Apoptosis of bcl-x-deficient telencephalic cells in vitro

Abstract

bcl-x is a member of the bcl-2 gene family, which is expressed at high levels in the embryonic brain. The targeted disruption of bcl-x results in massive cell death of immature neurons in the developing mouse brain (Motoyama et al., 1995). bcl-x-deficient mice die around embryonic day 13 (E13), probably secondary to their inability to produce mature red blood cells. To determine whether the death of immature neurons in the bcl-x- deficient brain is cell autonomous, we examined primary telencephalic cell cultures from E12.5 homozygous mutant (bcl-x(-/-)), heterozygous mutant (bcl- x(+/-)), and wild-type (bcl-x(+/+)) mice. bcl-x(-/-) telencephalic cells cultured in 0.5 or 2.0% fetal calf serum (FCS)-containing medium for 48 hr showed increased apoptosis, defined by abnormal bisbenzamide staining and terminal-deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), and decreased numbers of microtubule-associated protein-2- immunoreactive neurons compared with bcl-x(+/-) and bcl-x(+/+) cultures. Cycloheximide treatment of bcl-x(-/-) telencephalic cell cultures failed to prevent the increased cell death observed in low FCS-containing medium, suggesting a protein synthesis-independent apoptosis. There were no significant differences among bcl-x(-/-), bcl-x(+/-), and bcl-x(+/+) telencephalic cells grown for 48 hr in 5% FCS-containing medium or in a chemically defined serum-free medium (ITS). bcl-x(-/-) neurons generated in ITS showed increased susceptibility to subsequent serum deprivation. These results indicate that bcl-x is important for both neuron maturation and survival.