Myelination of the CNS is an orderly process initiated by development of the oligodendrocyte (OL). During an early proliferative phase, migratory glial precursor cells seed axonal fiber tracts in brain as they enter a specific lineage. Post-migratory differentiation heralds the end of proliferation and the onset of programmed gene expression to construct the myelin sheath. The regulation of these events is complex, involving the interplay of factors intrinsic to the cell with environmental cues encountered along its developmental course. The overall aim of this project is to define specific environmental signals that direct myelinogenesis at a crucial juncture of OL development, and resolve the cellular and molecular mechanisms by which they operate. Investigation will focus in vitro on the growth control of cells that have reached an intermediate progenitor step of the lineage in vivo. In the rat these cells are defined by a distinct surface antigenic phenotype; O4 antigen is expressed in the absence of galactocerebroside, a marker for further differentiation. Preliminary studies were designed to expose, in defined cell culture, growth requirements of 04+GalC- as they form and proliferate in vivo. Immunoselection technology was applied to isolate the progenitors directly from premyelinating brain. It was shown that cells at this stage are committed to terminal differentiation in vitro, but require extrinsic signals for both survival and proliferation. Culture medium conditioned by astrocytes from neonatal rat brain specifically prevents the death of progenitor cultures, but is neither mitogenic nor inhibitory of their subsequent differentiation program. Because the activity is potent, proteinaceous (approximately 10-30 kDa), and distinct from tested mitogens (bFGF and PDGF) it is hypothesized that OL viability is promoted by an unrecognized trophic factor. Further experimentation in vitro will analyze this factor with regard to its (a) cellular action (b) purification and biochemical characterization (c) expression into adulthood and (d) relationship to a genetic model of accelerated OL death in the hypomyelinating jimpy mouse. Progenitor proliferation is restored normally by neuron-conditioned culture medium, as well as basic fibroblast growth factor (FGF) which at higher doses is mitogenic for """"""""post-mitotic'. OL It is hypothesized that FGF plays a changing role during the OL lineage, with mitogenic as well as unrecognized functions. Further studies will characterize FGF receptors and related signal transduction pathways utilized by progenitors and differentiated oligodendrocytes. The capacity to culture both cell types in isolation and high yield enables pharmacological and biochemical analyses. These data are expected to contribute to our knowledge of the epigenetic control of myelinogenesis and will provide insight concerning a trophic basis for the etiology and prospective treatment of dysmyelinating diseases.
