Activation of vascular endothelial growth factor (VEGF) receptor-3 (VEGFR-3) by VEGF-C initiates lymphangiogenesis by promoting lymphatic proliferation and migration. However, it is unclear whether VEGFR-3 signaling is required beyond these initial stages, namely during the organization of new lymphatic endothelial cells (LECs) into functional capillaries. Furthermore, the role of VEGFR-2, which is also expressed on LECs and binds VEGF-C, is unclear. We addressed these questions by selectively neutralizing VEGFR-3 and/or VEGFR-2 for various time periods in an adult model of lymphangiogenesis in regenerating skin. While blocking either VEGFR-2 or VEGFR-3 with specific antagonist mAbs (DC101 and mF4-31C1, respectively) prior to lymphatic migration prevented lymphangiogenesis, blocking VEGFR-3 subsequent to migration did not affect organization into functional capillaries, and VEGFR-2 blocking had only a small hindrance on organization. These findings were confirmed in vitro using human LECs and anti-human antagonist mAbs (IMC-1121a and hF4-3C5): both VEGFR-2 and -3 signaling were required for migration and proliferation, but tubulogenesis in 3D cultures was unaffected by VEGFR-3 blocking and partially hindered by VEGFR-2 blocking. Furthermore, both in vitro and in vivo, while VEGFR-3 blocking had no effect on LEC organization, coneutralization of VEGFR-2, and VEGFR-3 completely prevented lymphatic organization. Our findings demonstrate that cooperative signaling of VEGFR-2 and -3 is necessary for lymphatic migration and proliferation, but VEGFR-3 is redundant with VEGFR-2 for LEC organization into functional capillaries.

SummaryWe report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.

The cellular and molecular events underlying the formation and differentiation of mesoderm to derivatives such as blood are critical to our understanding of the development and function of many tissues and organ systems. How different mesodermal populations are set aside to form specific lineages is not well understood. Although previous genetic studies in the mouse embryo have pointed to a critical role for the homeobox gene Mixlike (mMix) in gastrulation, its function in mesoderm development remains unclear. IntroductionThe formation of mesoderm is a pivotal process in the normal development of major tissues and organs of the mammalian embryo, including the hematopoietic, cardiovascular, and musculoskeletal systems. During early development, mesoderm is also a crucial component of extraembryonic structures such as the yolk sac, allantois, and placenta. 1,2 Mesoderm begins to form shortly after implantation during a process known as gastrulation. [3][4][5][6] Fate maps suggest that different mesodermal derivatives arise in an orderly manner from patterning of cell populations within distinct posteroanterior regions of the streak. 7,8 The most posterior region of the primitive streak is fated to form the extraembryonic mesoderm, while successively more anterior derivatives give rise to lateral, paraxial, and axial mesoderm. Despite intense effort, the mechanisms underlying the orderly allocation of mesoderm to its various lineages remain obscure. 9 A number of major signaling pathways, including transforming growth factor ␤ (TGF␤), fibroblast growth factor, 10 and Wnt, have been shown to play central roles during gastrulation. 4,6 While the functional relationships among these pathways in the induction and patterning of mesoderm remain to be defined, their activities are likely to be integrated, at least in part, through the downstream activities of nuclear transcription factors. Members of the Mix/Bix family of paired class homeobox genes are direct or indirect targets of the TGF␤ family members Nodal/activin [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]27 Xenopus Mix.1 (Xmix.1), the founding member of the Mix/Bix family, has been reported to ventralize mesoderm. 26 Several other Xenopus and zebrafish Mix/Bix genes have been implicated in the formation of endoderm or mesendoderm. 16,[18][19][20]22,24,25,28 Although Mix-related genes have been identified in all eukaryotic species analyzed, 29 only a single Mix gene has been found in mouse, chick, and humans. [29][30][31][32][33] The mouse Mix-like gene (here termed mMix; also known as Mml or Mixl1) is expressed in the posterior visceral endoderm prior to gastrulation and later in the primitive streak and nascent mesoderm. [32][33][34] The significance of this gene in gastrulation was revealed by analysis of mMix-deficient mouse embryos, which display numerous mesodermal and endodermal defects and arrest by embryonic day 9.5 (E9.5). 35 For example, the primitive streak is enlarged, a heart tube is absent and, in some mutants, the allantois is abno...

Members of the Xenopus and zebrafish Mix/Bix family of paired class homeodomain proteins play determining roles in both mesoderm and endoderm development and are induced by members of the TGFbeta/BMP family of signaling molecules. A single Mix gene has been identified in mouse, humans and chick. Prior to gastrulation, the mouse Mix (mMix) gene is expressed in the visceral endoderm and later in the primitive streak and nascent mesoderm, where it overlaps, in part, with T. Mix expression in ES-derived embryoid bodies is early and transient, overlapping partially with Flk1 activation around the time of formation of hemangioblasts. Both mMix mRNA and protein are found in a FACS-sorted population of T+Flk1+ cells from ES cell-derived embryoid bodies (EBs) which contains hemangioblasts. A complex embryonic lethal phenotype has been reported for Mix deficient embryos, including defects in allantoic (vascular) and cardiogenesis. Mesoderm forms in these embryos but is not patterned properly. Embryonic lethality occurs around E10.5–11, presumably as a result of the cardiovascular defects. We have generated inducible ES cell lines in which expression of Mix protein is responsive to doxycycline. Ectopic expression of Mix in EBs results in premature, enhanced expression of hemangioblast, angioblast and hematopoietic stem cell markers (mRNA and FACS analyses) and increased formation of stem/progenitor cells in clonogenic assays in methylcellulose. Together, the expression analyses, knockout phenotype, and gain-of-function studies in ES cells suggest that mMix functions early in induction and patterning of mesoderm, including formation of hematopoietic and endothelial lineages. Potential mMix target genes are being identified by microarray analyses of the inducible Mix ES lines. To examine mMix activities in vivo, we have generated null and conditional mMix knockout mice from several independently targeted ES cell lines. Analysis of these animals is in progress. Like Xenopus Mix.1, mouse Mix may represent an important connection between the TGFbeta/BMP pathway and hematopoietic/vascular development in the embryo.