Enlarged, sac-like X-galCstained structures were observed beneath the skin in the single heterozygotes and single-heterozygote littermate, suggesting that rather than forming sprouts, lymph sacs were growing in size in these embryos (Fig. receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, conversation between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C. Introduction Organ system development across higher order species requires formation of tubular networks. These networks can be found in the respiratory system (Affolter and Caussinus, 2008), in the vertebrate ureteric system (Costantini, 2006), and most prominently, in the circulatory system, including the blood and lymphatic vasculature (Horowitz and Simons, 2008). The architecture, and therefore function of such systems, is largely determined by one key topographical feature: branching, which occurs by the sprouting of new tubes from preexisting ones. Thus, the molecular mechanisms regulating sprouting are central to how a given branching system forms (Horowitz and Simons, 2008), yet our understanding of this process is limited. The lymphatic vasculature forms a hierarchical branching network that covers the skin and most internal organs of the body. The lymphatic system maintains tissue fluid balance by recovering fluid from the interstitial space (Alitalo et al., 2005). Unlike the circulatory system, the distal-most branches of the lymphatic vasculature are blind-ended capillaries that drain into larger-collecting lymphatics and return the lymph to the hematogenous system via the thoracic duct (Cueni and Detmar, 2006; Tammela et al., 2007). Imbalances in circulation of fluid or cells can result in lymphedema or disturbed immune responses. In the mouse, lymph vessel development begins around embryonic day 10 (E10) by sprouting from the cardinal veins in the jugular and perimesonephric area to form lymph sacs. From these lymph sacs, vessels subsequently grow by proliferation and centrifugal sprouting toward the skin and internal organs (Maby-El Hajjami and Petrova, 2008; Oliver and Srinivasan, 2008). After the initial differentiation and budding of lymphatic vessels, which is usually regulated IDH-C227 by Prox-1 and Sox-18 (Wigle et al., 2002; Fran?ois et al., 2008), their subsequent migration, growth, and survival are mainly controlled by VEGF-C (Karpanen and Alitalo, 2008; Maby-El Hajjami and Petrova, 2008). Homozygous mutants show a reduction of small lymphatic vessels and lymphatic capillaries, indicating that Nrp2 is not required for lymphatic development but modulates it (Yuan et al., 2002). Moreover, inhibition of Nrp2 using a monoclonal antibody that selectively blocks VEGF-C binding to Nrp2 resulted in a reduction of tumor lymphangiogenesis and metastasis, which is a result with significant clinical implications (Caunt et al., 2008). However, these Mouse monoclonal to CD8/CD45RA (FITC/PE) experiments did not address the mechanism by which Nrp2 mediates lymphangiogenesis in developmental or pathological contexts. In this study, we show that in vivo modulation of Nrp2 using blocking antibodies or genetic reduction of Nrp2 levels results in selective disruption of lymphatic sprout formation without affecting other aspects of lymphatic development. The inhibition of sprout formation appears to be a result of altered behavior of tip cells at the leading ends of lymphatic vessel sprouts. Finally, we show that Nrp2 genetically interacts with VEGFR3 and not VEGFR2, indicating that Nrp2 partners with VEGFR3 to mediate lymphatic vessel sprouting. Thus, like in the nervous system, where Nrp2 mainly regulates axon guidance, its function in the lymphatic vasculature appears to affect a particular step of formation of the lymphatic tree. However, although the guidance functions Nrp2 exerts in response to semaphorins in the nervous system are mainly repulsive and mediate growth cone collapse (Chen et al., 2000), they appear to be attractive in the vascular system, mediating tip cell extension and guided vessel sprouting in response to VEGF-C. Results Tail dermal lymphatics as a model system for studying developmental lymphangiogenesis The superficial dermal lymphatic network of the adult mouse tail consists of a hexagonal lattice of lymphatic capillaries (Hagendoorn et al., 2004). At each junction in this IDH-C227 matrix, there is a multiringed lymphatic vessel complex (hereafter referred to as lymphatic ring complexes [LRCs]) that connects the superficial network to collecting ducts. Initially, we performed a developmental analysis of lymphatic network formation. As the network largely forms after birth by sprouting from the major lateral lymphatic vessels that lie in the deeper dermis and the mature hexagonal pattern is established by postnatal day 10 (P10), we restricted IDH-C227 our analysis to this early postnatal time frame (Fig. 1). We found that grossly, the lymphatic vessels have an irregular, discontinuous pattern at early time points (P2; Fig. 1 A) with only few LRCs comprised of only single-ringed vessels (Fig. 1 E). Multiple lymphatic endothelial cells (LECs).
Enlarged, sac-like X-galCstained structures were observed beneath the skin in the single heterozygotes and single-heterozygote littermate, suggesting that rather than forming sprouts, lymph sacs were growing in size in these embryos (Fig
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