Double-labeling in situ hybridization analysis of mRNAs for carbonic anhydrase II and myelin basic protein: expression in developing cultured glial cells.

We applied in situ hybridization to analyze the location and the developmental changes in the distribution of the transcripts for carbonic anhydrase II (CAII) and myelin basic protein (MBP) in mouse primary cultured glial cells. Both mRNAs were localized to the oligodendrocyte using double-labeling in situ hybridization. No evidence for CAII transcripts in astrocytes was obtained, indicating that CAII is expressed only by oligodendrocytes in normal rodent glia. As early as 48 h after plating, CAII and MBP mRNAs are present in a few, small round cells. Message is present 2-4 days before levels of these proteins can be detected in similar primary glial cultures. The intensity of labeling for MBP and CAII mRNA positive cells increases significantly during the second week but then decreases after the end of the third week. Only the oligodendrocyte perikaryon and a few processes are positive during the first week. In contrast, at 14 days, a large number of cell processes in addition to the cell bodies are heavily stained for both mRNAs. Both mRNAs could be detected far away from the cell body, up to 250 microns in some cell processes. Some segments on a cell process accumulate higher levels of mRNA than other areas. These areas may correspond to the accumulation of free ribosomes and to starting points for the membrane sheets elaborated by cultured oligodendrocytes. The developmental profile for timing and distribution of these two messages mimics closely their in situ pattern.