2002, 22(19):6866. DOI:
Mol. Cell. Biol.
Zhao and Peter J. McKinnon
Helen R. Russell, Youngsoo Lee, Heather L. Miller, Jingfeng
Affected by Inactivation of the Bcl-2 Family
Murine Ovarian Development Is Not
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MOLECULAR AND CELLULAR BIOLOGY, Oct. 2002, p. 6866–6870
0270-7306/02/$04.00?0 DOI: 10.1128/MCB.22.19.6866–6870.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 22, No. 19
Murine Ovarian Development Is Not Affected by Inactivation of the
Bcl-2 Family Member Diva
Helen R. Russell, Youngsoo Lee, Heather L. Miller, Jingfeng Zhao, and Peter J. McKinnon*
Department of Genetics, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
Received 5 February 2002/Returned for modification 19 June 2002/Accepted 2 July 2002
Diva (also called Boo/Bcl-B) is a member of the Bcl-2 gene family and most likely functions during apoptosis.
Diva is highly expressed in the ovary, and both pro- and antiapoptotic functions have been ascribed to this
protein. To determine the role of Diva during murine development, we used gene targeting to inactivate Diva.
The Diva-null mice are born at the expected ratios, are fertile, and have no obvious histological abnormalities,
and long-term survival did not differ from littermate controls. Additionally, Diva was not required for apoptosis
occurring from genotoxic insult in the ovaries or other organs. Thus, Diva is not critical for the normal
development of the ovaries, or in its absence its function is subserved by another protein.
Cell death that occurs during normal organismal develop-
ment or results from disease or radiotherapy and chemother-
apy usually involves apoptosis, a genetically defined program
of cell elimination (1, 8). Apoptosis is critical for homeostasis,
and inappropriate control of apoptosis can result in a variety of
human pathologies, including cancer and neurodegeneration
(18, 22). The molecular basis of apoptosis is evolutionarily
conserved, and similar strategies for cell death are used in
organisms from nematodes to humans (8). The basic molecular
framework involves a stimulus activating one or more of a
variety of Bcl-2-related proteins (ced-9 in the nematode) that
in turn lead to the activation of caspases (ced-3) that act as
proteases to dismantle the dying cell (7, 8, 20).
The Bcl-2 family of proteins is functionally important in
apoptosis and often acts in a tissue-specific manner (1, 15, 21).
The canonical member of this family, Bcl-2, was first identified
as a component of a translocation in B-cell malignancies, and
when overexpressed was found to inhibit apoptosis in a variety
of biological systems (15). Other Bcl-2 family members act as
general effectors of cell death by either promoting or protect-
ing against cell death (1, 3). Pro-apoptotic members include
Bax, Bad, Bid, Bak, and Bik, while antiapoptotic members
include Bcl-2, Bcl-X, Mcl-1, Bcl-w, and A-1. Bcl-2 family pro-
teins can contain four conserved domains, designated Bcl-2
homology regions (BH1 to BH4) (1, 3, 15). The BH1 and BH2
motifs of the death antagonists (such as Bcl-2 and Bcl-X) and
the BH3 domain of the death agonists (such as Bax and Bak)
are important for homo- or heterodimerization between family
members and facilitate control of apoptosis (1, 3, 15, 30). The
BH4 domain, found in several antiapoptotic homologues, is
essential for the death-repressing activity (10). Some Bcl-2
family members share sequence homology only with the BH3
domain (11, 26). These BH3-domain-only proteins are thought
to activate multidomain Bcl-2 members to initiate apoptosis
As demonstrated in mice with null mutations for Bcl-2 fam-
ily members, this group of proteins plays important roles dur-
ing development and homeostasis (21). For example, Bcl-2
inactivation leads to polycystic kidney disease, while inactiva-
tion of Bax in the mouse resulted in hyperplasia of thymocytes
and male infertility due to spermatocyte hypoplasia (14).
Bax?/?animals also show a decrease in normal programmed
cell deaths in a number of nervous system tissues, including
peripheral ganglia and the trigeminal brainstem nuclear com-
plex, and neuronal cultures derived from Bax?/?animals are
resistant to a number of death-inducing agents (5, 6, 28, 29).
More dramatic effects are found in Bcl-X-null mice, where
embryonic survival requires the presence of this protein (17).
