The role of connexins in neuronal differentiation: Expression and function of Cx30, Cx36 and Cx43 in in vitro models of neuronal differentiation

Abstract

This study aimed to investigate the expression and function of three connexin subtypes: Cx30, Cx36 and Cx43, during neuronal differentiation. The study was performed using two in vitro models of differentiation: NT2/D1 and P19 EC cells. A temporal downregulation of Cx43 was confirmed in both cell lines during retinoic acidinduced neuronal differentiation using RT-PCR and immunoblotting. This preceded a timedependent increase in the neuronal doublecortin (DCX) protein. Temporal upregulation of Cx30 and Cx36 were observed by RT-PCR, with Cx30 exhibiting a transient peak in expression at the midway point of the timecourse of differentiation. Qualitative immunocytochemistry was used to examine the spatial pattern of protein distribution for Cx30 and Cx43 during differentiation. Cx43 was observed both intracellularly and on cell surfaces, while Cx30 was seen only as puncta on the plasma membrane. The temporal pattern of immunolabelling was similar to that seen using RT-PCR and immunoblotting. Spatially the Cx30 puncta were observed to localise on clusters of cells with DCX-positive processes in their vicinity; Cx43 was primarily seen on DCX-negative cells. Two different types of overexpression systems were generated for the Cx30 and Cx43 genes entailing either bicistronic or bidirectional reporter coexpression. These were compared in order to determine the better system for further study; bicistronic reporter expression following the Cx43 gene was weak, resulting in poor colocalisation. The bidirectional expression systems were chosen for further investigation. The systems were transiently transfected and induced to overexpress Cx30 and Cx43, and the effect of Cx overexpression on the expression of DCX was measured. Cx43 was seen to reduce the proportion of DCX-positive cells while Cx30 did not affect the proportion of cells specifying towards neuronal commitment. Whilst further investigation is warranted it can be concluded from this study that the downregulation of Cx43 is required for normal neuronal specification while the transient upregulation of Cx30 may play an as yet undetermined role in differentiation of neuronal populations.