Oncogene (1998) 16, 61 ± 68
 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice.
Pamela E Blackshear1, Susan M Goldsworthy2, Julie F Foley1, Kimberly A McAllister1,
L Michelle Bennett1, N Keith Collins1, Donna O Bunch1, Paula Brown1, Roger W Wiseman1
and Barbara J Davis1
1
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; 2Pathology Associates
International, Durham, North Carolina 27713, USA
The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell
cycle-dependent fashion in vitro and appear to be
regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization
expression patterns of Brca1 and Brca2 mRNA in vivo
in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues
including testes, ovaries, and hormonally altered ovaries.
Brca1 and Brca2 are expressed concordantly in
proliferating cells of embryos, and the mammary gland
undergoing morphogenesis and in most adult tissues.
The expression pattern of Brca1 and Brca2 correlates
with the localization of proliferating cell nuclear
antigen, an indicator of proliferative activity. In the
ovary, Brca1 and Brca2 exhibited a comparable
hormone-independent pattern of expression in oocytes,
granulosa cells and thecal cells of developing follicles. In
the testes, Brca1 and Brca2 were expressed in mitotic
spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes
revealed that the time course of Brca2 expression was
delayed in spermatogonia relative to Brca1. Thus, while
Brca1 and Brca2 share concordant cell-speci®c patterns
of expression in most proliferating tissues, these
observations suggest that they may have distinct roles
during meiosis.
Keywords: Brca1; Brca2; mitosis; meiosis
Introduction
Most hereditary predisposition to breast and ovarian
cancer can be attributed to germline mutations in the
BRCA1 or BRCA2 breast cancer susceptibilty genes
(Stratton, 1996). Germline mutations in the BRCA1
gene increase the risk for the development of earlyonset breast cancer and ovarian cancer (Miki et al.,
1994). Mutations in the BRCA2 gene result in an
increased risk for the development of breast cancer in
both women and men (Wooster et al., 1995; Stratton,
1996), a moderate increased risk for the development
of ovarian cancer in women (Stratton, 1996), and are
associated with pancreatic, cervical, and laryngeal
cancers (Wooster et al., 1995; Gudmundsson et al.,
1995). Inherited mutations in either BRCA1 or
BRCA2 are also associated with a signi®cant risk of
Correspondence: PE Blackshear
Received 13 June 1997; revised 15 August 1997; accepted 15 August
1997
colon cancer and predispose men to the development
of prostate cancer (Wooster et al., 1995; Stratton,
1996; Gudmundsson et al., 1995). Since inactivation of
both alleles of either BRCA1 or BRCA2 are key
features in neoplastic development in hereditary
cancers, these genes are believed to act as tumor
suppressor genes required for cell growth. Understanding how BRCA1 and BRCA2 function during
normal growth and development should help to de®ne
why their inactivation yields such profound predispositions to neoplasia.
Several studies have demonstrated a relationship
between BRCA1 and BRCA2 gene function and
normal growth control. For example, BRCA1 and
BRCA2 are induced at the G1/S boundary in normal
human mammary epithelial cell and breast cancer cell
lines stimulated to proliferate (Vaughn et al., 1996a, b;
Gudas et al., 1996). BRCA1 message and protein
expression are induced by the mitogenic activity of
hormones, such as in estrogen responsive cell lines
(Marks et al., 1997; Gudas et al., 1995). In human
breast cancer cell lines and spermatocytes, BRCA1
protein binds to Rad51, which functions in DNA
recombination and repair, supporting a role for
BRCA1 speci®cally during mitosis and meiosis (Scully
et al., 1997). Recent studies indicate a similar
interaction between Rad51 and the Brca2 gene
product (Sharan et al., 1997).
The mouse homologues, Brca1 and Brca2, appear
to function in normal growth control and their
similar expression patterns imply that their regulatory
pathways may be analogous. However, their phenotypic expressions in carcinogenesis associated with
loss of gene function is not completely redundant, as
demonstrated by the spectrum of tumors that
develop in mutation carriers. Therefore, we investigated the relationship between Brca1 and Brca2
tissue expression patterns during embryonic development, puberty, and in adult mice. Additionally, we
determined whether Brca2 was expressed independently of hormonal stimulation by comparing
expression of Brca2 in ovaries of cycling mice,
hypophysectomized mice, and in estrogen receptor
knock-out (ERKO) mice as previously determined for
Brca1 (Phillips et al., 1997). Our results demonstrate
that Brca1 and Brca2 have concordant patterns of
expression during embryogenesis, mammary gland
morphogenesis, and adult tissues associated with
proliferating cell populations, terminally differentiated neurons as well as hormonally altered
ovaries, but there are di€erences in their temporal
expression in the testis. Thus, although there is
considerable overlap, their regulatory pathways are
not identical.
