Background: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier by separating the neural retina from fenestrated capillaries in the choroid. The barrier depends upon tight junctions within the apical junctional complexes that bind neighboring cells. During development, permeability decreases as the apical junctional complex gradually matures. To investigate this process, the composition of the apical junctional complex was monitored during RPE development in chicken embryos.
Methods: Permeability was monitored by incubating freshly isolated RPE/choroid in medium containing horseradish peroxidase followed by histochemical staining and electron microscopy. The expression of the tight junction proteins, ZO-1 and occludin, was determined by immunofluorescence and immunoblotting. Development of the RPE apical junctional complex was to compared to the homologous complex that forms the outer limiting membrane of the neural retina.
Results: The apical junctional complex of the RPE was permeable to horseradish peroxidase until embryonic day 10-12. Two putative forms of ZO-1 had approximately the same molecular mass as mammalian ZO-1 and were present in the apical junctional complexes at different stages of development. We identified one form as ZO-1, because it was present in mature RPE and shared an epitope with the rodent isoforms, ZO-1 alpha+ and ZO-1 alpha-. The second form lacked this epitope but was identified by a polyclonal antibody to ZO-1. It was designated the ZO-1-like protein (ZO-1LP). On embryonic day 3, occludin and ZO-1LP were observed along the apical surface of the neuroepithelium that gave rise to the RPE and the neural retina. In the neural retina, occludin expression decreased just before inner segments were formed, but ZO-1LP expression continued in the outer limiting membrane throughout development. During RPE development, occludin expression was constant or increased slightly. By contrast, ZO-1LP was gradually replaced by ZO-1 and total ZO-1 immunoreactive proteins decreased more than 10x.
Conclusions: A gradual change in the composition of the apical junctional complexes accompanied the period of barrier formation. In RPE, ZO-1 gradually replaced ZO-1LP, but the decrease in ZO-1 expression suggests its functions during junction formation are not directly related to junction permeability. By contrast, occludin was lost and ZO-1LP retained where an adherens junction forms the permeable, outer limiting membrane.