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Somites are formed progressively from the presomitic mesoderm (PSM) in a highly regulated process according to a strict periodicity driven by an oscillatory mechanism. The Notch and Wnt pathways are key components in the regulation of this somitic oscillator and data from Xenopus and zebrafish embryos indicate that the Notch-downstream target Nrarp participates in the regulation of both activities. We have analyzed Nrarp/nrarp-a expression in the PSM of chick, mouse and zebrafish embryos, and we show that it cycles in synchrony with other Notch regulated cyclic genes. In the mouse its transcription is both Wnt- and Notch-dependent, whereas in the chick and fish embryo it is simply Notch-dependent. Despite oscillating mRNA levels, Nrarp protein does not oscillate in the PSM. Finally, neither gain nor loss of Nrarp function interferes with the normal expression of Notch-related cyclic genes., ((c) 2009 Wiley-Liss, Inc.)

Growth factor gradients regulate many developmental processes. VEGF-A is distributed in a graded fashion in growing tissues in order to direct sprouting of new vessels. Growth factor gradients can be formed by regulated production, retention, controlled release and degradation. VEGF-A production is controlled by hypoxia while its retention depends on the C-terminal heparin-binding motifs present in the longer splice-isoforms, VEGF164 and 188. This motif confers binding to the cell surface and the surrounding extracelluar matrix. The short isoform VEGF120 is diffusible and hence fails to direct endothelial tip cell migration. Conditional inactivation of heparan sulfate proteoglycans in the cells that produce VEGF results similarly in misguidance of the tip cells. Studying retinal developmental angiogenesis and pathological neovascularization side-by-side in the mouse retina, we find that endothelial tip cell guidance and stalk cell proliferation control are disrupted in neovascularization due to a loss of VEGF-A retention. The cause for this is proteolytic cleavage of VEGF-A by matrix metalloproteases (MMP) derived mostly from macrophages infiltrating the ischaemic retinal areas. Genetic or pharmacological inhibition of macrophage infiltration or MMP activity can rescue guided revascularization at the expense of pre-retinal neovascularization. Disruption of VEGF-A gradients provides a novel concept for the mechanism underlying pathological patterning in ocular disease.

Recent evidence indicates that sprouting angiogenesis in the central nervous system (CNS) is a guided process similar to the guidance of axons and insect tracheal tubes. Specialized tip cells of vessel sprouts navigate in response to local depots or gradients of vascular endothelial growth factor (VEGF-A). Neuropilin-1 (Nrp-1) is a transmembrane receptor with a repulsive function in axon guidance. Nrp-1 also binds the VEGF-A splice isoform VEGF165, stimulates angiogenesis, and is necessary for vascular development in the mouse. However, the morphogenetic events controlled by Nrp-1 in angiogenesis have not been defined. Here, we analyzed endothelial tip cell guidance in the CNS of Nrp-1-deficient mice. We focused our attention on the developing hindbrain, which is normally vascularized in a stereotyped manner. Initially, angiogenic sprouts extend along radial glia from the pial surface toward the ventricles, but in the subventricular zone (SVZ), they leave the radial path, turn laterally, and fuse to form a capillary plexus. Radial sprout elongation correlated with tip cell filopodia extensions along nestin-positive radial glial processes, but in the SVZ, the tip cell filopodia also extended perpendicular to the glial tracks and made contact with filopodia of the neighboring sprouts. In Nrp-1-deficient mice, the tip cell filopodia remained associated with the radial glia in the SVZ, which correlated with a failure of sprout turning and elongation across this region. As a result, the sprouts remained blind-ended forming glomeruloid tufts in the SVZ. These observations suggest that Nrp-1 plays an important role in allowing the endothelial tip cell filopodia to switch substrate and protrude in a new direction at a specific location in the developing brain., ((c) 2004 Wiley-Liss, Inc.)

Recruitment and adhesion of pericytes to endothelial cells represents a critical step in angiogenesis. We previously demonstrated the expression of neural (N)-cadherin at contact zones between pericytes and endothelial cells in embryonic chicken brain. To elucidate N-cadherin function in early angionenesis, we injected functionally blocking antibodies on embryonic days 4 and 5 into the tectal ventricle of chicken embryos. Brains were morphologically and immunocytochemically investigated on embryonic day 6. Blocking N-cadherin function resulted in defective pericyte adhesion, increased pericyte recruitment and disturbed vascular morphogenesis. Increased pericyte recruitment did not involve elevated pericytic proliferation. Concomitant disruption of ependymal adherens junctions and of endothelial-pericytic adhesion resulted in massive hemorrhaging in the basal forebrain, in misdirected endothelial sprouting, and ectopic vascularization. Morphological investigation of control embryos on embryonic days 4 and 5 indicated the initial involvement of pericytes in stabilization of angiogenic capillary sprouts. Together these results suggest that N-cadherin mediates adhesion, recognition, and signaling between pericytes and endothelial cells required for normal vascular morphogenesis.

The development of the blood-brain barrier depends upon the formation of a closely regulated system of adherens and tight junctions. A prerequisite for a functional junction system is the linkage of transmembrane adhesion receptors (cadherins) to the cytoskeleton via catenins. The localization of specific catenins at the adherens junction correlates with the stability of interendothelial contacts in vitro, but in vivo data are lacking thus far. Investigating brain angiogenesis in the chicken, we demonstrated that beta-catenin, but not plakoglobin, initially codistributed with N-cadherin at the ablumenal endothelial membrane at contact sites to perivascular cells, from where both antigens disappeared during blood-brain barrier maturation. In contrast, plakoglobin was most prominent at the interendothelial junction where only small amounts of beta-catenin were present. Western-blot analysis revealed a stronger developmental decrease of beta-catenin than plakoglobin, whereas N-cadherin was completely lost. beta-Catenin but not N-cadherin was reinduced in brain endothelial cells during dedifferentiation in vitro and localized to the interendothelial junctions. These first in vivo data support the hypothesis that endothelial beta-catenin and N-cadherin are transiently relevant for the contact of brain endothelial to perivascular cells. Plakoglobin seems not to interact with N-cadherin but is exclusively localized at interendothelial junctions providing evidence for its role in the formation of stable adherens junctions, which may play a role for the initiation, and/or stabilization of tight junctions. Dev Dyn 2000;217:86-98., (Copyright 2000 Wiley-Liss, Inc.)