Oligodendrocytes are the myelinating cells of the central nervous system. Unraveling the cellular mechanisms underlying oligodendrocyte proliferation and differentiation is important not only for understanding oligodendrocyte development and myelination but also for elucidating basic cellular mechanisms underlying white matter injury and demyelination.

To establish an animal model to study oligodendrocyte development and physiology in situ, the Section on Molecular and Cellular Neurobiology generated a transgenic mouse expressing the green fluorescent protein (GFP) under the control of the 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter. GFP+ cells could be easily visualized in live and fixed brain tissue throughout the mouse’s entire postnatal development. The spatiotemporal appearance of GFP+ cells in mouse embryos was consistent with the previously described origin of oligodendrocytes. Immunohistochemical analysis in brain tissue using different neural markers demonstrated that GFP expression was restricted to cells of the oligodendrocyte lineage. These cells included oligodendrocyte progenitors (OPs) and oligodendrocytes at distinct developmental stages. GFP+ OPs gave rise to differentiated oligodendrocytes in culture. Fluorescence-activated cell sorting was used to obtain a 100 percent pure population of GFP+ oligodendrocyte lineage cells. Electrophysiological patch clamp recordings of GFP+ cells in situ demonstrated that OP cells displayed large outward tetraethylammonium (TEA)-sensitive K+-currents and very small inward currents, whereas mature oligodendrocytes were characterized by the expression of large inward currents. In tissue slice cultures, the proliferation rate of GFP+ cells in developing white matter decreased with the age of the animals between postnatal day two and 20. TEA strongly inhibited proliferation of GFP+ cells in tissue slices. Our findings indicate that oligodendrocyte development and physiology can be studied in live tissue of CNP-GFP transgenic mice, that these mice represent a source of pure GFP+ oligodendrocyte lineage cells throughout development, and that (TEA)-sensitive K+-channels are involved in the regulation of OP proliferation in situ.

Proliferation and differentiation of oligodendroglial cells are tightly linked biological processes that control myelination in the central nervous system. The section analyzed expression of cdk2 and its partner cyclin E in vivo in cells purified from transgenic mice selectively expressing the green fluorescent protein in the oligodendroglial lineage. Cdk2 and cyclin E levels decreased during postnatal maturation, consistent with the time-course of oligodendrocyte progenitor (OP) proliferation and differentiation in vivo. To establish a causal link between cyclin E/cdk2 activity and OP cell proliferation, we used an in vitro transgenic approach that selectively targeted cdk2 in OP cells by overexpressing wild-type (wt) or dominant-negative (Dn) versions of this gene. Dn-cdk2 overexpression inhibited mitogen-induced OP cell proliferation, whereas overexpression of wt-cdk2 prevented reversible cell-cycle arrest associated with the activation of glutamatergic and b-adrenergic receptors and with K+ channel blockade. Thus, cdk2 activity plays a pivotal function in OP cell-cycle decisions occurring at G1/S checkpoint either in a pro- or antimitotic environment. Dn-cdk2- or wt-cdk2-mediated regulation of G1/S transition per se did not influence OP cell differentiation. Therefore, molecular mechanisms associated with initiation of OP differentiation are independent from cyclinE/cdk2 checkpoint and from the number of cell cycles that occur before the onset of differentiation.