Temporal expression of neuronal connexins during hippocampal ontogeny

Communication through gap junction channels provides a major signaling mech anism during early brain histogenesis, a developmental time during which ne ural progenitor cells are inexcitable and do not express ligand-gated chann el responses to the major CNS neurotransmitters. Expression of different ga p junction types during neurogenesis may therefore define intercellular pat hways for transmission of developmentally relevant molecules. To better und erstand the molecular mechanism(s) by which growth and differentiation of n eurons are modulated by gap junction channels, we have been examining the d evelopmental effects of a specific set of cytokines on differentiation and gap junction expression in a conditionally immortalized mouse embryonic hip pocampal neuronal progenitor cell line (MK31). When multipotent MK31 cells are in an uncommitted state, they uniformly express the neuroepithelial int ermediate filament class VI marker, nestin, are strongly coupled by gap jun ctions composed of connexin43 (Cx43) and express connexin45 (Cx45) at the m RNA level. As these cells undergo neuronal lineage commitment and exit from cell cycle, they begin to express the early neurofilament marker, NF66, an d coupling strength and expression of Cx43 begin to decline with concurrent expression of other connexin proteins, including Cx26, Cx33, Cx36, Cx40 an d Cx45. Terminal neuronal differentiation is heralded by the expression of more advanced neurofilament proteins, increased morphologic maturation, the elaboration of inward currents and action potentials that possess mature p hysiological properties, and changing profiles of expression of connexin su btypes, including upregulation of Cx36 expression. These important developm ental transitions are regulated by a complex network of cell cycle checkpoi nts. To begin to examine the precise roles of gap junction proteins in trav ersing these developmental checkpoints and in thus regulating neurogenesis, we have focused on individual members of two classes of genes involved in these seminal events: ID (inhibitor of differentiation)-1 and GAS (growth a rrest-specific gene)5, When MK31 cells were maintained in an uncommitted st ate, levels of ID-1 mRNA were high and GAS5 transcripts were essentially un detectable. Application of cytokines that promote neuronal lineage commitme nt and cell cycle exit resulted in down-regulation of ID-1 and upregulation of GAS5 transcripts, whereas additional cytokine paradigms that promoted t erminal neuronal differentiation resulted in the delayed down-regulation of GAS5 mRNA. Stable MK31 transfectants were generated for ID-1 and GAS5. In basal conditions, cellular proliferation was enhanced in the ID-1 transfect ants and inhibited in the GAS5 transfectants when compared with control MK3 1 cells. When cytokine-mediated neurogenesis was examined in these transfec ted cell lines, constitutive expression of ID-1 inhibited and constitutive expression of GAS5 enhanced initial and terminal stages of neuronal differe ntiation, with evidence that terminal neuronal maturation in both transfect ant lines was associated with decreased cellular viability, possibly due to the presence of conflicting cell cycle-associated developmental signals. T hese experimental reagents will prove to be valuable experimental tools to help define the functional interrelationships between changing profiles of connexin protein expression and cell cycle regulation during neuronal ontog eny in the mammalian brain. The present review summarizes the current state of research involving the t emporal expression of such connexin types in differentiating hippocampal ne urons and speculates on the possible role of these intercellular channels i n the development and plasticity of the nervous system. In addition, we des cribe the functional properties and expression pattern of the newly discove red neuronal-specific gap junctional protein, Cx36, in the developing mouse fetal hippocampus and in the rat retina and brain. (C) 2000 Elsevier Scien ce B.V. All rights reserved.