Tissue-specific effects are observed in other cases; for example,
Bcl-W inactivation results in infertility due to arrested sperm
development associated with a gradual loss of germ cells and
Sertoli cells from the testis (23, 24). In this report we have
investigated the consequences of inactivating the Bcl-2-related
protein Diva (12, 25). This gene contains several BH domains
(BH1, BH2, and BH4), with some contention existing regard-
ing the presence of a BH3 domain (12, 13, 25), and it can
modulate apoptosis in vitro (2, 12, 13, 16, 19, 25). Diva is also
relatively restricted in expression, with high levels of expres-
sion confined to the ovary (12, 25). Here we report that Diva-
null mice are fertile, respond normally to apoptotic stimuli,
and do not have any obvious developmental defects.
MATERIALS AND METHODS
Gene targeting. The murine Diva gene contains two exons within a 3.24-kb
region of genomic DNA. Diva genomic DNA from strain 129Ola was obtained by
isolation of a P1 clone (Genome Systems, St. Louis, Mo.) containing the entire
3.24-kb genomic Diva DNA. A BamHI/BspEII fragment (?8 kb) immediately 5?
to Diva exon 1 was cloned into the BglII site of pNTK1901 (Stratagene, San
Diego, Calif.) to generate pDiva-1, and a 2.5-kb BamHI/BglII fragment imme-
diately 3? of Diva exon 2 was cloned into the BamHI site of pDiva-1 to generate
pDiva-KO. This construct was linearized with SalI and electroporated into W9.5
embryonic stem (ES) cells. Targeted ES cells were identified by Southern blot
analysis of EcoRV-digested ES genomic DNA using a genomic BglII/EcoRV
fragment 3? of Diva. The probe, ?200 bp in length, was generated by PCR using
mouse genomic DNA as a template with the following primers: forward, 5? AGA
TCT ACT GAA CTC AGC, and reverse, 5? ATA TCT GAG AAG CCA AGG.
EcoRV digestion of G418-resistant ES cells identified a 4-kb fragment in tar-
geted clones due to loss of an EcoRV site in the mutant allele that was readily
distinguishable from the endogenous 2.9-kb Diva genomic EcoRV fragment.
* Corresponding author. Mailing address: Department of Genetics,
St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis,
TN 38105. Phone: (901) 495-2700. Fax: (901) 526-2907. E-mail:
on June 9, 2013 by guest
FIG. 1. Inactivation of mouse Diva. (A) Diva was inactivated by replacing both exons 1 and 2 with a neomycin selection cassette driven by the
PGK promoter derived from pNTK901. Selected restriction sites relevant to the generation and analysis of Diva inactivation are indicated.
Homologous recombination removed an EcoRV site from the Diva locus, resulting in a 4-kb mutant Diva allele fragment after EcoRV digestion
of genomic DNA; Southern blot analysis was done using a probe encompassing the genomic region contained in a 3? BglII/EcoRV fragment
(probe). (B) The Southern blot shown was probed with Diva cDNA, is a representative analysis of mice derived from mating Diva heterozygotes,
and shows that exons 1 and 2 containing the Diva ORF are absent from Diva?/?mice. (C) Northern blot analysis shows that the Diva message
(1.2 kb) is present in the WT but not Diva?/?ovaries, while no Diva signal is detected in the testis. The control probe (reticulon, 1.6 kb; GenBank
no. AF133669) was used to ensure RNA integrity in samples used for Northern analysis.
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Resulting clones were injected into C57BL/6 blastocysts and then implanted into
the uteri of pseudopregnant F1 B/CBA foster mothers and allowed to develop to
term. Male chimeras were selected for high percentage of agouti coat color and
were mated to C57BL/6 females to obtain germ line transmission. The presence
of the mutated allele was confirmed by Southern blot analysis or PCR, and
heterozygous F1 males and females were interbred to generate F2 animals for
subsequent study. Genotyping of Diva mutant mice was done from tail DNA
using PCR with the following primers to identify the Diva wild-type (WT) allele:
GDP 1, 5? CAG ACG ATT GCCC CGG C, and GDP 4, 5? GGT AAC ATC
AGC ATC ACA GAA TGC. The Neormarker gene was identified using the
following primers: Neo 4, 5? CGG GAG CGG CGA TAC CGT AAA GC, and
Neo 7, 5? GAA GCG GGA AGG GAC TGG CTG CTA.
