Morphogenesis of a vascular network requires dynamic vessel growth and regression. To investigate the cellular mechanism underlying this process, we deleted focal adhesion kinase (FAK), a key signaling mediator, in endothelial cells (ECs) using Tie2-Cre mice. Targeted FAK depletion occurred efficiently early in development, where mutants exhibited a distinctive and irregular vasculature, resulting in hemorrhage and lethality between embryonic day (e) 10.5 and 11.5. Capillaries and intercapillary spaces in yolk sacs were dilated before any other detectable abnormalities at e9.5, and explants demonstrate that the defects resulted from the loss of FAK and not from organ failure. Time-lapse microscopy monitoring EC behavior during vascular formation in explants revealed no apparent decrease in proliferation or migration but revealed increases in cell retraction and death leading to reduced vessel growth and increased vessel regression. Consistent with this phenotype, ECs derived from mutant embryos exhibited aberrant lamellipodial extensions, altered actin cytoskeleton, and nonpolarized cell movement. This study reveals that FAK is crucial for vascular morphogenesis and the regulation of EC survival and morphology.

During development, pancreatic endocrine cells are specified within the pancreatic epithelium. They subsequently delaminate out of the epithelium and cluster in the mesenchyme to form the islets of Langerhans. Neurogenin3 (Ngn3) is a transcription factor required for the differentiation of all endocrine cells and we investigated its role in their delamination. We observed in the mouse pancreas that most Ngn3-positive cells have lost contact with the lumen of the epithelium, showing that the delamination from the progenitor layer is initiated in endocrine progenitors. Subsequently, in both mouse and chick newly born endocrine cells at the periphery of the epithelium strongly decrease E-cadherin, break-down the basal lamina and cluster into islets of Langerhans. Repression of E-cadherin is sufficient to promote delamination from the epithelium. We further demonstrate that Ngn3 indirectly controls Snail2 protein expression post-transcriptionally to repress E-cadherin. In the chick embryo, Ngn3 independently controls epithelium delamination and differentiation programs. Developmental Dynamics 240:589-604,

β1 integrin (encoded by Itgb1) is established as a regulator of angiogenesis based upon the phenotypes of complete knockouts of β1 heterodimer partners or ligands and upon antibody inhibition studies in mice. Its direct function in endothelial cells (ECs) in vivo has not been determined because Itgb1-/- embryos die before vascular development. Excision of Itgb1 from ECs and a subset of hematopoietic cells, using Tie2-Cre, resulted in abnormal vascular development by embryonic day(e) 8.5 and lethality by e10.5. Tie1-Cre mediated a more restricted excision of Itgb1 from ECs and hematopoietic cells and resulted in embryonic lethal vascular defects by e11.5. Capillaries of the yolk sacs were disorganized, and the endothelium of major blood vessels and of the heart was frequently discontinuous in mutant embryos. We also found similar vascular morphogenesis defects characterized by EC disorganization in embryonic explants and isolated ECs. Itgb1-null ECs were deficient in adhesion and migration in a ligand-specific fashion, with impaired responses to laminin and collagens, but not to fibronectin. Deletion of Itgb1 reduced EC survival, but did not affect proliferation. Our findings demonstrate thatβ1 integrin is essential for EC adhesion, migration and survival during angiogenesis, and further validate that therapies targeting β1 integrins may effectively impair neovascularization.

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. Here, we demonstrate that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-offunction mutations show small aortae and large cardinal veins. Notch does not affect the overall number of endothelial cells but balances the proportion of arterial to venous endothelial cells, thereby modulating the relative sizes of both vessel types. Loss of ephrin B2 or its receptor EphB4 also leads to enlarged aortae and underdeveloped cardinal veins; however, endothelial cells with venous identity are mislocalized in the aorta, suggesting that ephrin B2/EphB4 signaling functions distinctly from Notch by sorting arterial and venous endothelial cells into their respective vessels. Our findings provide mechanistic insight into the processes underlying artery and vein size equilibration during angiogenesis.

Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type betaB1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (betaB1(DeltaN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens betaB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.