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D.C. Darland, M. Saint–Geniez, J. Chen, R. Benezra, P.A. D'Amore; Expression of Id 1 Protein in Developing Retinal Vasculature . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4758.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The goal of this study was to examine expression of the dominant negative helix–loop–helix (HLH) protein, Id 1, in developing retinal vasculature. Id proteins have previously been suggested to regulate cell fate decisions in a variety of cell types. Less is known about the role of Id proteins in promoting retinal endothelial cell (EC) differentiation. Four mammalian genes coding four proteins with partially overlapping developmental expression patterns have been identified. Id protein expression is associated with proliferating, undifferentiated cells. We sought to identify a role for Id1 protein in mediating EC differentiation by examining Id 1 expression in nascent, differentiating and mature retinal vessels. Methods: Eyes from P10.5 mice were fixed overnight in 4% paraformaldehyde in phosphate–buffered saline (PBS) and the retinas microdissected. Retinas were incubated with 3% donkey serum, 0.1% saponin, 0.1% Triton X–100 in PBS overnight at 4°C then washed once with PBS. Retinas were incubated with primary antibodies to Id 1 (Biocheck Inc and Santa Cruz), NG2 (Chemicon), SM22 alpha (Abcam) or isotype–specific IgG. Primary antibody binding was revealed with host–appropriate secondary antibody coupled to cy3 (Jackson Immunolabs, diluted 1:200). EC were localized with labeling for lectin B4 coupled to FITC (Vector Labs). Results were visualized using a Leica confocal and images were generated using Adobe Photoshop. Results: We observed Id 1 protein localized to the nucleus of EC in newly formed capillaries of P10.5 mice, indicating that the Id 1 protein was actively inhibiting transcription by HLH proteins. At P10.5 the primary retinal vasculature is largely established, but a front of differentiating cells is present and the secondary and tertiary vasculature are still forming. Immunolocalization of Id 1 protein revealed a punctate cytoplasmic pattern in the hyaloid vessels that were undergoing regression at this developmental time point. Id 1 labeling was low but detectable in some arterioles and venules, but not detected in arteries or veins. NG2– and SM22 alpha–positive pericytes were associated with nascent vessels and differentiating EC. Conclusions: Our results reveal a novel expression pattern for Id 1 in the retinal vasculature and suggest a role for Id 1 in controlling EC differentiation and maturation. Therefore, Id 1 action may be an interesting potential target for regulating vessel formation during developmental or pathologic angiogenesis.
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