Initial reports describing Diva found high expression of this gene in the granulosa cells of the ovary and in the epididymis of the testis, although expression was lower in the testis than in the ovary (
12,
25). Additionally, in situ hybridization showed widespread Diva expression in the developing nervous system and the ovary (
12). We used Northern blot analysis and PCR to confirm the spatial and temporal distribution of
Diva mRNA. Northern blot analysis of a number of adult mouse tissues and various stages throughout mouse development found a detectable signal only in the ovary; no signal was found in any other tissues even after extended exposure (data not shown). However, Diva mRNA was detected using PCR from first-strand cDNA in all tissues examined including developing postnatal day 5 (P5) brain and adult mouse brain, liver, and kidney (data not shown). Therefore, Diva mRNA is abundant in the ovary and at levels only detected by PCR in other tissues.
To determine the biological role of Diva, we used gene targeting to inactivate mouse Diva. This gene (GenBank no. NM013479, NM013479, and AF102501) is located on chromosome 9 and contains two coding exons. Inactivation of Diva was achieved by replacing an ~3-kb genomic region containing both exons with a Neor selection cassette to delete the entire Diva open reading frame (ORF) (Fig. ). Targeting of ES cells occurred at a frequency of approximately 1/25 (Fig. ), and two of these targeted ES lines were used to generate chimeras and, subsequently, Diva heterozygous mice. Interbreeding of Diva heterozygotes generated Diva-null mice, which were born at the expected frequency of 1/4. Southern blot analysis using a Diva cDNA probe also showed an absence of Diva coding sequence in Diva−/− mice, while both WT and Diva+/− mice contained Diva ORF sequence (Fig. ). We further confirmed that Diva expression was disrupted in the Diva−/− mice using Northern blot analysis; Diva mRNA of 1.2 kb was identified in RNA obtained from WT and heterozygous, but not homozygous Diva−/−, ovaries (Fig. ).
Although gene targeting resulted in the complete removal of the genomic DNA encoding
Diva, these mice were fertile and had no obvious behavioral defects or affected organs, and long-term survival was indistinguishable from that of WT littermates. Survival of
Diva−/− mice was monitored for up to 2 years and histological analysis of the mice at various ages showed no gross anatomical defects in the
Diva−/− ovaries compared to WT ovaries (Fig. and e). As
Diva expression was reported in the developing nervous system (
12), we performed histological analysis of the nervous system at various ages up to 8 months but found no discernible differences in any brain regions compared to littermate controls. Immunohistochemical studies using a variety of markers failed to reveal any differences between Diva-null and control mice up to 8 months of age in a number of tissues, including the ovaries (data not shown).
Because
Diva was highly expressed in the ovary and Diva-null animals were fertile and showed no differences from control littermates, we reasoned that
Diva−/− mice might be deficient in apoptosis. Diva has been implicated in apoptosis involving Apaf-1 and caspase-9 (
12,
25), which are components known to be associated with genotoxic stress-induced apoptosis. Furthermore, apoptosis induced by Diva can be inhibited by a dominant-negative mutant of caspase-9 (
12). Consistent with this, Diva can interact with Apaf-1 and displace Bcl-X from the Apaf-1/Bcl-X complex, suggesting that inhibition of Bcl-X function by Diva may occur through competitive binding to Apaf-1 (
12,
25).
To determine if
Diva−/− mice were differentially sensitive to genotoxic stress compared to WT littermates, we examined ionizing radiation (IR)-induced apoptosis in these mice. Pronounced apoptosis as determined histologically was observed at 6 h following IR in granulosa cells in both Diva-null and WT controls (Fig. , c, f, and g). However, while no differences were found between
Diva−/− and WT mice, there is a clear genetic basis for ovarian radiation-induced apoptosis as p53
−/− null mice were completely resistant to IR-induced apoptosis in the ovary (Fig. ). Because
Diva was detected in the developing brain we also examined IR-induced apoptosis in various developing nervous system tissues of
Diva−/− and WT controls. Widespread IR-induced apoptosis was found throughout susceptible regions of the developing nervous system (
5), including the cerebellar external granule layer (Fig. and c), the hippocampal dentate gyrus (Fig. ), and the retina (Fig. ), and was identical in both
Diva−/− and WT controls. Therefore, while other Bcl-2-related members can modulate the response to radiation (
5), Diva is not required for IR-induced apoptosis in the developing nervous system.
The Bcl-2 family is important for regulating apoptosis as determined by extensive in vitro analysis and mouse knockout models for many of these molecules (
21). Diva (Boo/Bcl-B) has been ascribed both pro- and antiapoptotic roles (
2,
12,
13,
16,
19,
25), although because of the relative tissue-restricted expression of this gene, it is likely that cellular context will be important for Diva-regulated apoptosis. Furthermore, as Diva has been shown to interact with a number of different Bcl-2 family members, including Bcl-X and Bax (
12,
13), it is likely that these associations also modulate Diva function. In addition to Diva, other Bcl-2 family proteins have been found in the ovary, including Mcl-1, Bok, Bod, and Bad, suggesting the potential for functional modulation by interaction between these various anti- and pro-apoptotic factors (
9). Recent data have shown that the interplay between Bcl-2 family members can determine the outcomes of apoptotic signals whereby multidomain Bcl-2-related proteins influence the activity of the pro-apoptotic BH3-domain-only proteins (
4). Moreover, activation of either Bax or Bak is a critical determinant for apoptosis in many instances (
27). Thus, perhaps the apparent lack of a phenotype in the Diva-null mice and the physiological role of Diva may be understood with further genetic manipulation such as the generation of mice with other apoptotic control genes inactivated in concert with Diva.
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