Developmental regulation and structural organization of connexins in epidermal gap junctions.

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).