation in Western blots).</p><p>3) Although the flow cytometry data in <xref ref-type="fig" rid="fig5">Figure 5C</xref> indicates reduced surface expression of VEGFR2 this seems only to be the case for around 50% of the cells. On the other hand, the Western blot data (<xref ref-type="fig" rid="fig5">Figure 5 A</xref>) indicates a much stronger reduction of around 90% (contradicting the flow cytometry data somewhat). This demonstrates that also intracellular VEGFR2 must be dramatically reduced and not only surface VEGFR2</p><p>4) VEGFR2 levels were only studied in lung endothelial cells. Yet the main vascular phenotype in Nrp1 mutants is found in the brain and retinal vasculature.</p><p>5) The Nrp1Y297A mice also developed normally but that study found abnormalities in postnatal animal, such as abnormal retinal vasculature development, reduced neovascularisation in the oxygen induced retinopathy model and reduced tumour growth. These postnatal phenotypes should be also checked in the Nrp1D320K mice.</p><p>In summary, this study has been executed very nicely but the main drawback is that it ends prematurely. When I got to <xref ref-type="fig" rid="fig5">Figure 5</xref> I was expecting several more figures looking into receptor distribution/signalling in cultured cells etc. and in vivo phenotypes under stress conditions.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03720.019</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>Is the Nrp1 VEGF binding mutant mouse really normal? A close look at the retinal vasculature (e.g. at P7, P9, and adulthood) and at health/fertility at later ages would be useful. You might consider stressing the system, for example with the oxygen induced retinopathy model (your colleague Lois Smith at Children's Hospital is the expert) or with a test of tumor angiogenesis, both of which tie into the applied aspects of this work as related to the efficacy of anti-Nrp1 antibodies. Comparing the phenotypes to those reported by <xref ref-type="bibr" rid="bib6"><italic>Fantin et al (2014)</italic></xref> would be useful.</italic></p><p>We thank the reviewers for this valuable suggestion. We have performed new experiments and now provide additional data describing the phenotype of the <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant.</p><p>1) We generated a viability table detailing the survival of <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants in comparison to their littermates across several different developmental stages (E14.5, P7 and adult, <xref ref-type="fig" rid="fig3s2">Figure 3 – figure supplement 2E</xref>). In contrast to the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph, <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants are present at appropriate Mendelian ratios and exhibit a normal survival rate.</p><p>2) We grossly examined several organ systems (brain, heart, lung, and kidney) at P9 and adulthood and now include organ weights in <xref ref-type="fig" rid="fig3s2">Figure 3–figure supplement 2B,C</xref>. Together with the whole mount images and body weight graph presented in <xref ref-type="fig" rid="fig3">Figure 3D-F</xref>, our results clearly demonstrate that <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants experience normal body growth.</p><p>3) We also generated whole mount images to illustrate the normal morphology of the <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> heart at P9 (<xref ref-type="fig" rid="fig3s2">Figure 3–figure supplement 2A</xref>) and noted in the text that <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants do not have any of the cardiac abnormalities observed in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph.</p><p>4) We have also noted in the text that <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants are fertile (pg 8) and in contrast to the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph, <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants maintained appropriate NRP1 protein levels in several adult organ systems (<xref ref-type="fig" rid="fig3s2">Figure 3–figure supplement 2D</xref>).