Flk1, the gene encoding the vascular endothelial growth factor receptor 2 (VEGFR-2), is a well-known marker for vascular and hematopoietic progenitors and is indispensable for normal hematopoiesis and vasculogenesis. Here we show that Flk1 expression in the early mouse embryo marks a broad spectrum of mesodermal progenitors exiting the primitive streak as well as later mesodermal cell types including some cardiomyocytes, portions of the somites, and all extraembryonic mesoderm cells. These findings made use of an Flk1-lacZ knock-in allele in which the neomycin selection cassette was removed, which resulted in full replication of the endogenous expression of Flk1. Targeted IntroductionThe mouse Flk1 gene encodes the major signaling receptor, vascular endothelial growth factor receptor 2 (VEGFR-2), for vascular endothelial growth factor A (VEGF-A), and is essential for development of the vascular and hematopoietic systems in the early embryo. 1-4 VEGF signaling through VEGFR-2 continues to play key roles in controlling blood-vessel development throughout embryogenesis and into the adult. 5,6 In the early embryo and in differentiating embryonic stem (ES) cells, Flk1 expression seems to mark a common progenitor for both blood and endothelium, the so-called hemangioblast. 7,8 Recently, it has become evident that the expression of Flk1 may mark progenitors with broader potential than just endothelial formation and hematopoiesis. Single Flk1 ϩ cells derived from differentiating ES cells can produce cells expressing smooth muscle cell or cardiomyocyte markers in vitro. 9,10 In addition, multipotential cell lines derived from either the embryonic dorsal aorta 11 or from adult bone marrow stroma 12 have been shown to express Flk1. However, these experiments do not address whether Flk1 expression also marks cells with broad potential in vivo, nor the functional importance of Flk1 for differentiation of lineages other than endothelium and blood lineages.In situ expression analysis of endogenous Flk1 13 or of a Flk1-lacZ knock-in allele 3 has shown that Flk1 is expressed in some nonendothelial embryonic cell types, such as the posterior lateral plate mesoderm and the allantois. In addition, sorted Flk1 ϩ cells from E9.5 day embryos have been shown to produce cells expressing smooth muscle cell markers in vitro. 14 Lineage tracing of Flk1-expressing cells using Cre recombinase-mediated cell marking indicated that Flk1-expressing cells could also contribute progeny to the cardiac and skeletal muscle cell lineages, 15 a finding not predicted by the expression of the endogenous gene. Here we show that the expression of a Flk1-lacZ knock-in allele can also be detected in some cardiomyocytes and in parts of the somites, as well as all extraembryonic mesoderm cells arising from the primitive streak, provided that the neomycin selection cassette is removed from the genome. In addition, deletion of a region in intron 1 that has been proposed to direct endothelial expression produced no alteration in either endothelial or broader mes...

Vascular and nervous systems, two major networks in mammalian bodies, show a high degree of anatomical parallelism and functional crosstalk. During development, neurons guide and attract blood vessels, and consequently this parallelism is established. Here, we identified a noncanonical neurovascular interaction in eye development and disease. VEGFR2, a critical endothelial receptor for VEGF, was more abundantly expressed in retinal neurons than in endothelial cells, including endothelial tip cells. Genetic deletion of VEGFR2 in neurons caused misdirected angiogenesis toward neurons, resulting in abnormally increased vascular density around neurons. Further genetic experiments revealed that this misdirected angiogenesis was attributable to an excessive amount of VEGF protein around neurons caused by insufficient engulfment of VEGF by VEGFR2-deficient neurons. Moreover, absence of neuronal VEGFR2 caused misdirected regenerative angiogenesis in ischemic retinopathy. Thus, this study revealed neurovascular crosstalk and unprecedented cellular regulation of VEGF: retinal neurons titrate VEGF to limit neuronal vascularization. PAPERFLICK: