Sprouting angiogenesis is critical to blood vessel formation, but the cellular and molecular controls of this process are poorly understood. We used time-lapse imaging of green fluorescent protein (GF-P)-expressing vessels derived from stem cells to analyze dynamic aspects of vascular sprout formation and to determine how the vascular endothelial growth factor (VEGF) receptor flt-1 affects sprouting. Surprisingly, loss of flt-1 led to decreased sprout formation and migration, which resulted in reduced vascular branching. This phenotype was also seen in vivo, as flt-1 ؊/؊ embryos had defective sprouting from the dorsal aorta. We previously showed that loss of flt-1 increases the rate of endothelial cell division. However, the timing of division versus morphogenetic effects suggested that these phenotypes were not causally linked, and in fact mitoses were prevalent in the sprout field of both wild-type and flt-1 ؊/؊ mutant vessels. Rather, rescue of the branching defect by a soluble flt-1 (sflt- 1 IntroductionThe first step in the formation of most blood vessels is the production of an intricately branched vascular plexus. [1][2][3] This plexus is subsequently pruned and remodeled and in some cases coalesced to form larger vessels. The primary branched plexus forms by several processes, including the initial assembly of vascular precursor cells called vasculogenesis, and the subsequent migration of endothelial cells from the parent vessel called sprouting angiogenesis. The sprouts that form migrate until they reach another sprout or vessel, whereupon they most often form connections that are essential for elaborating and expanding the branched network. Although the formation of vascular sprouts has been described historically, 4-6 surprisingly little is known of the cellular processes and molecular controls of sprout formation, and even less is known about how these processes are integrated with other ongoing cellular events such as cell division.The vascular sprouts that form during embryonic development extend from primitive vessels. Individual endothelial cells send out filopodia, then migrate away from the parent vessel without breaking all contacts with surrounding cells, so that eventually multiple cells comprise the sprout. Cell numbers in the sprout are also increased by cell division behind the leading tip of the sprout. 6,7 Formation of a branched vascular plexus depends on the regulated expression of vascular endothelial growth factor-A (VEGF-A) by nonendothelial cells. This signal modulates intracellular signaling pathways that regulate endothelial cell division, migration, and survival. 8 This regulation is dose-dependent, as modest changes in the amount of available VEGF-A in either direction compromise vascular development, and loss of even one copy of the Vegf-A gene leads to vascular disruption and embryonic lethality. 9-13 Availability also seems to be regulated by the production of different isoforms of VEGF-A via alternative splicing. These isoforms have differing affinities for matrix componen...
New blood vessel formation requires the coordination of endothelial cell division and the morphogenetic movements of vessel expansion, but it is not known how this integration occurs. Here, we show that endothelial cells regulate division orientation during the earliest stages of blood vessel formation, in response to morphogenetic cues. In embryonic stem (ES) cell-derived vessels that do not experience flow, the plane of endothelial cytokinesis was oriented perpendicular to the vessel long axis. We also demonstrated regulated cleavage orientation in vivo, in flow-exposed forming retinal vessels. Daughter nuclei moved away from the cleavage plane after division, suggesting that regulation of endothelial division orientation effectively extends vessel length in these developing vascular beds. A gainof-function mutation in VEGF signaling increased randomization of endothelial division orientation, and this effect was rescued by a transgene, indicating that regulation of division orientation is a novel mechanism whereby VEGF signaling affects vessel morphogenesis. Thus, our findings show that endothelial cell division and morphogenesis are integrated in developing vessels by flow-independent mechanisms that involve VEGF signaling, and this cross talk is likely to be critical to proper vessel morphogenesis. IntroductionBlood vessels form and expand in both development and disease, via processes that include vasculogenesis, angiogenesis, and intussusception (reviewed in Risau, 1 Eichmann et al, 2 Coultas et al 3 ). Sprouting angiogenesis is the coordinated migration of groups of endothelial cells from vessels and their subsequent fusion to form new interconnections. In this way, simple vascular tubes are ramified and extended to form a primitive vascular plexus. This vessel plexus forms at numerous sites in the embryo, including the yolk sac, the head mesenchyme, and surrounding the neural tube. The primitive vascular plexus is then remodeled under the influence of blood flow and interactions with mural cells. Thus, the initial pattern of vessels serves as a template for remodeling that leads to a mature vasculature.During formation of the primitive vascular plexus, several cellular processes must be regulated and integrated. Specifically, endothelial cells respond to some morphogenetic cues by sprouting, while actively dividing to expand the pool of endothelial cells. One level of integration occurs via the signaling pathways that promote angiogenesis, because many affect both endothelial cell division and morphogenesis. The VEGF signaling pathway is an example of this mode of integration, because it regulates both cell division and branching morphogenesis (reviewed in Rousseau et al, 4 Kliche and Waltenberger, 5 Ferrara et al,6 Nagy and Senger, 7 and Shibuya and Claesson-Welsh 8 ). VEGF-A (VEGF) binds 2 highaffinity receptors on endothelial cells, flk-1 (VEGFR-2) and flt-1 (VEGFR-1), and perturbation of VEGF signaling by genetic deletion of either receptor affects both endothelial cell division and morphogenesis. ...
Blood vessel formation requires the integrated regulation of endothelial cell proliferation and branching morphogenesis, but how this coordinated regulation is achieved is not well understood. Flt-1 (vascular endothelial growth factor [VEGF] receptor 1) is a high affinity VEGF-A receptor whose loss leads to vessel overgrowth and dysmorphogenesis. We examined the ability of Flt-1 isoform transgenes to rescue the vascular development of embryonic stem cell–derived flt-1−/− mutant vessels. Endothelial proliferation was equivalently rescued by both soluble (sFlt-1) and membrane-tethered (mFlt-1) isoforms, but only sFlt-1 rescued vessel branching. Flk-1 Tyr-1173 phosphorylation was increased in flt-1−/− mutant vessels and partially rescued by the Flt-1 isoform transgenes. sFlt-1–rescued vessels exhibited more heterogeneous levels of pFlk than did mFlt-1–rescued vessels, and reporter gene expression from the flt-1 locus was also heterogeneous in developing vessels. Our data support a model whereby sFlt-1 protein is more efficient than mFlt-1 at amplifying initial expression differences, and these amplified differences set up local discontinuities in VEGF-A ligand availability that are important for proper vessel branching.
Checkpoint pathways inhibit cyclin-dependent kinases (Cdks) to arrest cell cycles when DNA is damaged or unreplicated. Early embryonic cell cycles of Xenopus laevis lack these checkpoints. Completion of 12 divisions marks the midblastula transition (MBT), when the cell cycle lengthens, acquiring gap phases and checkpoints of a somatic cell cycle. Although Xenopus embryos lack checkpoints prior to the MBT, checkpoints are observed in cell-free egg extracts supplemented with sperm nuclei. These checkpoints depend upon the Xenopus Chk1 (XChk1)-signaling pathway. To understand why Xenopus embryos lack checkpoints, xchk1 was cloned, and its expression was examined and manipulated in Xenopus embryos. Although XChk1 mRNA is degraded at the MBT, XChk1 protein persists throughout development, including pre-MBT cell cycles that lack checkpoints. However, when DNA replication is blocked, XChk1 is activated only after stage 7, two cell cycles prior to the MBT. Likewise, DNA damage activates XChk1 only after the MBT. Furthermore, overexpression of XChk1 in Xenopus embryos creates a checkpoint in which cell division arrests, and both Cdc2 and Cdk2 are phosphorylated on tyrosine 15 and inhibited in catalytic activity. These data indicate that XChk1 signaling is intact but blocked upstream of XChk1 until the MBT.
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