</p><p>5) We examined the retinal vasculature in the <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant at P9 and adulthood (<xref ref-type="fig" rid="fig6">Figure 6</xref>). Through this analysis, we uncovered that <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants have normal vessel density and do not develop the endothelial tufts observed in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> mutant. <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants do exhibit some of the angiogenesis phenotypes previously reported in <xref ref-type="bibr" rid="bib28">Pan et al., 2007</xref> and <xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref> such as reduced vascular extension and reduced retinal arteries at P9. This mild retinal phenotype suggests that the retinal vasculature is uniquely sensitive to the loss of VEGF-NRP1 binding. Interestingly, the vascular extension phenotype is resolved in adulthood and the retinal abnormalities in the <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant are substantially less severe than those described in <xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref> suggesting that some of the vascular defects reported in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> mutant are secondary to aberrant NRP1 protein levels rather than a lack of VEGF-NRP1 binding.</p><p>6) Finally, we performed femoral artery ligation surgeries to induce hind limb ischemia in <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants and control littermates. In this challenge situation, the <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant initially exhibited reduced blood flow recovery to the injured hind limb post, but eventually caught up to control levels by seven days post-surgery (<xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref>).</p><p><italic>The decrease in VEGFR2 levels (and surface VEGFR2), which is presented as the mechanism by which the Nrp1 KO exerts its effects, could use more experimental backing. Checking the level of VEGFR2 phosphorylation in response to VEGF and an assessment of VEGFR2 trafficking with vs. without Nrp1 vs. with the Nrp1 VEGF binding mutant (in cell culture) would be very helpful</italic>.</p><p>We agree with these sentiments and have examined the level of VEGFR2 phosphorylation in lung lysates following VEGF treatment in the <italic>Tie2-Cre,Nrp1</italic><sup><italic>fl/-</italic></sup> and <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants. We discovered that <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants have a modest reduction in VEGFR2 phosphorylation at the tyrosine residue 1175 (Y1175) upon VEGF treatment without a change in total VEGFR2 protein (<xref ref-type="fig" rid="fig5">Figure 5C,D</xref>; <xref ref-type="fig" rid="fig5s2">Figure 5–figure supplement 2A,B</xref>). In contrast, the <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/-</italic></sup> mutant exhibits a significant reduction in VEGFR2 phosphorylation and VEGFR2 protein levels (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>; <xref ref-type="fig" rid="fig5s2">Figure 5–figure supplement 2C,D</xref>).</p><p>In regards to the suggestion for cell culture experiments, we have found several <italic>in vitro</italic> studies using multiple cell culture systems already examining the impact of NRP1 on VEGFR2 trafficking that demonstrate how NRP1 is essential for the proper presentation, recycling, and degradation of VEGFR2.</p><p>1) <xref ref-type="bibr" rid="bib31">Shintani et al., 2006</xref> used siRNA knock-down and adenovirus infection of <italic>Nrp1</italic> in human umbilical vein endothelial cells (HUVEC) to show that VEGFR2 protein level is decreased in the absence of NRP1 while <italic>Vegfr2</italic> mRNA levels are unaffected by NRP1 siRNA. Moreover, the overexpression of <italic>Nrp1</italic> led to an increase in VEGFR2 protein level as well as VEGFR2 cell surface expression.</p><p>2) Similarly, Holmes et al., 2008 found that NRP1 siRNA consistently and significantly reduced VEGFR2 expression upon VEGF induced receptor-mediated endocytosis, but found no significant changes in <italic>Vegfr2</italic> mRNA levels.</p><p>3) <xref ref-type="bibr" rid="bib1">Ballmer-Hofer et al., 2011</xref> used porcine aortic endothelial cell (PAEC) lines stably expressing VEGFR2, NRP1, or both in conjunction with immunostaining to visually follow VEGFR2 trafficking in the presence and absence of NRP1. In PAECs expressing only VEGFR2, VEGF stimulation triggered VEGFR2 to be internalized and accumulate in Rab7 vesicles for degradation. However, in the presence of NRP1, VEGFR2 is stabilized in Rab11 vesicles to be recycled back to the cell surface.