Neuronal differentiation requires a biphasic modulation of gap junctional intercellular communication caused by dynamic changes of connexin43 expression

Abstract

It was suggested that gap junctional intercellular communication (GJIC) and connexin (Cx) proteins play a crucial role in cell proliferation and differentiation. However, the mechanisms of cell coupling in regulating cell fate during embryonic development are poorly understood. To study the role of GJIC in proliferation and differentiation, we used a human neural progenitor cell line derived from the ventral mesencephalon. Fluorescence recovery after photobleaching (FRAP) showed that dye coupling was extensive in proliferating cells but diminished after the induction of differentiation, as indicated by a 2.5-fold increase of the half-time of fluorescence recovery. Notably, recovery half-time decreased strongly (five-fold) in the later stage of differentiation. Western blot analysis revealed a similar time-dependent expression profile of Cx43, acting as the main gap junction-forming protein. Interestingly, large amounts of cytoplasmic Cx43 were retained mainly in the Golgi network during proliferation but decreased when differentiation was induced. Furthermore, down-regulation of Cx43 by small interfering RNA reduced functional cell coupling, which in turn resulted in a 50% decrease of both the proliferation rate and neuronal differentiation. Our findings suggest a dual function of Cx43 and GJIC in the neural development of ReNcell VM197 human progenitor cells. GJIC accompanied by high Cx43 expression is necessary (1) to maintain cells in a proliferative state and (2) to complete neuronal differentiation, including the establishment of a neural network. However, uncoupling of cells is crucial in the early stage of differentiation during cell fate commitment.