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The ALK homozygous embryos die at midgestation, exhibiting severe vascular abnormalities characterized by excessive fusion of capillary plexes into cavernous vessels and hyperdilation of large vessels.These vascular defects are associated with enhanced expression of angiogenic factors and proteases and are characterized by decient differentiation and recruitment of vascular smooth muscle cells.Taken together, our results suggest that the balance between the ALK and ALK signaling pathways in endothelial cells plays a crucial role in determining vascular endothelial properties during angiogenesis.T ransforming grow th factorb is a multifunction protein and is involved in diverse biological processes, including grow th, dif ferentiation, and inf lammation. To date, seven type I receptors have been identified and designated as activ in receptorlikekinase. A LK is expressed in blood vessels during embr yogenesis. Various concentrations ofwildtype cDNA were used for the calibration cur ve for each primer set.Af ter cycles of PCR reaction, the relative amount of gene transcripts was calibrated by the comparisonwithwildtype cur ve. In addition, the PCR products were separated on the agarose gel andvisualizedwith ethidium bromide. A ll experiments were repeated at least three times, and similar results were obtained each time.The endocardium and myocardium were present in the mutant embr yos but appeared to be immature as comparedwith thewildtype heart. To investigate the molecular basis underlying the vascular defects in A LK mutant embr yos, we analyzed the expression of a set of genes known to be involved in the blood vessel formation by a quantitative RTPCR method. The plasminogenplasmin system has been implicated in proteolysis of perivascular matrix during angiogenesis, urok inasetype PA were sign ificantly elevated in the A LK embr yos. These results indicate that the A LK signaling is required for dif ferentiation and proper localization of VSMC to the perivascular Columbin region.Visualized amplied PCR products by the ethidium bromide Pyridoxal phosphate staining.The same amount of cDNA and methods were used for PCR amplication of each gene.After cycles of amplication and quantitative analyses, PCR products were separated on an agarose gel.Conversely, neo gene primers amplied cDNA from y and y embryos but not from the wildtype embryos.The mutant embryo is severely growth retarded and expresses tgSM in the anterior part of the dorsal aorta and somites.The in vivo mechan ism by which these two phases are regulated has not been clearly defined.A LK embr yos exhibited hyper fusion and hyperdilation of blood vessels.Molecular studies in A LK embr yos indicate that the A LK signaling represses angiogen ic factors and plasminogen activators, which are marker genes for the activation phase of angiogenesis.Furthermore, we show that A LK is also required for the dif ferentiation and recruitment of vascular smooth muscle cells.Therefore, the balance between A LK and A LK may be crucial for controlling the properties of endothelium during angiogenesis.Vascular phenotypes appear to be identicalwithin each group but dif ferent, although overlapping, between these two groups.After transfection, cells were treated with for hr.Luciferase activities were then measured and plotted.In all assays, luciferase activities are plotted in arbitrary units.A LK signaling pathway.

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