Brca1 and Brca2 expression
PE Blackshear et al
62
Results
Brca1 and Brca2 expression during mouse embryogenesis
During embryonic development, in situ hybridization
studies demonstrated that Brca2 was ubiquitously
expressed throughout the three germ layers of day
7.5 embryos which evolved into a tissue speci®c
pattern through day 19. The expression pattern of
Brca2 was analogous to that of Brca1 at the same
time points, and in most instances, correlated with
PCNA positive staining (Figure 1). In gestational day
14 embryos, intense in situ hybridization staining,
which represents relatively high expression for both
Brca1 and Brca2 transcripts, was localized to
neuroectodermal tissues, including the ventricular
zone, and medial and lateral walls of the cerebral
hemispheres, trigeminal ganglia, primodial spinal
nerve roots and ganglia, Rathke's pouch, and
neuroepithelium of the eye. These neuroectodermal
cells also demonstrated PCNA positivity except for
the medial and lateral walls of the cerebral hemispheres. Similarly high staining intensity, and thus
relative expression, was observed in ectodermallyderived tissues including the stratum basalis of the
epidermis, tongue, otic cochlea, ameloblasts of the
tooth bud, and lens of the eye. High staining intensity
was also observed in mesodermally-derived tissues
including somites, brown fat, hematopoetic cells in the
liver, and cartilage. Within the same embryo section,
moderate staining intensity was localized to mesodermally-derived tissues of the genital ridge, including the
overlying mesothelium and the Mullerian ducts, and
mesenchymal tissues of the limb. Low staining
intensity was detected in the heart, which is of
mesodermal origin. In endodermally-derived tissues,
moderate staining intensity was found in hepatocytes
and intestinal epithelium. In the lung (endodermal
origin), the alveolar buds stained moderately for both
1A
1B
transcripts while the bronchial epithelial cells stained
with low intensity. By day 19, when most tissues had
reached their de®nitive prenatal morphology, both
Brca1 and Brca2 were expressed with the same
relative staining intensity as described for the day 14
embryos except there was no evidence of Brca1 or
Brca2 expression in the adrenal gland or urinary
bladder.
Brca1 and Brca2 expression in adult mouse tissues
The expression patterns of Brca1 was similar to Brca2
in the adult mouse. In situ hybridization of Brca1 and
Brca2 speci®cally localized expression to several tissues,
which in most instances, correlated with PCNA
positive staining (not shown). Brca1 and Brca2
expression was widely disseminated in lymph nodes
containing primary follicles and was speci®cally
con®ned to germinal centers of secondary follicles
which contain proliferating B lymphocytes. Brca1 and
Brca2 expression was localized to pancreatic acinar and
islet cells and also correlated well with PCNA labeling
(not shown). RNase protection assays con®rmed Brca1
and Brca2 expression in the pancreas. Expression of
Brca1 and Brca2 was detected in uterine endometrial
glands, the glandular mucosa of the stomach, duodenal
crypt epithelial cells, lymphocytes of the periarterial
sheaths and lymphatic nodules in the spleen, the outer
rim of the thymic cortex which contains proliferating T
lymphocytes, and in epithelial cells in the outer root
sheath of the hair follicles. Staining for Brca1 and
Brca2 transcripts was low in hepatocytes, myocardial
cells, and proximal tubular epithelial cells of the
kidney. The neurons were the only cells that expressed
both Brca1 and Brca2 but the expression pattern was
not correlated with cell proliferation. Low expression
in the brain was con®rmed by RNase protection assay.
Brca1 and Brca2 expression was not detected in lung or
skeletal muscle.
