The earliest blood vessels in the mammalian embryo are formed when endothelial cells (ECs) differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing ECs. Here we show that the earliest precursors of erythrocytes, megakaryocytes and macrophages, the yolk sac-derived erythro-myeloid progenitors (EMPs), provide a complementary source of ECs that are recruited into pre-existing vasculature. Whereas a first wave of yolk sac-resident EMPs contributes ECs to the yolk sac endothelium, a second wave of EMPs colonises the embryo and contributes ECs to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognised source of ECs, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing ECs and the incorporation of ECs derived from hematopoietic precursors.
Key Points NRP1 promotes brain angiogenesis cell autonomously in endothelium, independently of heterotypic interactions with nonendothelial cells. NRP1 plays a key role in endothelial tip rather than stalk cells during vessel sprouting in the brain.
Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1 Y297A/Y297A mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF 164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1 Y297A/Y297A mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth. KEY WORDS: NRP1, VEGF, Angiogenesis, Arteriogenesis, Retina, Hindbrain INTRODUCTIONNRP1 is a transmembrane receptor for the VEGF 165 isoform (VEGF 164 in mice) and the neuronal guidance cue SEMA3A, with essential roles in both vascular and neuronal development (reviewed by Pellet-Many et al., 2008;Raimondi and Ruhrberg, 2013). Accordingly, Nrp1-null mice die before birth with severe cardiovascular and neuronal defects (Kitsukawa et al., 1997;Kawasaki et al., 1999 Centre for Cardiovascular Biology and Medicine, BHF Laboratories, Division of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK. *These authors contributed equally to this work ‡ These authors contributed equally to this work § Authors for correspondence (I.Zachary@ucl.ac.uk; c.ruhrberg@ucl.ac.uk) This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.Received 25 August 2013; Accepted 3 November 2013 whereas mice carrying a mutated extracellular domain that abolishes SEMA3A, not VEGF 164 , binding show defective nerve, but not blood vessel, patterning (Gu et al., 2003;Vieira et al., 2007). These and other genetic, biochemical and cell biological data support a model in which VEGF 165 binding induces complex formation between NRP1 and VEGFR2 (KDR -Mouse Genome Informatics) to enhance VEGFR2 signalling during EC migration in vitro (e.g. Soker et al., 2002;Wang et al., 2003;Evans et al., 2011) and arteriogenesis in vivo (Lanahan et al., 2013).The extracellular NRP1 a1/a2 and b1/b2 domains are crucial f...
The formation of the cardiovasculature, consisting of both the heart and blood vessels, is a critical step in embryonic development and relies on three processes termed vasculogenesis, angiogenesis, and vascular remodeling. The transmembrane protein NRP1 is an essential modulator of embryonic angiogenesis with additional roles in vessel remodeling and arteriogenesis. NRP1 also enhances arteriogenesis in adults to alleviate pathological tissue ischemia. However, in certain circumstances, vascular NRP1 signaling can be detrimental, as it may promote cancer by enhancing tumor angiogenesis or contribute to tissue edema by increasing vascular permeability. Understanding the mechanisms of NRP1 signaling is, therefore, of profound importance for the design of therapies aiming to control vascular functions. Previous work has shown that vascular NRP1 can variably serve as a receptor for two secreted glycoproteins, the VEGF-A and SEMA3A, but it also has a poorly understood role as an adhesion receptor. Here, we review current knowledge of NRP1 function during blood vessel growth and homeostasis, with special emphasis on the vascular roles of its multiple ligands and signaling partners.
The mouse embryo hindbrain is a robust and adaptable model for studying sprouting angiogenesis. It permits the spatiotemporal analysis of organ vascularization in normal mice and in mouse strains with genetic mutations that result in late embryonic or perinatal lethality. Unlike postnatal models such as retinal angiogenesis or Matrigel implants, there is no requirement for the breeding of conditional knockout mice. The unique architecture of the hindbrain vasculature allows whole-mount immunolabeling of blood vessels and high-resolution imaging, as well as easy quantification of angiogenic sprouting, network density and vessel caliber. The hindbrain model also permits the visualization of ligand binding to blood vessels in situ and the analysis of blood vessel growth within a natural multicellular microenvironment in which endothelial cells (ECs) interact with non-ECs to refine the 3D organ architecture. The entire procedure, from embryo isolation to imaging and through to results analysis, takes approximately 4 d.
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