Histology. Ovaries were obtained from 2-month-old mice 6 h after 18 Gy of
whole-body ionizing radiation from a cesium irradiator (delivered at a rate of 1.2
Gy/min) and were placed in 10% formalin. Histology of unirradiated tissues was
done using age-matched Diva-null mice and littermate controls. Ovaries were
paraffin embedded, sectioned into 8-?m sections with an HM325 microtome
(Microm), and hematoxylin and eosin stained according to standard procedures.
For studies using nervous system tissues, mice were used 5 days after birth (P5;
day of birth is P0) and irradiated with 18 Gy. Nervous system tissues were
collected after fixation by transcardial perfusion with 4% paraformaldehyde,
cryoprotected in 20% sucrose–phosphate-buffered saline, and cryosectioned
(12-?m coronal sections) using an HM500 M cryostat (Microm). Neutral red
staining was performed with 1% neutral red (Aldrich Chemical) in 0.1 M acetic
acid (pH 4.8) for 1 min followed by dehydration in ethanol and mounting with
Permount (Fisher). In all cases, experiments were done in triplicate and com-
parative studies of Diva-null mice used WT littermates as controls.
RESULTS AND DISCUSSION
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 chromo-
some 9 and contains two coding exons. Inactivation of Diva was
achieved by replacing an ?3-kb genomic region containing
both exons with a Neorselection cassette to delete the entire
Diva open reading frame (ORF) (Fig. 1A). Targeting of ES
cells occurred at a frequency of approximately 1/25 (Fig. 1A),
and two of these targeted ES lines were used to generate
chimeras and, subsequently, Diva heterozygous mice. Inter-
breeding 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. 1B). 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 heterozy-
gous, but not homozygous Diva?/?, ovaries (Fig. 1C).
FIG. 2. Radiation-induced apoptosis in Diva-null ovaries. Ovaries from WT (a) or Diva?/?(e) mice are histologically indistinguishable, as was
apoptosis after ionizing radiation treatment (b, c, f, and g). However, p53?/?ovaries (d, h) were completely resistant to radiation-induced
apoptosis. Panels b to d and f to h represent two different comparative views through the ovaries. Panels a and e are unirradiated WT and Diva?/?
mice, respectively. Arrows identify pyknotic cells indicative of apoptosis. Magnification, ?200.
6868RUSSELL ET AL.MOL. CELL. BIOL.
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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 lit-
termates. 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. 2a and e). As Diva expression
was reported in the developing nervous system (12), we per-
formed 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. Immunohisto-
chemical 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
Because Diva was highly expressed in the ovary and Diva-
null animals were fertile and showed no differences from con-
trol littermates, we reasoned that Diva?/?mice might be de-
ficient 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 apopto-
sis. 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. Pro-
nounced apoptosis as determined histologically was observed
at 6 h following IR in granulosa cells in both Diva-null and WT
controls (Fig. 2b, 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 ap-
optosis in the ovary (Fig. 2d and h). Because Diva was detected
in the developing brain we also examined IR-induced apopto-
sis in various developing nervous system tissues of Diva?/?and
WT controls. Widespread IR-induced apoptosis was found
throughout susceptible regions of the developing nervous sys-
tem (5), including the cerebellar external granule layer (Fig. 3b
and c), the hippocampal dentate gyrus (Fig. 3e and f), and the
FIG. 3. Radiation-induced apoptosis in the Diva-null developing nervous system. The nervous system of Diva?/?mice was histologically
indistinguishable from that of WT controls. Apoptosis 6 h after IR in the P5 Diva?/?cerebellar external granule layer (EGL; b and c), dentate
gyrus (DG; e and f), and retina (h and i) was similar to that in WT tissue. Apoptosis was assessed by the presence of pyknotic nuclei (arrows) by
use of neutral red staining (5). Magnification, ?400.
VOL. 22, 2002Diva KNOCKOUT MOUSE 6869
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retina (Fig. 3h and i), and was identical in both Diva?/?and Download full-text
WT controls. Therefore, while other Bcl-2-related members
can modulate the response to radiation (5), Diva is not re-
quired for IR-induced apoptosis in the developing nervous
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 ex-
pression 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 addi-
tion 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 mul-
tidomain Bcl-2-related proteins influence the activity of the
pro-apoptotic BH3-domain-only proteins (4). Moreover, acti-
vation of either Bax or Bak is a critical determinant for apo-
ptosis 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.
These studies were supported by the NIH (NS-37956, NS-39867 and
CA-21765) and the American Lebanese and Syrian Associated Char-
ities (ALSAC) of St. Jude Children’s Research Hospital.
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