1C
1D
Br
Brca2+
Brca2(negative control)
Brca1+
PCNA
Figure 1 In situ hybridization analysis of Brca1 and Brca2 in a gestational day 14 embryo from a CD-1 mouse. (a) Mid-sagittal
section of formalin-®xed, paran embedded embryo with Brca2 cRNA digoxigenin-labeled antisense (+) probe. Transcript is
visualized as a blue product from alkaline phosphatase-conjugated antidiogoxigenin antibody reacted with NBT-BCIP detection
system. The counterstain is Nuclear Fast Red. (b) Serial section of embryo shown in (a) hybridized with Brca2 sense (7) probe to
serve as a negative control. (c) Brca1 cRNA digoxigenin-labeled antisense probe hybridized to same embryo as in (a) but di€erent
plane of section. (d) Serial section of embryo shown in (c) stained for nuclear PCNA protein and visualized as brown by a DAB
color reaction. Bar=1 mm for 1(a ± d). Symbols: (Br)-brain, (Tg)-trigeminal ganglia, (Ln)-lung, (Lv)-liver
Brca1 and Brca2 expression
PE Blackshear et al
Brca1 and Brca2 expression during mammary gland
ductular morphogenesis in mice
Because the loss of either BRCA1 or BRCA2 gene
function leads to the development of human breast
cancer, we determined the cell type speci®c in vivo
expression pattern of Brca1 and Brca2 during normal
mammmary gland development (Figure 2). During
postnatal ductular proliferation and glandular development during pregnancy, Brca1 and Brca2 mRNA
expression was localized to both epithelial and stromal
cells (®broblasts). Mammary glands from prepubertal,
2-week-old mice consisted of primordial ducts containing few epithelial cells that expressed Brca1 and Brca2,
or that were PCNA immunopositive (not shown).
Mammary glands from pubertal, 5-week-old virgins
consisted of terminal end buds and branching epithelial
ducts and ductules in which Brca1 and Brca2 were
expressed in the epithelial cells of terminal end buds,
ductal epithelial cells, and the immediately adjacent
stromal ®broblasts (Figure 2a). Mammary glands from
sexually mature, virgin mice consisted of minimally
branching ducts in which staining for Brca1 and Brca2
mRNA was detected in few epithelial and stromal cells
(Figure 2b). During early pregnancy to day 13.5 when
Brca2+
Brca2-
lateral alveolar buds were forming, Brca1 and Brca2
were prominently expressed in the epithelial cells and
adjacent mammary stromal ®broblasts (Figure 2c).
However, Brca1 and Brca2 expression was no longer
apparent in adjacent stromal cells at day 14 of
pregnancy (not shown). By gestational day 19 when
mammary alveoli were well developed, the expression
of Brca1 and Brca2 was primarily con®ned to epithelial
cells of the ducts and were present in a few alveolar
epithelial cells (not shown). In lactating mice, the
alveolar epithelial cells were well di€erentiated with
minimal to undetectable expression of Brca1 or Brca2
(Figure 2d). At one week post-weaning while the
alveoli were regressing, there was a notable reexpression of epithelial cell and stromal cell Brca1
and Brca2 transcripts (Figure 2e). These expression
patterns correlated well with PCNA immunopositivity
(Figure 2a ± e).
RNase protection assays using Brca1 and Brca2
probes on total RNA isolated from mammary glands
collected from similar time points showed an increase
in transcript levels of both genes associated with
mammary gland morphogenesis (Figure 3). Mammary
glands from 4 week old virgins were used to determine
the level of expression associated with terminal end
Brca1+
PCNA
2A
5 wk
2B
12 wk
2C
gd 13.5
2D
Ld 9
2E
1 wk reg
Figure 2 In situ hybridization analysis of Brca1 and Brca2 compared with PCNA immunohistochemistry during mammary gland
growth, di€erentiation and regression in the CD-1 mouse. Tissues were hybridized with Brca2 antisense probe (left panel); Brca2
sense probe (middle left panel); Brca1 antisense probe (middle right); or stained for PCNA by immunohistochemistry (right panel).
(a) Terminal endbud in 5-week-old virgin (5 wks). Bar=37mm. (b) Duct from adult nulliparous mouse (12 wks). Bar=37mm. (c)
Beginning alveolar buds, gestational day 13.5 (gd 13.5). Bar=37mm. (d) Lactation day 9 (Ld 9). Bar=37mm. (e) Mammary gland
regression one week post-weaning (1 wk reg). Bar=37mm
63
Brca1 and Brca2 expression
PE Blackshear et al
64
bud formation which occurs in 3 ± 5 week old virgins.
