Risek B, Klier F G, Gilula N B
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Dev Biol. 1994 Jul;164(1):183-96. doi: 10.1006/dbio.1994.1190.
The developmental regulation of gap junctions was analyzed in the developing rat epidermis by immunohistochemical and ultrastructural methods. The molecular composition of gap junction plaques was examined by laser scanning confocal microscopy following immuno-double labeling with monoclonal and polyclonal antibodies specific for alpha 1 (Cx43) and beta 2 (Cx26) connexins, respectively. During early fetal development (embryonic period), gap junctions were identified as large junctional plaques consisting of alpha 1 and beta 2 connexins. Ultrastructurally, gap junctions were detected in the two-layered epidermis between the subapical borders of peridermal cells, at the periderm/basal layer interface, and between the basal cells. The first "switch" in the utilization of alpha 1 and beta 2 connexins was observed at the onset of epidermal stratification, when beta 2 expression was down-regulated in the periderm and in the upper part of the intermedium. Gap junctions were also detected ultrastructurally in all layers of the stratified, nondifferentiated epidermis at E16. Junctional sizes included small plaques (0.05 micron 2) in the periderm, medium-size plaques (1 micron 2) in the upper part of the intermediate layer, and very large plaques (25 microns 2) in the basal layer. The second "switch" in the utilization of gap junction components coincided with epidermal differentiation (> E18), when beta 2 was preferentially expressed in the differentiated granular and upper spinous layers. alpha 1 connexin was present in the less differentiated spinous layer and in the proliferating basal layer. Gap junctions were no longer detectable in the periderm following differentiation (keratinization) of the epidermis (E18-E20). An analysis of immuno-double-stained sections by laser scanning confocal microscopy revealed domains of potentially mixed and segregated antigens within large junction plaques. These results indicated that large gap junction plaques (> 1 micron in size) can contain segregated domains of connexons, which contain a single protein (homooligomer).
采用免疫组织化学和超微结构方法，对发育中的大鼠表皮中缝隙连接的发育调控进行了分析。在用分别针对α1（Cx43）和β2（Cx26）连接蛋白的单克隆抗体和多克隆抗体进行免疫双标记后，通过激光扫描共聚焦显微镜检查缝隙连接斑的分子组成。在胎儿早期发育（胚胎期），缝隙连接被鉴定为由α1和β2连接蛋白组成的大的连接斑。超微结构上，在周皮细胞顶下边界之间的两层表皮、周皮/基底层界面以及基底细胞之间检测到缝隙连接。在表皮分层开始时观察到α1和β2连接蛋白利用的首次“转换”，此时周皮和中间层上部的β2表达下调。在E16时，在分层的未分化表皮的所有层中也通过超微结构检测到缝隙连接。连接斑大小包括周皮中的小斑（0.05平方微米）、中间层上部的中等大小斑（1平方微米）和基底层中的非常大的斑（25平方微米）。缝隙连接成分利用的第二次“转换”与表皮分化（>E18）同时发生，此时β2优先在分化的颗粒层和上部棘层中表达。α1连接蛋白存在于分化程度较低的棘层和增殖的基底层中。表皮分化（角质化）后（E18-E20），周皮中不再能检测到缝隙连接。通过激光扫描共聚焦显微镜对免疫双染切片的分析揭示了大连接斑内潜在混合和分离抗原的区域。这些结果表明，大的缝隙连接斑（尺寸>1微米）可以包含连接子的分离区域，连接子包含单一蛋白质（同型寡聚体）。