</p><p>4) <xref ref-type="bibr" rid="bib14">Hamerlik et al., 2012</xref> examined human glioblastoma multiforme cells isolated from patients and found that shRNA mediated knock-down of NRP1 resulted in dramatically decreased VEGFR2 protein levels accompanied by a lower surface presentation of VEGFR2 and a decrease in cell viability. Furthermore, cell surface protein biotinylation and immunofluorescence staining with confocal microscopy confirmed the co-localization of VEGFR2-NRP1 with the early/recycling endosome</p><p>These <italic>in vitro</italic> studies are consistent with our <italic>in vivo</italic> results and provide clear mechanistic data related to how NRP1 regulates VEGFR2. Our current study provides the first <italic>in vivo</italic> evidence that NRP1 controls VEGFR2 levels at the cell membrane and offers the first <italic>in vivo</italic> phenotypic characterization linking NRP1 regulated VEGFR2 surface expression to vascular development.</p><p><italic>Is the VE cadherin level in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref> <italic>lower in the absence of Nrp1? If so, does this suggest other roles for Nrp1? Also</italic>, <italic>the decrease in VEGFR2 by Western blot seems more dramatic than the decrease shown by FACS. Is the lower level of VEGFR2 shown by FACS in the absence of Nrp1 really insufficient for VEGF signaling? Or is something more subtle at play (defective trafficking?)?</italic></p><p>To further clarify the western blot data shown in <xref ref-type="fig" rid="fig5">Figure 5</xref>, we have quantified the intensity of the bands from all of our blots and determined that VE-cadherin levels are not significantly reduced relative to Tubulin (see quantification below). In addition, we have quantified the total VEGFR2 protein levels in our Western blots (<xref ref-type="fig" rid="fig5">Figure 5 B,D</xref>) and selected a more representative blot to reflect this quantification. We also examined VEGFR2 phosphorylation in the <italic>Tie2-Cre,Nrp1</italic><sup><italic>fl/-</italic></sup> mutants to determine if the reduction in VEGFR2 levels in the absence of NRP1 is sufficient to alter VEGF signaling. Indeed, we found a significant reduction in VEGFR2 phosphorylation (almost absent) and included them in <xref ref-type="fig" rid="fig5s2">Figure 5–figure supplement 2</xref>.</p><p>Reviewer #1:</p><p><italic>1) The degree of surface VEGFR2 reduction is much less than that of total VEGFR2 when Npn1 was deleted in ECs (compare</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5D</italic></xref> <italic>with 5A), suggesting that the loss of Npn1 effect on surface VEGFR2 might be secondary to a reduction of total VEGFR2. Since the exiting data cannot distinguish between maintaining surface VEGFR2 levels versus regulating total VEGFR2 levels, the mechanism depicted in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5G</italic></xref> <italic>is not supported by actual data. The diagram should be modified to reflect a reduction in total VEGFR2, and the related text descriptions throughout the manuscript should be revised accordingly</italic>.</p><p>To reconcile the level of total versus surface VEGFR2 protein, we have gone back and quantified the VEGFR2 levels by Western blot and found that the reduction in VEGFR2 is more in line with the results observed in the FACS analysis. We have updated <xref ref-type="fig" rid="fig5">Figure 5</xref>, to include a more representative Western blot and a graph quantifying these results. Moreover, since the decrease in total VEGFR2 is comparable to the reduction in surface VEGFR2 seen via FACS analysis, we believe our model accurately reflects the data presented in this manuscript now.</p><p><italic>2) Since the reduction of surface VEGFR2 is modest (∼ 30%,</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5D</italic></xref><italic>) in the Npn1 null ECs, the functional significance of reducing VEGFR2 levels has not been clearly established. This question can be addressed by including a phospho-VEGFR2 blot or ELISA in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref>.</p><p>This point is a great suggestion, and we have performed experiments to quantify phospho-VEGFR2 upon VEGF treatment. In particular, we have examined the level of VEGFR2 phosphorylation in lung lysates following VEGF treatment in the <italic>Tie2-Cre,Nrp1</italic><sup><italic>fl/-</italic></sup> and <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants.