Spatial and temporal patterns of distribution of the gap junctional protein connexin43 during retinal regeneration of adult newt

Abstract

Newts possess the ability to regenerate a functional retina after complete removal of the original retina. We performed immunoblot and immunohistochemical analyses of newt retinas at different stages of regeneration by using an antibody against a gap junction channel protein, connexin43 (Cx43). The specificity of the antibody was shown on immunoblots as well as immunohistochemical staining pattern in the normal retina. Punctate Cx43 immunolabeling was detected intensely between proliferating cell nuclear antigen-immunoreactive progenitor cells in the regenerating retinas, and the amount of this labeling tended to be prominent along both scleral and vitreal sides. The amount of Cx43 became less abundant as regeneration proceeded. This temporal loss of Cx43 during regeneration was also shown on the immunoblot analysis. Furthermore, the loss of Cx43 was observed in a spatial manner in the peripheral retina, where progenitor cells clustered at the ciliary marginal zone (CMZ) are adding new cells of all types in order toward the central retina. Immunolabeling often extended longitudinally throughout the retina when regenerating retinas became thick. Double immunolabeling with Cx43 and glial fibrillary acidic protein indicated the overlapping between the Cx43 and Müller cell processes. At the beginning of the synaptic formation, immunolabeling almost disappeared in the entire retina. However, in the completely regenerated retina, Cx43 reappeared in the distal end of Müller cells and pigment epithelial cells in the same pattern as in the normal retina. The above observations lead us to speculate that Cx43-mediated gap junctions may play an important role in regenerating events. Possible roles of Cx43 during regeneration are discussed.