Brca1 and Brca2 expression increased in early
pregnancy, peaked at gestational day 10.5, and
decreased to nearly undetectable levels during lactation associated with alveolar epithelial di€erentiation.
At 1 week post weaning, during mammary gland
regression, expression returned to levels seen in glands
of virgin mice and during early pregnancy. These
expression levels correlated with the intensity of
staining as well as the change in volume of stained
cells associated with cell proliferation as demonstrated
by in situ hybridization (Figures 2 and 3).
4d). The expression pattern from a normal cycling
adult was compared to prepubertal ovaries from 2
week old virgins, the ovaries from hypophysectomized
mice, and ovaries from mice lacking the estrogen
Brca1 and Brca2 expression in mouse ovary
Since Brca1 is expressed independently of hormonal
stimulation in the mouse ovary (Phillips et al., 1997),
we determined whether Brca2 expression was dependent or independent of hormonal stimulation, including estrogen, by comparing expression of Brca2 in
ovaries of cycling mice, hypophysectomized mice, and
in ERKO mice that do not have a normal response to
estrogen. In the normal adult ovary, Brca1 and Brca2
transcripts were localized speci®cally to granulosa cells,
thecal cells and oocytes of developing follicles (Figure
4a ± c) as well as luteal cells of recently formed corpora
lutea (not shown) and surface epithelium. The
expression pattern of Brca1 and Brca2 correlated
closely with PCNA immunohistochemistry (Figure
Brca2+
Brca2-
4A
4B
Figure 3 Relative expression for Brca1 and Brca2 during
mammary growth, di€erentiation and regression as determined
by RNase protection assay. Each data point represents percentage
of Brca1 (^) and Brca2 (*) expression +/- standard error in the
mouse mammary gland from 4 week virgin females (4 wk virgin),
at gestational day 9 (gd 9), gestational day 10.5 (gd 10.5),
gestational day 13 (gd 13), gestational day 19 (gd 19), lactation
day 9 (Ld 9), and 1 week regression (1 wk reg)
Brca1+
4C
PCNA
ERKO/Brca2+
4D
4E
Figure 4 In situ hybridization analysis of Brca1 and Brca2 compared to PCNA positive staining in the ovary from adult CD-1
mouse and ERKO mouse: (a) Tissue was hybridized with Brca2 cRNA digoxigenin-labeled antisense probe. (b) Serial section of
ovary shown in (a) hybridized with Brca2 sense probe to serve as a negative control. (c) Adjacent section of ovary shown in (a)
hybridized with Brca1 cRNA digoxigenin-labeled antisense probe. (d) Adjacent section of ovary shown in (c) stained for PCNA by
immunohistochemistry. (e) Ovary section from an ERKO mouse hybridized with Brca2 cRNA digoxigenin-labeled antisense probe.
Bar=75mm (a ± e). Symbols: (G)-granulosa cells and (T)-theca cells of developing follicles, (P)-primary follicles
Brca1 and Brca2 expression
PE Blackshear et al
receptor (ERKO). Ovaries from the 2-week-old virgins
contained primordial, small and medium sized follicles
with no corpora lutea as expected in the prepubertal
state. Brca1 and Brca2 were expressed in the oocytes,
granulosa cells and thecal cells (not shown) as in the
adult cycling mice. Brca2 was also expressed in
oocytes, thecal cells, and granulosa cells of small
growing follicles in hypophysectomized mice (not
shown). Ovaries from homozygous mutant ERKO
(7/7) mice contained variably sized follicles and
hemorrhagic follicular cysts lined by granulosa cells
but corpora lutea were absent. Brca2 expression again
was localized to the oocytes, granulosa cells of small
and medium sized follicles and thecal cells (Figure 4e).
Expression of Brca1 and Brca2 in ovaries from wildtype ERKO (+/+) animals was indistinguishable from
cycling adult CD-1 mice.
