Oncogene (2002) 21, 1009 ± 1016
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Expression of the PAX2 oncogene in human breast cancer and its role in
progesterone-dependent mammary growth
Gary B Silberstein*,1, Gregory R Dressler2 and Katharine Van Horn1
1
Department of Molecular, Cell and Developmental Biology, Sinsheimer Laboratories, University of California, Santa Cruz,
California, CA 95064, USA; 2Department of Pathology, University of Michigan, Ann Arbor, Michigan, MI 48109, USA
In this study, we ®rst describe expression of the paired
domain transcription factor PAX2 in the normal and
cancerous human breast, then demonstrate in a murine
model a novel function for PAX2 in the regulation of
progesterone stimulation of secondary ductal growth. In
human mammary tissue, PAX2 expression was coincident with sub-populations of mammary ductal cells,
some of which possessed an undi€erentiated histiotype,
and was also found in 450% of the human breast
tumors surveyed (n=38). In the mouse, mammary
parenchyma with a targeted deletion of PAX2 developed
normal ductal systems when grafted into wild-type host
mammary fat pads, but failed to undergo higher order
side-branching and lobular development in response to
progesterone. A previously unsuspected PAX2/WT1
(Wilms' tumor suppressor gene) regulatory axis in the
mammary gland was also indicated. Using RT ± PCR, a
signi®cant reduction in WT1 mRNA expression was
detected in the PAX2 mutant glands compared to wildtype counterparts and double-antibody immunohistochemistry detected the co-localization of PAX2 and
WT1 in the nuclei of normal and cancerous breast cells.
These data indicate a role for PAX2 (and possibly WT1)
in the regulation of the progesterone response of the
mature mammary gland. The potential contribution of
PAX2 to breast tumor pathogenesis is discussed.
Oncogene (2002) 21, 1009 ± 1016. DOI: 10.1038/sj/onc/
1205172
Keywords: breast cancer; mammary; PAX2; progesterone; Wilms' tumor
known to regulate several mammotrophic growth factor
pathways, including insulin-like growth factor, epidermal growth factor, and transforming growth factor beta
(Dey et al., 1994; Drummond et al., 1992; Vicanek et al.,
1997; Werner et al., 1993). WT1 is also expressed in the
kidney, where it is positively regulated by the paireddomain transcription factor PAX2 and is required for
the metanephric mesenchyme to respond to signals
inducing cell proliferation (Dehbi et al., 1996; Kreidberg
et al., 1993). During embryonic kidney development
PAX2 expression is transient whereas persistent expression of the gene in Wilms' kidney tumors (Dressler and
Douglass, 1992), renal cell carcinoma (Gnarra and
Dressler, 1995), and polycystic kidneys (Ostrom et al.,
2000) suggests multiple roles in maintaining the dedi€erentiated or proliferative state and preventing apoptosis
of the renal epithelium. Deregulated expression of PAX2
can transform cells in vitro and promote tumor
formation in nude mice, indicating a direct role for
PAX2 in tumorigenesis (Maulbecker and Gruss, 1993;
Stuart and Gruss, 1996). PAX2 expression in the
mammary gland and its potential relationship with
WT1 in that tissue has not been previously investigated,
in part due to the death of PAX2 mutant mice within the
®rst hours after birth. Here we report the unexpected
expression of PAX2 in normal human breast tissue, as
well as in a high percentage of breast tumors. To begin to
understand a functional role, we have rescued the
mammary anlagen from PAX2-null mouse embryos to
wild-type host mammary glands and now report that
PAX2 is necessary for progesterone-dependent growth
of lateral ductal branches.
Introduction
Previously, we demonstrated that the WT1 Wilms'
tumor suppressor gene was expressed in normal human
breast tissue as well as in a high percentage of breast
tumors (Silberstein et al., 1997). The WT1 protein may
act as an activator or suppressor of transcription and is
*Correspondence: GB Silberstein, Sinsheimer Laboratories,
University of California, Santa Cruz, CA 95064, USA;
E-mail: [email protected]
Received 11 October 2001; revised 5 November 2001; accepted 7
November 2001
Results
Expression of PAX2 in the normal and cancerous human
breast
The expression of PAX2 in the human breast was
investigated by immunohistochemistry and in situ
hybridization. Using a polyclonal anti-PAX2 antibody
(Dressler and Douglass, 1992), nuclear staining was
detected in cells from the two primary developmental
lineages comprising myoepithelial and luminal cells in
alveolar (Figure 1a) as well as ductal (Figure 1b)
elements of the tissue. These cells are arranged in two
PAX2 mammary expression and function
GB Silberstein et al
1010
Figure 1 PAX2 expression in normal and cancerous human mammary tissue. The nuclei of immuno-positive cells stained brown;
counter stain: methyl green. No counter stain was used for the in situ hybridization, (a) Lobule cluster (cross- and grazing sections).
PAX2 positive cells with oval as well as polygonal nuclear morphology (arrows within the lumens, L) lined the lobule lumens and
were often adjacent to unstained cells. Immuno-positive myoepithelial cells (arrows on periphery of lobules) and immuno-negative
counterparts (arrowheads) were also detected (Bar=20 mm). (b) Duct (longitudinal section of one side of a duct; high
magni®cation). The arrangement of PAX2 immuno-positive and -negative cells mimicked pattern seen in lobules with strongly
staining luminal cells often adjacent to non-staining cells (boxes). Myoepithelial cells (outside dotted line) stained lighter than ductal
cells (small arrow) or were negative for PAX2 (arrowhead). (Bar=5 mm). (c) In situ hybridization localization of PAX2 mRNA in
normal mammary duct (small arrow) and side-branch (large arrow). Control hybridization with sense-strand probe (inset).