</p><p><italic>3) Data from</italic> <xref ref-type="bibr" rid="bib6"><italic>Fantin et al. 2014</italic></xref> <italic>indicated that either severely reduced NRP1 levels and/or abolishment of NRP1-VEGFA binding plays an important role in postnatal angiogenesis. This is an important issue in the angiogenesis field. The authors have the proper tool and data to address this question. They can contribute to the angiogenesis community by mentioning some aspects of postnatal development in their Npn1VEGFR- mice. For example, they can include a table that documents mouse numbers at different age after birth to demonstrate if there is reduced viability overtime, instead of only giving a statement about a single time point</italic>. <italic>They can also point out if they have seen overt phenotypes reported by</italic> <xref ref-type="bibr" rid="bib6"><italic>Fantin et al. 2014</italic></xref><italic>, for example, have they ever seen evidence of cardiac failure in some of their knockin mice?</italic></p><p>We thank the reviewer for this suggestion. Indeed, we have the proper <italic>in vivo</italic> tools to delineate whether the phenotypes described in <xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref> originate from the severe reduction in NRP1 levels or the abolishment of VEGF-NRP1 binding. We have expanded the phenotypic analysis of our <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant by including a table that documents survival at different developmental stages to demonstrate the sustained viability of the <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant. We have also examined organ weight, heart morphology, and retinal angiogenesis. As described above, we do not observe any of the impaired growth, cardiac failure, or postnatal fatality described in <xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref> demonstrating that their results arise from reduced NRP1 levels in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph rather than a lack of VEGF-NRP1 binding.</p><p><italic>4) Some mechanistic data related to how NPN1 regulates VEGFR2 level will significantly elevate this manuscript to a different level. The authors should consider including one or two in vitro analysis similar to the NCAM / FGFR1 study (</italic><xref ref-type="bibr" rid="bib10"><italic>Francavilla et al 2009</italic></xref><italic>)</italic>.</p><p>As described in detail above, previous <italic>in vitro</italic> work in multiple cell culture systems has used gain of function (overexpression) and loss of function (RNAi knock-down) to demonstrate that NRP1 is essential for the proper presentation, recycling and degradation of VEGFR2. These <italic>in vitro</italic> studies are consistent with our <italic>in vivo</italic> results and provide clear mechanistic data related to how NRP1 regulates VEGFR2. Our current study provides the first <italic>in vivo</italic> evidence that NRP1 controls VEGFR2 levels at the cell membrane and offers the first <italic>in vivo</italic> phenotypic characterization linking NRP1 regulated VEGFR2 surface expression to vascular development.</p><p>Reviewer #2:</p><p><italic>1) The authors are not giving a fair representation of the literature. In the Abstract they report: “Npn1 is essential for vascular morphogenesis, but how Npn1 functions to guide vascular development remains elusive”. This statement is unfair considering several previous papers: Lanahan et al Dev Cell 2013; Fantin et al Development 2014 or Raimondi et al J Exp Med 2014</italic>.</p><p><italic>Furthermore, similarly to what is reported here,</italic> <xref ref-type="bibr" rid="bib6"><italic>Fantin et al., 2014</italic></xref> <italic>already generated mice expressing Npn1 with a mutation that prevents VEGF binding. Homozygous mutated mice were viable and no severe and lethal cardiovascular phenotypes were observed implying that Npn1 could have VEGF-independent role during vascular development. Although the experimental approaches used in the two papers are different, the final conclusion remains the same</italic>.</p><p>We have updated this sentence in the Abstract to provide a fairer representation of the previously published work. As Reviewer 1 points out, the phenotypes described in <xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref> have two potential interpretations: they could originate from the severe reduction in NRP1 levels or from the abolishment of VEGF-NRP1 binding. Our <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant maintains appropriate levels of NRP1 protein (<xref ref-type="fig" rid="fig3">Figure 3C</xref>, Figure 3–figure supplement 3D) and provides the proper <italic>in vivo</italic> tool to delineate between these two alternatives. We have now assessed the phenotypes described in <xref ref-type="bibr" rid="bib6">Fantin et al., 2014</xref> and while some are linked to VEGF-NRP1 binding (retinal vasculature extension and arteriogenesis) others most likely result from the reduced NRP1 levels observed in the <italic>Nrp1</italic><sup><italic>Y297A/Y297A</italic></sup> hypomorph rather than from lack of VEGF-NRP1 binding (cardiac failure, retinal vascular density, and retinal endothelial tufts). A detailed point-by-point description of these results is provided above.