Introduction Connexins are a multigenic family of proteins that can be distinguished on the basis of their molecular weight. Six oligomerized connexins form a hemichannel (or connexon) that can bind to another hemichannel present in the membrane of an adjacent cell, resulting in a complete intercellular channel. These channels, which accumulate at specific membrane areas defined as gap junctions, allow cells to share small molecules (<1 kDa) such as metabolites and ions. Such an exchange of signals (or intercellular coupling) has been implicated in various cellular mechanisms, including ionic homeostasis, electrical synchronization, proliferation and migration (Bruzzone et al., 1996; Kumar and Gilula, 1996). Connexins are expressed in the three main cell types of the central nervous system (CNS), that is, astrocytes, oligodendrocytes and neurons. Increasing evidence suggests that connexins regulate different aspects of neuronal networks and neuron-glia interactions, in both the developing and mature brain (for a review, see Dermietzel and Spray, 1998). This would require the establishment of communication compartments that isolate groups of coupled cells engaged in a coordinated activity from other populations that participate in distinct processes. Such a dynamic regulation of cell-cell communication seems particularly well suited to respond to the instructive cues operating during CNS development (Kandler and Katz, 1995; Nadarajah et al., 1997; Naus and Bani- Yaghoub, 1998). Thus, in vivo studies have shown that, in the developing neocortex, neuroblasts and proliferating cells located in the ventricular zone are coupled, whereas differentiating or migrating neurons are not (LoTurco and Kriegstein, 1991; Bittman et al., 1997). Evidence for coupling territories has also been obtained in other areas of neurogenesis, such as the subventricular zone and rostral migratory stream (Miragall et al., 1997; Menezes et al., 2000). Furthermore, there is an inverse correlation between the expression of connexin43 (Cx43) and Cx26, and cell proliferation (Miragall et al., 1997; Bittman and LoTurco, 1999), suggesting that coupling and cell cycle of neural progenitors may be interdependent. Studies in vitro have shown that Cx43 expression and coupling are downregulated as differentiation proceeds in either immortalized rodent neuroblasts (Rozental et al., 1998) or cell lines derived from rat peripheral neurotumoral cells (Donahue at al., 1998). In a human teratocarcinoma cell line consisting of precursors able to differentiate into post-mitotic neurons, Cx43 protein expression is decreased after neuronal 3241 Embryonic neural progenitors isolated from the mouse striatal germinal zone grow in vitro as floating cell aggregates called neurospheres, which, upon adhesion, can be induced to differentiate into the three main cell types of the central nervous system (CNS), that is, astrocytes, neurons and oligodendrocytes. To study the possible role of connexins and junctional communication during differentiation of neural progenitors, we assessed cell-to- cell communication by microinjecting Lucifer Yellow into neurospheres at various times after adhesion. Cells located in neurospheres were strongly coupled, regardless of the differentiation time. Microinjections performed on the cell layers formed by differentiated cells migrating out of the neurosphere established that only astrocytes were coupled. These observations suggest the existence of at least three distinct communication compartments: coupled proliferating cells located in the sphere, uncoupled cells undergoing neuronal or oligodendrocytic differentiation and coupled differentiating astrocytes. A blockade of junctional communication by 18-β-glycyrrhetinic acid (βGA) reduced, in a concentration-dependent manner, the viability of undifferentiated neural progenitor cells. This effect appeared to be specific, inasmuch as it was reversible and that cell survival was not affected in the presence of the inactive analog glycyrrhyzic acid. Addition of βGA to adherent neurospheres also decreased cell density and altered the morphology of differentiated cells. Cx43 was strongly expressed in either undifferentiated or differentiated neurospheres, where it was found both within the sphere and in astrocytes, the two cell populations that were dye coupled. Western blot analysis further showed that Cx43 phosphorylation was strongly increased in adherent neurospheres, suggesting a post-translational regulation during differentiation. These results point to a major role of cell-to-cell communication and Cx43 during the differentiation of neural progenitor cells in vitro. Key words: Astrocyte, CNS, Connexin, Gap junction, Neuron, Oligodendrocyte Summary Cell coupling and Cx43 expression in embryonic mouse neural progenitor cells Nathalie Duval 1 , Danielle Gomès 1 , Viviane Calaora 1 , Alessandra Calabrese 2 , Paolo Meda 2 and Roberto Bruzzone 1, * 1 Institut Pasteur, Unité de Neurovirologie et Régénération du Système Nerveux, 75015 Paris, France 2 Département de Morphologie, Centre Médical Universitaire, 1211 Geneva, Switzerland *Author for correspondence (e-mail: bruzzone@pasteur.fr) Accepted 11 June 2002 Journal of Cell Science 115, 3241-3251 (2002) © The Company of Biologists Ltd Research Article