In vitro connexin expression and function in postnatal neural progenitors

Abstract

The studies presented in this thesis were performed to gain insight into the role of connexins (Cxs) and Cx-mediated communication in regulating postnatal neural precursor cell (NPC) fate. Cxs are protein subunits that oligomerize into hexamers to form hemichannels, permitting the passage of molecules between the cytoplasm and the extracellular space. Docking of two hemichannels elaborated by adjacent cells form a single gap junction channel allowing direct cell-cell communication. Here, I hypothesized that Cx-mediated gap junctions and open hemichannels in nonjunctional membranes represent two robust signalling mechanisms by which NPCs may communicate with neighbouring instructive cells and the extracellular environment. In Chapter 2 of this thesis, I show that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45 and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are expressed by primary postnatal hippocampal precursor cells grown as neurospheres. The specificity of immunodetection was confirmed using cultures derived from null-mutant mice controls. I also found that culture conditions affect Cx expression and that the repertoire of Cxs expressed by NPCs changes over the course of differentiation in vitro. NPCs grown as monolayers on laminin-coated coverslips expressed Cx26, Cx29, Cx30, Cx36, Cx37, Cx40, Cx43 and Cx45 protein but not Cx32 and Cx47. Every Cx protein examined except Cx32 was found in discrete cell lineages following differentiation to neuronal and glial lineages. In Chapter 3, a series of dye uptake assays were performed that established inducible hemichannel activity in NPCs cultured as adherent neurospheres. I found that this hemichannel activity was lost when neurosphere-derived cells were maintained as proliferating monolayers or induced to differentiate. Conversely, functional gap junction biochemical coupling, as assessed by passage of the anionic dye lucifer yellow, increased following differentiation. Finally, one Cx (Cx29) was chosen to assess the impact of altering Cx expression on NPC fate. In Chapter 4, Cx29 null-mutation was found to reduce spontaneous astrocytic differentiation following growth factor withdrawal. Together, these observations provide further support for the hypothesis that the repertoire of Cx proteins dictates, in part, a progenitor cell's intrinsic capacity to respond to its microenvironment.