</p><p><italic>2) The reduction in VEGFR2 levels in Npn1 mutants is indeed striking but does not correspond to the findings of many other authors where the uncoupling or strong reduction on Npn1 did not modify VEGFR2 or VEGFR1 expression (see, as an example, Fantin et al. Development 2014). In addition, while in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5 A</italic></xref> <italic>the drop in receptor expression is almost complete; in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5 D</italic></xref> <italic>it is around 35% only</italic>.</p><p><italic>Since Title, Abstract and Discussion are focused on this finding the authors should present more solid data to support their conclusions. Last but not least, if the decrease in VEGFR2 is in the range presented in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5 A</italic></xref> <italic>the phenotype of Npn1 KO should be comparable to that of VEGFR2 KO and this is not the case</italic>.</p><p>We agree with this sentiment and have gone back to quantify the VEGFR2 levels in our Western blots. Indeed, the decrease in VEGFR2 levels is more on par with the FACS analysis and we have replaced the Western blot image with a more representative data. Furthermore, we have included our quantification in <xref ref-type="fig" rid="fig5">Figure 5</xref>.</p><p><italic>3) The authors do not investigate the possible effects of their Npn1 mutant on neurons. Taking into account that some types of neurons rely on VEGF165 and Npn1 signaling, these observations are needed for a more complete interpretation of the mutant phenotype</italic>.</p><p>First, we looked at the most robust neural phenotype that has been linked to VEGF-NRP1 function. Specifically, facial motor neurons express NRP1, and their migration during development is dependent on the presence of VEGF164 (Schwarz et al., 2004). According to the literature, <italic>Nrp1</italic><sup><italic>-/-</italic></sup> mutants have a defect in which the migration of the facial motor neurons is disrupted. The phenotype was thought to be a result of VEGF-NRP1 binding in neurons because <italic>Nrp1</italic><sup><italic>Sema-</italic></sup> and <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/-</italic></sup> mutants do not have this phenotype while VEGF-120 mice phenocopy the <italic>Nrp1</italic><sup><italic>-/-</italic></sup>. With <italic>in situ</italic> hybridization for <italic>Isl1</italic>, we analyzed the migratory path of motor neurons at E12.5. To our surprise, we did not see a migration defect in the <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant. However, we also failed to see a migration defect in the neuronal-specific NRP1 knock-out (<italic>Nestin-Cre,Nrp1</italic><sup><italic>fl/fl</italic></sup>) demonstrating that NRP1 is not required in neurons for this phenotype. In this regard, our data bring the claims of previous work into question and resolving this discrepancy is beyond the scope of our current manuscript. In addition, we also examined gonadotropin-releasing hormone (GnRH) neurons because Carboni et al., 2011 showed that VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signaling pathways through NRP1. However, as with the facial motor neurons, we did not detect any GnRH survival phenotype in our <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants.</p><p><italic>4) Introduction. Most of previously published data converge in saying that the domain of Npn1 responsible for the binding of Sema 3a is the b1 region and not the a1 (see for instance</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref> <italic>Nat. Rev. Immunol. 2103 by Kikutani). This should be introduced and discussed in a better way in the text</italic>.</p><p>We have already stated that “A previous structure-function analysis revealed that the b1 domain of NRP1 is necessary and sufficient for VEGF binding (<xref ref-type="bibr" rid="bib12">Gu et al., 2002</xref>). However, this b1 region is also required for SEMA3-NRP1 interactions so a series of <italic>Nrp1</italic> variants containing smaller deletions in the b1 domain were engineered with site-directed mutagenesis to identify a region specific for VEGF-NRP1 binding (<xref ref-type="fig" rid="fig1">Figure 1A</xref>)” in the Results section.