Oligodendrocytes or their putative progenitors were the only cells found to be immunoreactive to polyclonal antisera against the enzyme 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in developing and mature brains of rats and mice, as visualized by light and electron microscopy. Prior to myelination (day 6), oligodendrocytes of the corpus callosum have reticular networks of CNP-containing filopodia, in addition to abundant CNP throughout the cytoplasm. Some glioblast-like cells of the subventricular zone are also immunoreactive to anti-CNP, suggesting that, as progenitors of oligodendroglia, they express this myelination-related protein as one of the earliest events in myelinogenesis. Following the commencement of myelination (day 15), many oligodendrocytes lose much of their lacelike network of fine projections, possessing, instead, larger CNP-filled processes that extend to myelin-bearing fibers. CNP was always found only in the cytoplasm-containing compartments of the cells and myelin sheaths; neither lamellae nor cellular membranes were immunostained. These data support our contention that CNP is not an intrinsic membrane protein, despite its strong interaction with membrane components when cells are disrupted. In mutant (mld) mice (day 25), the many distended and uncompacted oligodendroglial processes that invest axons with only a few turns of membrane contained cytoplasmic CNP, accounting for the elevated levels of CNP activity previously noted in tissue fractions.

The gene encoding 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is one of the earliest myelin genes to be expressed in the brain. It is expressed at basal levels in some non-neural tissues but at much higher levels in the nervous system, and its relevance and mechanism are unknown. Using transgenic mice, we examined the expression pattern conferred by a 4-kilobase (-kb) 5'-flanking sequence of the mouse CNP gene coupled to the bacterial lacZ reporter gene. Here we report that this 4-kb fragment contains sufficient information to direct expression of the transgene to the tissue and/or cell type, in which CNP is normally expressed. In the central nervous system (CNS), CNP-lacZ expression was regulated in a temporal manner, consistent with endogenous CNP expression. Transgene expression was detected in embryonic brain and spinal cord in immature oligodendrocytes, and it significantly increased with age. In adult mice, beta-galactosidase activity (which appeared to be oligodendrocyte specific) was found essentially in white matter areas of the CNS. Moreover, the transgene was expressed in peripheral nervous system, testis, and thymus-tissues that normally express CNP. Taken together, our results provide strong evidence that cis-acting regulatory elements, necessary to direct spatial and temporal expression of the transgene in oligodendrocytes, are located within the 4-kb 5'-flanking sequence of the mouse CNP gene. This promoter could be a valuable tool to target specific expression of other transgenes to oligodendrocytes, and may provide important new insights into myelination or dysmyelination.

We have shown that the developmental regulated appearance of the two myelin-associated glycoprotein (MAG) polypeptides in normal mouse brain myelin does not reflect the developmental pattern of differential splicing of primary gene transcripts as determined earlier by RNase protection assays. Contrary to expectation, the large (L-MAG) and small (S-MAG) polypeptides are present in about equal amounts at a relatively early stage of myelination, day 24 or earlier. In quaking (qk) mutant mouse brain myelin, both MAG polypeptides are evident at all ages examined; the relatively greater abundance of S-MAG compared to L-MAG at early ages (days 18 and 22) confirms our earlier observation on in vitro translations of mRNA. At later ages (day 27 and beyond) both isoforms are present in approximately equal amounts. The L-MAG but not the S-MAG polypeptide can be phosphorylated by kinases that are endogenous to isolated qk myelin, analogous to the phosphorylation we have observed in vivo in normal mice and in their isolated myelin.

Myelin-associated glycoprotein (MAG), a myelin-specific protein, is expressed as two isoforms, designated as L-MAG and S-MAG. Both share identical extracellular and transmembrane domains but differ in their cytoplasmic domains. L-MAG is expressed earlier during myelination than S-MAG. These features, as well as others, suggest that the isoforms have different functions. To confirm this hypothesis, both isoforms were expressed transiently and stably in Madin-Darby canine kidney (MDCK) epithelial cells, and the localization of the isoforms was studied. In both transiently and stably transfected cells, L-MAG sorted primarily to the basolateral membrane. In single transfected cells, S-MAG sorted primarily to the apical membrane. When groups of adjacent cells became transiently transfected, S-MAG accumulated at areas of cell-cell contact within the basolateral membrane. In stably transfected cells S-MAG sorted to the basolateral membrane. The data suggest that L-MAG contains an invariable basolateral sorting signal, but that the sorting of S-MAG is dependent upon extrinsic factors, such as coexpression by adjacent (contacting) cells. As MDCK cells sort the MAG isoforms differently, these data support the hypothesis that the MAG isoforms do perform different functions.

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