Brca1 and Brca2 expression in mouse testes
The human BRCA1 and mouse Brca2 proteins interact
with the Rad51 protein during mitosis and meiotic
recombination (Scully et al., 1997; Sharan et al., 1997)
and BRCA1 has been shown to be expressed in meiotic
male germ cells (Zabludo€ et al., 1996). We examined
the mouse testes at various stages of development to
speci®cally identify and correlate Brca1 and Brca2
expression in mitotic and meiotic germ cells. Both
transcripts were localized in the spermatogonia and
spermatocytes of meiotic prophase (Figure 5a ± c).
Sertoli cells and Leydig interstitial cells were consistently negative for Brca1 and Brca2 transcripts. Brca1
expression was more readily apparent during the early
stages of spermatogenesis (spermatogonia and preleptotene spermatocytes) than Brca2 in similarly staged
seminiferous tubules. Brca1 and Brca2 expression was
most intense in the late pachytene and diplotene
spermatocytes. In contrast, PCNA positive staining
was prominent in spermatogonia and pre-leptotene
spermatocytes, which comprise the mitotic phase of
germ cell development. In addition, PCNA positive
nuclear staining was prominent in leptotene and
zygotene spermatocytes with minimal to no staining
in early pachytene spermatocytes dependent on the
stage of spermatogenesis for a given seminiferous
tubule (Figure 5d).
In e€ort to evaluate the di€erence in temporal
expression between Brca1 and Brca2 in the testes, we
Brca2+
Brca2-
5B
5A
Brca1+
PCNA
4D
5C
5D
Figure 5 In situ hybridization analysis of Brca1 and Brca2 compared with PCNA immunohistochemistry in testes from adult CD-1
mouse: (a) Tissue was hybridized with Brca2 cRNA digoxigenin-labeled antisense probe. Leptotene spermatocytes (arrow head) and
pachytene spermatocytes (arrow) (b) Serial section of testes shown in (a) hybridized with Brca2 sense probe. (c) Adjacent section of
testes shown in (a) hybridized with Brca1 cRNA digoxigenin-labeled antisense probe. (d) Adjacent section of testes shown in (a)
stained for PCNA. Bar=37mm (a ± d)
65
Brca1 and Brca2 expression
PE Blackshear et al
66
examined the expression of these genes during the ®rst
wave of spermatogenesis in male postnatal mice when
spermatogenesis is synchronous. Northern analysis of
RNA isolated from the testes of mice starting at
postnatal day 8 demonstrated that both genes were
expressed as early as postnatal day 8 when the testes
contains 27% spermatogonia and 73% Sertoli cells
(Bellve et al., 1977). However, the intensity of
expression appeared signi®cantly greater for Brca1
than for Brca2 on postnatal day 8, 10 and 12 (Figure
6). By day 14 when the testes contains 12%
spermatogonia and 36% preleptotene, leptotene, and
zygotene spermatocytes, and 15% of newly emergent
pachytene spermatocytes, both Brca1 and Brca2
expression were signi®cantly up-regulated to a comparable degree. Northern analysis of mixed germ cells,
pachytene spermatocytes, and round spermatids con®rmed the expression of Brca2 in pachytene spermatocytes that contributed to the increased intensity
observed in the testes on postnatal day 14 (not shown).
Discussion
Brca1 and Brca2 mRNAs are expressed in a
concordant cell-type speci®c manner associated with
cell proliferation. Both genes are coordinantly expressed from gestational day 7.5 to day 19 of
embryogenesis, a process which is characterized by
rapid cell growth and morphogenesis. In adulthood,
Brca1 and Brca2 transcripts are localized to tissues that
maintain high intrinsic rates of proliferation such as
the ovary, testes, intestinal crypts, and the mammary
a
Days Post-natal
8 10 12 14 16 18 20 22 24 26 28 30
Brca 1
28S
b
Brca 2
28S
c
Actin
18S
Figure 6 Expression of Brca1 and Brca2 during postnatal
development of mouse testes. Northern analyses using the same
®lter hybridized with probes for Brca1 and Brca2, and b-actin are
shown in panels a, b, and c, respectively. Days of age are shown
above each lane. Fifteen ug of total RNA was loaded per lane.
