Alterations of endothelial cell markers inop/op AGM culture.

(A) Morphology of adherent cells in wild-type and op/op AGM culture. AGM cultures were prepared from wild-type and op/op embryos, and photographs of the adherent cells were taken before FACS analysis was performed. Adherent cells proliferated in both cultures, and no significant difference in cell number was observed. Cells appeared healthy, and no dead cells were accumulated in either culture. (left) Wild-type. (right)op/op. (B) Expression of endothelial cell markers on adherent cells. Adherent cells were harvested from 3 independent cultures with the same genotype by using cell dissociation buffer. For FACS analysis, these cells were mixed and were incubated with mouse serum for blocking nonspecific binding of antibody. Antibodies against VE-cadherin, Flk-1, ICAM-2, and PCLP-1 were then added to the cells. FITC-conjugated anti-rat IgG was used to stain the cells. Numbers in figures indicate the percentage of positive cells. The experiment was performed twice, and generally the same results were obtained. Note that the expression of endothelial cell markers VE-cadherin, Flk-1, and ICAM-2 was reduced in op/op AGM culture, whereas the expression of a hemangioblast marker, PCLP-1, was unchanged. (C) Expression of Flt-1 in AGM culture. Adherent cells in AGM culture at the eighth day were used to prepare total RNA. RNA (10 μg) was electrophoresed and hybridized with DIG-labeled Flt-1 probe. Flt-1 expression was detected in each genotype. Arrowhead shows the specific band for the Flt-1 gene. Lane 1, wild type; lane 2,op/+; lane 3, op/op. (D) Incorporation of DiI-Ac-LDL. After 8 days of incubation, 10 μg/mL of DiI-Ac-LDL was added to the AGM culture and was incubated for 4 hours. Using a confocal microscope, the DiI-Ac-LDL–positive cells were detected in the clusters of endothelial cells of wild-type (i) and op/op(ii). i, wild-type; ii, op/op.