Williams C D, Rizzolo L J
Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06520-8062, USA.
Anat Rec. 1997 Nov;249(3):380-8. doi: 10.1002/(SICI)1097-0185(199711)249:3<380::AID-AR9>3.0.CO;2-Y.
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.
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.
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.
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.
视网膜色素上皮（RPE）通过将神经视网膜与脉络膜中的有孔毛细血管分隔开来形成外血视网膜屏障。该屏障依赖于顶端连接复合体中的紧密连接，紧密连接将相邻细胞连接在一起。在发育过程中，随着顶端连接复合体逐渐成熟，通透性降低。为了研究这一过程，在鸡胚RPE发育过程中监测了顶端连接复合体的组成。
通过将新鲜分离的RPE/脉络膜在含有辣根过氧化物酶的培养基中孵育，随后进行组织化学染色和电子显微镜观察来监测通透性。通过免疫荧光和免疫印迹法测定紧密连接蛋白ZO-1和闭合蛋白的表达。将RPE顶端连接复合体的发育与形成神经视网膜外限制膜的同源复合体进行比较。
直到胚胎第10 - 12天，RPE的顶端连接复合体对辣根过氧化物酶具有通透性。两种假定形式的ZO-1的分子量与哺乳动物的ZO-1大致相同，并且在发育的不同阶段存在于顶端连接复合体中。我们将其中一种形式鉴定为ZO-1，因为它存在于成熟的RPE中，并与啮齿动物异构体ZO-1α +和ZO-1α -共享一个表位。第二种形式缺乏该表位，但通过针对ZO-1的多克隆抗体鉴定。它被命名为ZO-1样蛋白（ZO-1LP）。在胚胎第3天，在产生RPE和神经视网膜的神经上皮顶端表面观察到闭合蛋白和ZO-1LP。在神经视网膜中，闭合蛋白的表达在内段形成之前就降低了，但ZO-1LP的表达在整个发育过程中在外限制膜中持续存在。在RPE发育过程中，闭合蛋白的表达保持恒定或略有增加。相比之下，ZO-1LP逐渐被ZO-1取代，并且总的ZO-1免疫反应性蛋白减少了10倍以上。
顶端连接复合体组成的逐渐变化伴随着屏障形成期。在RPE中，ZO-1逐渐取代ZO-1LP，但ZO-1表达的降低表明其在连接形成过程中的功能与连接通透性没有直接关系。相比之下，在黏着连接形成可渗透的外限制膜的地方，闭合蛋白消失而ZO-1LP保留。