Exposure times were 12 days for Brca1 and Brca2 and 5 days for
b-actin. Brca1 (a) is expressed at day 8 and continues throughout
the prepubertal period with maximal intensity at day 14 and 16
coincident with appearance of the ®rst wave of pachytene
spermatocytes. Brca2 (b) is only expressed at a low level on day
8 and expression increases to reach a maximum on days 14, 16
and 18 coincident with the appearance of pachytene spermatocytes. Actin expression (c) is shown as an RNA loading control
gland during pregnancy. Our results are similar to that
reported by Conner and co-workers which established
tissue distribution of Brca1 and Brca2 in mouse tissues
by RT ± PCR (Conner et al., 1997), and other in situ
hybridization studies (Marquis et al., 1995; Lane et al.,
1995; Rajan et al., 1996, 1997). Additionally, our
studies demonstrate that the expression patterns of
Brca1 and Brca2 are closely correlated in most tissues
with PCNA nuclear staining, used as a marker for
proliferating cells in S-phase. Exceptions to this
correlation between Brca1 and Brca2 expression and
PCNA positivity are found in the testes and terminally
di€erentiated neurons. Moreover, during the mitotic
phase of spermatogenesis, Brca1 expression appears to
precede Brca2 expression. Thus, our data comparing
Brca1 and Brca2 in the testes provides the ®rst
evidence of temporal di€erences in the expression
pattern of these genes during spermatogenesis.
Spermatogenesis is characterized by waves of mitotic
divisions of the spermatogonia stem cells of which a
subset commits to meiotic divisions to form the haploid
spermatozoa. The stages of the ®rst meiosis occur as a
distinct progression from leptotene through diplotene.
The second meiotic division occurs rapidly in secondary
spermatocytes which in turn give rise to haploid
spermatids which then di€erentiate into spermatozoa.
The initial wave of spermatogenesis is synchronized and
the stages readily distinguishable in the postnatal
mouse. In the adult, the mitotic and meiotic cells can
be distinguished by morphology and cellular association. Thus, the testes proved useful for delineating the
di€erences between Brca1 and Brca2 in proliferating cell
populations. In a previous study Brca1 expression in the
testes was found to be limited to meiotic cells (Zabludo€
et al., 1996). In contrast, our data demonstrates that
Brca1 and Brca2 are expressed in mitotic spermatogonia
in addition to meiotic spermatocytes through late
pachytene and diplotene stages. Perhaps the di€erence
in results are due to the relative sensitivity of the
techniques employed in each study. Most recently in
vivo studies indicate that Brca2 expression is upregulated during spermatogenesis from day 12 onward
in postnatal mice while Brca1 expression remains
relatively constant (Rajan et al., 1997). We ®nd by in
situ hybridization and Northern analysis that Brca1
expression was greater than Brca2 during the mitotic
phase of spermatogenesis, but that both genes had a
comparable increase in intensity during late meiotic
prophase. Therefore, we suggest that Brca1 expression
is up-regulated prior to Brca2 during the mitotic phase
of spermatogenesis. The lag in Brca2 expression in
mitotic cells during spermatogenesis implies that the
regulation of these gene are not identical and that they
may even be interdependent. For example, each gene
may be independently regulated or the induction of
Brca2 may require the initial expression of Brca1 during
spermatogenesis.
The expression patterns of Brca1 and Brca2 during
spermatogenesis also suggests these genes function in
the cell beyond DNA replication, since Brca1 and
Brca2 transcripts are expressed for longer periods than
the PCNA protein. Additionally, Brca1 and Brca2
expression patterns parallel that of the mouse rad51
protein which is expressed in spermatogonia and early
and mid-meiotic prophase spermatocytes (Yamamoto
et al., 1996). Human BRCA1 and mouse Brca2
Brca1 and Brca2 expression
PE Blackshear et al
proteins bind to Rad51 which is thought to be involved
in repair of DNA double strand breaks, and may act to
monitor the process of DNA replication and recombination (Scully et al., 1997; Sharan et al., 1997).
Therefore, our results substantiate a role for Brca1
and Brca2 in meiotic as well as mitotic recombination
and replication.
