GFAP gene expression during development of astrocyte.

Abstract

Glial fibrillary acidic protein (GFAP) is expressed exclusively in astrocytes in the central nervous system. In order to characterize individual cultured cells in which the GFAP promoter is active and to identify the regulatory mechanisms of GFAP expression in these cells, we have developed a unique assay system for promoter activity using retrovirus vectors. Retrovirus containing the mouse GFAP promoter fused to the lacZ gene were used to infect mixed glial cultures. The infected cells, in which the GFAP promoter was active, were visualized by X-Gal staining. From these experiments, we found that a 256 bp fragment 5' of the transcription initiation site was sufficient to confer astrocyte-specific expression of GFAP. The GFAP promoter became active about 3 days before GFAP protein can be detected immunohistochemically, which indicates that detection of GFAP promoter activity can be used to identify astrocyte progenitors. We have also established immortalized astrocyte cell lines in which we detect GFAP promoter activity. Immorto mouse is a transgenic mouse generated by the introduction of thermolabile SV40 T Ag, tsA58. A mixed glial culture prepared from 2-day-old Immorto mouse brain was incubated at 32 degrees C, at which temperature most of the cells expressed T Ag. The culture was then infected with retrovirus containing GFAP promoter-lacZ, and the infected cells were selected. Using the fluorescence-activated cell sorter with fluorescein di-beta-D-galactopyranoside as a substrate (FDG-FACS), these cells were separated into two groups: FDG(+), in which the GFAP promoter was active, and FDG(-), in which it was inactive. Mature astrocyte cell lines were established from the FDG(+) cells by colony isolation. The FDG(-) cells were cloned by colony isolation and cultured at 32 or 39 degrees C. At the latter temperature the expression of T Ag was suppressed and cell differentiation was induced in most cells. The cells which became positive for X-Gal staining only after switching to 39 degrees C were collected as immature astrocyte cell lines. These immortalized cell lines should be useful to investigate the molecular mechanisms of astrocyte differentiation.