REMODELING OF JUNCTIONAL COMPLEXES DURING THE DEVELOPMENT OF THE OUTER BLOOD-RETINAL BARRIER

Background: The retinal pigment epithelium (RPE) forms the outer blood -retinal barrier by separating the neural retina from fenestrated capi llaries in the choroid. The barrier depends upon tight junctions withi n the apical junctional complexes that bind neighboring cells. During development, permeability decreases as the apical junctional complex g radually matures. To investigate this process, the composition of the apical junctional complex was monitored during RPE development in chic ken embryos. Methods: Permeability was monitored by incubating freshly isolated RPE/choroid in medium containing horseradish peroxidase foll owed 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 j unctional 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 approximate ly the same molecular mass as mammalian ZO-1 and were present in the a pical junctional complexes at different stages of development. We iden tified one form as ZO-1, because it was present in mature RPE and shar ed an epitope with the rodent isoforms, ZO-1 alpha+ and ZO-1 alpha-. T he 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 s urface of the neuroepithelium that gave rise to the RPE and the neural retina. In the neural retina, occludin expression decreased just befo re 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 p roteins 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 for mation are not directly related to junction permeability. By contrast, occludin was lost and ZO-1LP retained where an adherens junction form s the permeable, outer limiting membrane. (C) 1997 Wiley-Liss, Inc.