The expression of Brca1 and Brca2 in the nervous
system suggests a further complexity of function. An
earlier study investigating the tissue distribution of
Brca1 using Northern analysis of adult mouse tissues
reported low but detectable expression in brain (Lane et
al., 1995). However, Rajan and co-workers recently
found only Brca2 is expressed in the adult brain,
embryonic brain and spinal cord, whereas Brca1 is
found in particular areas of embryonic brain but not the
adult brain (Rajan et al., 1997). Our in situ hybridization studies in embryonic and adult brains, and RNase
protection assays of adult brain demonstrate that both
Brca1 and Brca2 transcripts are expressed in neural
tissues. Thus, while Brca1 and Brca2 appear to function
in association with cell proliferation in other tissues, in
the brain these gene products may be involved in
neuronal growth and maintenance perhaps by interaction with proteins that are common to both neurons
and mitotic cells, such as some structural proteins
(Cleveland, 1990; Kuriyama and Nislow, 1992).
Since loss of function of the BRCA1 and BRCA2
genes results in a dramatic predisposition to breast and
ovarian cancer in humans, a central question focuses
on the biology of these tissues and why they are more
susceptible to tumor formation. In the mammary
gland, we have demonstrated that Brca1 and Brca2
transcripts are coordinately expressed and that they are
readily detected in both epithelial and stromal cells
during periods of ductal and glandular proliferation.
Mammary ductal elongation and alveolarization are
dependent on both epithelial and stromal cell
proliferation and epithelial and stromal cell interaction
(Daniel and Silberstein, 1987; Howlett and Bissell,
1993). The localization of Brca1 and Brca2 transcripts
in both epithelial and stromal cell populations during
mammary gland development is consistent with the
hypothesis that both of these cell populations are
involved in the development of mammary cancers
(Smith et al., 1991). Additionally, the temporal
expression of Brca1 and Brca2 during mammary
gland development clearly correlates with ductular
proliferation and morphogenesis. These results are
consistent with in vitro studies that show expression
of BRCA1 and BRCA2 mRNA is regulated by the cell
cycle and that these two genes are induced with similar
kinetics at the G1/S boundary in normal human
mammary epithelial cells and breast cancer cell lines
(Vaughn et al., 1996a,b; Gudas et al., 1996).
Furthermore, recent in vitro and in vivo studies
demonstrate that Brca1 and Brca2 are coordinately
regulated in mouse mammary epithelial cells and the
mammary gland during puberty and pregnancy (Rajan
et al., 1996; 1997) which is consistent with our results.
The presence of signi®cant Brca1 expression in
hormonally responsive tissues such as the testes,
mammary gland, ovary and uterus led to the hypothesis
that Brca1 may be hormonally regulated (Marquis et al.,
1995). Moreover, Brca1 and Brca2 may be regulated
di€erently by sex hormones (Rajan et al., 1997).
However, the expression of Brca2, like Brca1 (Phillips
et al., 1997), is expressed in the ovary independently of
various hormonal stimulations. The similar pattern of
expression of both genes suggests that their expression is
closely related to the cell cycle. In vitro studies have also
demonstrated that BRCA1 expression is up-regulated
due to mitogenic e€ects of several hormones such as
estrogen, rather than a direct interaction with the gene
(Marks et al., 1997; Gudas et al., 1995).
In summary, we show that Brca2, like Brca1, is
widely expressed in the embryo and a variety of adult
tissues associated with the cell cycle. We also
demonstrate that Brca1 and Brca2 have similar in
vivo, hormone independent-patterns of cell speci®c
expression during normal growth and di€erentiation
in the ovary. Moreover, Brca1 and Brca2 are expressed
beyond the mitotic phase of spermatogenesis during
meiotic recombination which is consistent with
proposed functions for these breast cancer susceptibility genes related to maintaining genome integrity.
Although Brca1 and Brca2 appear to be coordinately
regulated in many tissues, the temporal divergence in
expression between Brca1 and Brca2 in the testes
suggests that the regulatory pathway for these two
genes may not be identical.
Materials and methods
Animals
Young virgin and timed pregnant CD-1 mice were obtained
from Charles River (Raleigh, NC). The morning of a
vaginal plug was counted as day 0.5 post-coitus. The
following tissues were frozen or ®xed for 24 h in 10%
neutral bu€ered formalin and then embedded in paran:
fetuses (from day 7.5, 9, 10.5, 13, 13.5, 14, 14.5, 19 postcoitus): the fourth mammary gland (from day 7.5, 9, 10.5,
13, 13.5, 14, 14.5, 19 post-coitus; lactation day 9; and 2-day,
1-week and 4-weeks post weaning; and from 2-week old, 5week old, 12-week old, and 16-week old virgins); ovary,
uterus, liver, spleen, kidney, stomach, small and large
intestine, pancreas, heart, lung, brain and skeletal muscle.
