Temporal expression of mouse glial fibrillary acidic protein mRNA studied by a rapid in situ hybridization procedure.
A rapid and sensitive in situ hybridization technique is described for the detection of mRNA sequences in 6-8-micron cryostat sections. The method incorporates the use of alpha-thio-35S-labelled nucleoside triphosphates for the generation of high-specific-activity DNA probes and a high-stringency washing procedure that virtually eliminates background without unduly compromising histological integrity. Whereas signal resolution is less than that observed using 3H probes, 35S-labelled probes are well-suited for experiments where resolution at the cellular level is required. The method has been applied to a study of the developmental regulation of glial fibrillary acidic protein (GFAP) mRNA expression in developing mouse brain. GFAP-specific sequences are first detectable after the second postnatal day, and thereafter rise to a level that is maintained throughout development and into adulthood. The distribution of GFAP-encoding sequences broadly reflects the known distribution of astrocytes, but the levels of mRNA within these cells vary by a surprisingly large amount depending on their location. For example, in adult animals, the astrocytes of the glial limitans contain an abundance of GFAP-specific mRNA that is higher than corresponding levels in astrocytes in the cerebellar white matter, whereas these cells in turn contain considerably more GFAP-specific mRNA than astrocytes in the gray matter of the cerebrum. Unexpectedly, parallel RNA blot transfer experiments show the existence of some GFAP-encoding mRNA size heterogeneity that is restricted to the first postnatal week.
S A
SA
Lewis
N J
NJ
Cowan
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001921:Brain / Q000254:growth & development / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics*; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000096:biosynthesis*; D014404:Tubulin / Q000235:genetics; D000509:alpha-Fetoproteins / Q000235:genetics
D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D014404:Tubulin; D000509:alpha-Fetoproteins; D004247:DNA