</p><p>Reviewer #3:</p><p><italic>Furthermore, the authors found that the Nrp1D320K mutation caused a dramatic reduction of VEGFR expression. This is a very intriguing observation and it would be interesting to know the reason for this and what the functional consequences are. The study does not explore this but the authors conclude (in the Discussion) that “Nrp1 regulates angiogenesis by controlling the amount of VEGFR2 expression at the cell surface and consequently the level of VEGFR2-VEGF signaling</italic>.<italic>”</italic></p><p><italic>There are several problems with this statement</italic>:</p><p><italic>1) It is logically wrong. Since the Npn1VEGF mice have reduced VEGFR2 levels but develop normally, Nrp1 obviously is not regulating angiogenesis via VEGFR2 levels, otherwise the mice would have a Nrp1 KO phenotype</italic>.</p><p>Based on the critique, it is apparent that Reviewer 3 misunderstood the results from our study. We clearly show that <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mice have normal VEGFR2 levels, as seen in <xref ref-type="fig" rid="fig5">Figure 5</xref> via Western blot and FACS analysis. In contrast, <italic>Tie2-Cre,Nrp1</italic><sup><italic>fl/-</italic></sup> mutants have reduced VEGFR2 levels which is consistent with the <italic>Nrp1</italic><sup><italic>-/-</italic></sup> phenotype shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>.</p><p><italic>2) The authors have not actually shown that VEGFR2 signalling is reduced. This would have to be shown more directly (e.g. VEGFR2 phosphorylation in Western blots)</italic>.</p><p>We have performed additional experiments to determine the level of VEGFR2 phosphorylation in lung lysates following VEGF treatment in the <italic>Tie2-Cre,Nrp1</italic><sup><italic>fl/-</italic></sup> and <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutants. Please see our detailed description above.</p><p><italic>3) Although the flow cytometry data in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5C</italic></xref> <italic>indicates reduced surface expression of VEGFR2 this seems only to be the case for around 50% of the cells</italic>. <italic>On the other hand, the Western blot data (</italic><xref ref-type="fig" rid="fig5"><italic>Figure 5 A</italic></xref><italic>) indicates a much stronger reduction of around 90% (contradicting the flow cytometry data somewhat). This demonstrates that also intracellular VEGFR2 must be dramatically reduced and not only surface VEGFR2</italic></p><p>As previously described above, we have reassessed VEGFR2 levels in our Western blots and now have included quantification in <xref ref-type="fig" rid="fig5">Figure 5</xref>. Importantly, we have determined that the decrease in VEGFR2 observed in the <italic>Tie2-Cre,Nrp1</italic><sup><italic>fl/-</italic></sup> mutant is comparable to the reduction seen in our FACS analysis</p><p><italic>4) VEGFR2 levels were only studied in lung endothelial cells. Yet the main vascular phenotype in Nrp1 mutants is found in the brain and retinal vasculature</italic>.</p><p>We examined VEGFR2 levels in lung endothelial cells for practical purposes, in that we were able to harvest the greatest number of endothelial cells from this organ to perform our analysis. The <italic>Tie2-Cre;Nrp1</italic><sup><italic>fl/-</italic></sup> mutants are embryonic lethal and finding a tissue with an adequate source of cells was of paramount importance. We attempted to analyze brain endothelial cells via FACS, but the tissue was difficult to dissociate and formed clumps during the immunolabeling process. These clumps were unable to be analyzed via the FACS equipment and resulted in a significant loss of endothelial cells from our samples.</p><p><italic>5) The Nrp1Y297A mice also developed normally but that study found abnormalities in postnatal animal, such as abnormal retinal vasculature development, reduced neovascularisation in the oxygen induced retinopathy model and reduced tumour growth. These postnatal phenotypes should be also checked in the Nrp1D320K mice</italic>.</p><p>We appreciate this constructive criticism and have now included a broader phenotypic analysis of the <italic>Nrp1</italic><sup><italic>VEGF-</italic></sup> mutant. In particular, we have examined the retinal vasculature at two time points (P9 and adult), challenged the mutant with a hindlimb ischemia assay, and included additional information about fertility, organ size, and viability.</p></body></sub-article></article>