RNA probe preparation
Brca1 and Brca2 cRNA probes were generated from a 143
base pair cDNA fragment containing exon 7 of the murine
Brca1 gene (nucleotides 418 ± 560, Accession # U32446) and
a 163 base pair cDNA fragment from exon 11 of the murine
Brca2 gene (nucleotides 3283 ± 3445, Accession # U89652).
These PCR products were subcloned into the EcoRV site of
pBluescript1 (Stratagene, La Jolla, CA). The resulting
Brca1 plasmid was linearized by digestion with either
BamHI or NotI, and the Brca2 plasmid was linearized
with either HindIII or BamHI (New England Biolabs).
RNase protection assays were performed on mouse testis
and mammary gland tissues as previously described
(McAllister et al., 1997). RNase protection analysis of
mammary gland was performed with pooled mammary
gland tissue from one to six mice to obtain 40 mg of total
RNA for each time point. The percent relative expression for
Brca1 and Brca2 was obtained by taking the raw counts from
two samples of pooled total RNA for each data point and
normalizing their value relative to the highest expression at
gestation day (gd) 10.5 and then averaged. The relative
percent for gd 10.5 is set at 100%.
The digoxigenin-labeled probes were prepared as previously described (Phillips et al., 1997). For in situ
hybridization, the tissue sections were de-paranized,
67
Brca1 and Brca2 expression
PE Blackshear et al
68
treated with 0.2 M HCL, 0.3% Triton-X 100, proteinase K
and acetylated. The fetuses were treated with 20mg/ml
proteinase K for 5 min at 378C; all other tissues were
treated with 10 mg/ml for 12 min at 378C. The RNA probes
were hybridized overnight at 558C for mammary glands and
fetuses and 488C for all other tissues. The hybridized probe
was detected using an alkaline phosphatase-conjugated
antidigoxigenin as previously described (Phillips et al., 1997).
To determine relative expression of transcripts during
embryonic development, staining intensity for speci®c
transcripts was compared for each organ or anatomical
region within the same slide from at least three separate
experiments and scored by two pathologists (PEB and BJD) as
high, moderate, low, or no signal detected. Localization of
Brca1 and Brca2 expression during mammary gland development and in adult tissues was determined by identi®cation of
signal from at least three separate experiments and compared
to appropriate, concurrently run negative controls.
The proliferating cell nuclear antigen (PCNA) protein was
detected by immunohistochemistry with 3,3'-diaminobenzidine
(DAB) as previously described (Foley et al., 1993) in adjacent
sections of tissues stained for Brca1 or Brca2. The dilution of
primary antibody was 1 : 200 and secondary antibody was
1 : 800. PCNA immunopositivity was determined by visualizing the number of cells with dark brown to black nuclear Sphase staining per tissue area in adjacent sections.
Northern analysis
The testes of prepubertal CD-1 mice from Charles River
(Raleigh, NC) were collected on post-natal days 8 ± 30 and
frozen in liquid nitrogen. Mixed spermatogenic cells, pachytene
spermatocytes (91.5%), and round spermatids (92%) were
isolated from adult CD-1 mice as described previously (Romrell
et al., 1976; Bellve et al., 1977; O'Brien et al., 1989). Total RNA
was isolated using Trizol reagent (Life Technologies, Grand
Island, NY). RNA samples were separated on a 1.2% agarose/
6.6% formaldehyde gel and transferred by capillary action to
Genescreen membranes (New England Nuclear Research
Products, Boston, MA) according to the manufacturer's
instructions. Northern blotting was performed as described
previously (Welch et al., 1995).
Acknowledgements
The authors wish to thank E Mitch Eddy of the National
Institute of Environmental Health Sciences (NIEHS) for
his expert advice and the donation of mouse testes RNA;
Debbrah A O'Brien of the University of North Carolina at
Chapel Hill for her gift of isolated germ cells; Kenneth S
Korach and John F Couse of NIEHS for the ERKO ovary
sections; Gina Goulding, Cindy R Moomaw and Norris
Flagler for their excellent technical assistance.
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