Loss of BH3-only protein Bim inhibits apoptosis of hemopoietic cells in the fetal liver and male germ cells but not neuronal cells in bcl-x-deficient mice

Abstract

Members of the Bcl-2 family include pro- and antiapoptotic proteins that regulate programmed cell death of developing tissues and death in response to cellular damage. In developing mice, the antiapoptotic Bcl-x(L) is necessary for survival of neural and hematopoietic cells, and consequently, bcl-x-deficient mice die around Day 13.5 of embryogenesis. Furthermore, adult bcl-x(+/-) heterozygous male mice have reduced fertility because of testicular degeneration. Bax, a multi-BH (Bcl-2 homology) domain proapoptotic member of the Bcl-2 family, is regulated by Bcl-x(L) and is required for the neuropathological abnormalities seen in bcl-x-deficient embryos. The BH3 domain only subgroup of the Bcl-2 family includes proapoptotic members that are essential for the initiation of apoptotic signaling. In this study, we investigated the role for Bim, a BH3 domain only protein, in the embryonic lethality and increased developmental cell death in bcl-x-deficient animals and the perturbed testicular function in bcl-x(+/-) adults. Our studies show that bim deficiency attenuates hematopoietic cell death in the fetal liver of bcl-x-deficient animals, indicating that Bim contributes to programmed cell death in this cell population. In addition, we found that testicular degeneration of adult bcl-x(+/-) males was rescued by concomitant Bim deficiency. However, concomitant Bim deficiency had no effect on the embryonic lethality and widespread nervous system abnormalities caused by bcl-x deficiency. Our work identifies Bim as an important regulator of bcl-x deficiency-induced cell death during hematopoiesis and testicular development.

Figure 1

Bim loss has no effect on neurodegeneration caused by bcl-x deficiency. (A) Dorsal root ganglia (DRG) in bcl-x^+/o bim^+/o E12.5 embryos contained few apoptotic cells as determined by hematoxylin and eosin staining. (B) In bcl-x^−/− bim^+/o embryos, many cells with fragmented, condensed nuclei were visible in the DRG (arrows). (C) Numerous apoptotic cells were also visible in bcl-x^−/− bim^−/− embryos (arrows). (D) Ventral spinal cord (SC) of bcl-x^+/o bim^+/o E12.5 embryos showed occasional apoptotic cells (arrow). (E) In contrast, large numbers of apoptotic cells and degenerative changes were noted in bcl-x^−/− bim^+/o embryos (arrows). (F) Loss of Bim in bcl-x^−/− bim^−/− embryos did not alleviate these defects (arrows). Bars: A–C = 20 μm; D–F = 50 μm.

Figure 2

Bim loss does not alter terminal deoxynucleotidyltransferase-mediated dUPT nick end labeling (TUNEL) reactivity in bcl-x–deficient spinal cord. (A) Spinal cord in bcl-x^+/o bim^+/o E12.5 embryos contained few apoptotic cells as determined by TUNEL staining. Previous reports have described significant apoptosis characterized by TUNEL positivity in spinal cord of bcl-x^−/− bim^+/o mice (Motoyama et al. 1995 and data not shown). (B) Concomitant Bim deficiency (bcl-x^−/− bim^−/−) did not rescue this phenotype and resulted in significant numbers of TUNEL-positive neurons (indicated by arrows). Bar = 50 μm.

Figure 3

Bim loss significantly reduces the abnormal apoptosis of hematopoietic cells in the fetal liver but not spinal cord caused by bcl-x deficiency. TUNEL staining was performed on sections from E12.5 embryos, and TUNEL-positive cells within multiple ×100 fields were quantitated. Fields were assessed in spinal cord (left) and liver (right). ns, not significant. *p<0.005.

Figure 4

Bim loss reduces testicular hypoplasia in bcl-x happloinsufficient adult mice. Testis weights from multiple adult male mice of the indicated genotypes were assessed, and average testis weight (g)/total body weight (kg) was calculated. Testes of adult bcl-x^+/− mice were 57% smaller than those of bcl-x^+/+ mice. This size difference was reduced to 27% in bcl-x^+/− bim^+/− adults and eliminated in bcl-x^+/− bim^−/− adults. ns, not significant. *p<0.005.

Figure 5

Bim loss rescues testicular degeneration caused by loss of one allele of bcl-x. (A) Hematoxylin and eosin–stained testis from bcl-x^+/− bim^+/+ showed significant degenerative changes with vacuole formation and disrupted testicular morphology. (B) Vacuolar degenerative changes were not observed in bcl-x^+/o bim^+/o adult testis, although some parenchymal loss was evident. (C) Adult bcl-x^+/− males lacking both alleles of bim (bcl-x^+/− bim^−/−) had normal testicular morphology. Bar = 50 μm.