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The tetraspanin/transmembrane 4 superfamily gene superfamily encodes proteins that span the plasma membrane four times. Tetraspan proteins are implicated in proliferation, motility, and differentiation in various cell types, and in some cells they may link plasma membrane proteins into signalling complexes. Using a subtractive cDNA library prepared from oligodendrocytes and their progenitor cells, we have identified Tspan-2 as a member of this superfamily. In situ hybridization analysis revealed robust expression in cells of the oligodendrocyte lineage in comparison with the Plp gene, a well-characterized marker for myelin-forming glia in the CNS. Rat Tspan-2 mRNA is restricted to the nervous system and is detectable by northern blot shortly after birth in the CNS. Subsequently the gene is up-regulated strongly between postnatal day 3 and 10, and expression levels continue to rise up to postnatal day 22. These data indicate that Tspan-2 is likely to play a role in signalling in oligodendrocytes in the early stages of their terminal differentiation into myelin-forming glia and may also function in stabilizing the mature sheath.

To study the biology and repair capacities of mouse oligodendroglial cells, we established cultures of cells purified from neonatal wild-type and 9.6-kb MBP-LacZ transgenic newborn mice cerebral hemispheres as free-floating aggregates in the continuous presence of neurblastoma conditioned medium (N1-B104). In vitro analysis indicated that the initial cell preparations were enriched in oligodendrocyte pre-progenitors that expressed PSA-NCAM and GAP-43 but not GD3, O4, NF68 or glial fibrillary acidic protein (GFAP) markers. These pre-progenitors required increased concentrations of insulin and progesterone to allow their survival in vitro. With time in culture, spheres composed of oligodendrocyte pre-progenitors became oligospheres enriched in oligodendrocyte progenitors expressing GAP-43 and GD3. As well as conserving bipotentiality in vitro, these spheres were able to form myelin in vivo after transplantation into the neonatal shiverer mouse brain. Thus, the oligosphere strategy is a powerful method for generating large populations of mouse oligodendrocyte pre-progenitors and progenitors. The ability to generate oligospheres from transgenic mice will be instrumental in the further dissection of the molecular and cellular mechanisms of myelination and remyelination of the central nervous system. J. Neurosci. Res. 58:735–751, 1999. © 1999 Wiley-Liss, Inc.

Myelin sheaths develop in the central nervous system (CNS) as elaborations of the processes of oligodendrocytes. Although many details of the spiral wrapping of oligodendrocyte processes around axons and their subsequent transformation into myelin sheaths are known from thin-section electron-microscopic studies, the three-dimensional architecture of the myelin-forming cells is incompletely understood. To characterize this aspect of oligodendrocyte development, we labeled thick (100- to 300-microns) sections of developing murine CNS with oligodendrocyte marker antibodies, recorded individual cells in serial optical sections by confocal microscopy, and created whole-cell reconstructions of oligodendrocytes before and during the initiation of myelination. We distinguish three stages in the maturation of oligodendrocytes, which at all three stages are labeled by the O4, O1 and Ranscht monoclonals and by antibodies against the myelin-specific proteins CNP and myelin basic protein. Premyelinating oligodendrocytes, present before axonal ensheathment begins, emit multiple irregular processes which have predominant radial orientation. These processes, which generally terminate within 50 microns of the cell body, have a surface area 3-8 times or more that of the cell body itself and may represent a mechanism for sampling the local environment of each cell and for identifying target axons. Transitional cells have initiated one or more myelin sheaths; these cells progressively reduce the number of their radial processes as they increase the number of their myelin internodes. The radial processes of each transitional cell are most reduced in parts of the process arbor where ensheathment has begun, suggesting directional control in the elaboration or stability of the radial processes. Mature myelin-bearing oligodendrocytes entirely lack the radial processes and instead emit a few sparsely branching processes which connect cell bodies with myelin internodes. Three-dimensional analysis of the earliest stages in myelin sheath formation reveals two distinct phases. The initiating event in the formation of myelin internodes is the growth of thin unbranched processes, termed 'initiator processes', along axons. The second phase, spiral ensheathment of target axons, begins through the elaboration from each initiator process of lamellar extensions which extend circumferentially around the target axon and thereby form the first turn of its myelin sheath.

qkI, a newly cloned gene lying immediately proximal to the deletion in thequakingviablemutation, is transcribed into three messages of 5, 6, and 7 kb. Antibodies raised to the unique carboxy peptides of the resulting QKI proteins reveal that, in the nervous system, all three QKI proteins are expressed strongly in myelin-forming cells and also in astrocytes. Interestingly, individual isoforms show distinct intracellular distributions: QKI-6 and QKI-7 are localized to perikaryal cytoplasm, whereas QKI-5 invariably is restricted to the nucleus, consistent with the predicted role of QKI as an RNA-binding protein. Inquakingviablemutants, which display severe dysmyelination, QKI-6 and QKI-7 are absent exclusively from myelin-forming cells. By contrast, QKI-5 is absent only in oligodendrocytes of severely affected tracts. These observations implicate QKI proteins as regulators of myelination and reveal key insights into the mechanisms of dysmyelination in thequakingviablemutant.