(Bar=15 mm). (d) Ductal carcinoma in situ. PAX2 positive cells (arrows); PAX2 negative cells (arrowheads). (Bar=20 mm). Inset:
PAX2 mRNA in situ hybridization. Cells expressing PAX2 mRNA (arrows) surround PAX2 non-expressing cells (arrowheads).
(Bar=25 mm). (e) In®ltrating ductal carcinoma. In®ltrating tumor cells had large, anaplastic nuclei (large arrows) or small nuclei
with compact chromatin (small arrows); both populations were negative for PAX2 protein. Inset: PAX2 positive cells (arrows) in
vicinity of main tumor (Bar=20 mm)
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PAX2 mammary expression and function
GB Silberstein et al
circular layers and form a tube-within-a-tube. The
innermost cells line the lumen, forming the ductal or
acinar tube proper, and give rise to secretory structures
at pregnancy, while the myoepithelium forms the
epithelial boundary with the stroma and is comprised
of contractile cells that facilitate milk extrusion
(Silberstein, 2001).
The histiotype of certain PAX2 positive cells with
large, oval nuclei and di€use chromatin (Figure 1b)
was consistent with that of putative mammary stem
cells (Smith and Medina, 1988; Stingl et al., 1998;
Toker, 1967; Williams and Daniel, 1983). PAX2
positive luminal cells with this nuclear morphology
coexisted with cells not ®tting this histiotype (Figure
1a). The not-infrequent side-by-side arrangement of
PAX2 positive and negative cells (Figure 1b, boxes)
may have functional signi®cance as paracrine signaling
is known to be important in progesterone-dependent
lateral branching (Brisken et al., 1998, 2000). Myoepithelial cells also exhibited a mixed, PAX2 positive
and negative staining pattern (Figure 1a,b), with some
myoepithelial cells having less immunoreactivity than
adjacent luminal cells (Figure 1b). The localization of
PAX2 mRNA in mammary tissue was detected by in
situ hybridization and re¯ected PAX2 immuno-staining. In the pictured section of a branched duct PAX2,
for example, PAX2 mRNA was found throughout the
structure, with possibly higher amounts in the tips of
developing branches (Figure 1c). No signal was seen
with a sense probe (Figure 1c, inset).
Once we established the expression of PAX2 in the
normal breast, mammary tumors were examined.
Carcinoma in situ is one of the earliest histologically
identi®able stages in breast tumor progression, in
which proliferating cells occlude the lumen of a duct
(Figure 1d). In this example, the tumor cell mass
consists primarily of PAX2 positive cells. The pattern
of PAX2 mRNA expression re¯ected the immunohistochemistry, with small groupings of cells exhibiting
little or no PAX2 mRNA interspersed with cells
expressing high levels (Figure 1d, inset). An in®ltrating
ductal carcinoma (Figure 1e) consisted of at least two
cell populations, one with large, anaplastic nuclei, the
second with smaller nuclei that were also abnormal;
both cell types were PAX2 negative. A nearby, possibly
less advanced element of this tumor, contained a
mixture of PAX2 negative and positive cells (inset
Figure 1e).
In a more comprehensive survey, mammary carcinomas from 38 patients were grouped based on
whether specimens had high or low numbers of
PAX2 positive epithelial cells (Table 1). All histopathological types and grades of tumor were represented, and patients ranged in age from 19 to 88 years.
PAX2 positive tumors were observed in 53% of these
cases, with ®ve tumors having more or less equally
mixed populations of PAX2 negative and positive cells.
No correlation was noted between PAX2 staining and
ductal versus lobular tumors. Pathological grading and
estrogen/progesterone receptor expression status was
available for a subset of the tumors (Table 2). Lower
Table 1 Survey of PAX2 protein expression in normal and
cancerous breast tissue
Tissue
Tumor
Vicinal d
PAX2 positive a
PAX2 negative b
PAX2-neg/pos c
40% (15/38)
20
47% (18/38)
1
13% (5/38)
-
1011
a
Tumors with 80% or more of cells immunopositive for PAX2,
estimated by inspection. bPAX2 immunostaining was absent in
greater than 90% of the tumor cells. cTumors with an approximately
equal mixture of PAX2 negative and positive cells. dTissue that was
excised from the vicinity of a tumor, contained normal-appearing
structures and cells that exhibited ductal and lobular PAX2 staining
patterns as in Figure 1a,b. Tumors studied: n = 38; classes
represented: 27 in®ltrating ductal carcinomas, seven in®ltrating
lobular carcinomas, medullary, colloid and adenocarcinoma, one
each, and one unclassi®ed
grade and steroid receptor positive tumors were
roughly equally divided between PAX2 positive and
negative tumors. High grade and receptor negative
tumors were more likely to be PAX2 negative; 86% of
Grade 3 tumors and 75% of receptor negative tumors
were PAX2 negative. WT1 and PAX2 expression were
strongly correlated; 72% of PAX2 positive tumors
were WT1 positive, while 85% of WT1 negative
tumors were also PAX2 negative. This study added
18 more cases to the patient population originally
screened for WT1 expression (Silberstein et al., 1997).
All 18 cases showed some degree of WT1 staining,
supporting recent ®ndings that a much higher
percentage of breast tumors are WT1-positive than
was originally reported (Loeb et al., 2001).
Transplantation rescue and progesterone insensitivity of
PAX2-null mammary glands
To investigate the function of PAX2 in the mammary
gland, we utilized the PAX2 null allele generated by
homologous recombination. Mice homozygous for the
PAX2 null allele die post-natally due to exencephaly
and renal insuciency (Torres et al., 1996). PAX2 null
mammary anlagen were therefore rescued by grafting
into wild-type mammary fat pads in immune compromized mice. PAX2 null mammary outgrowths were
then divided and re-transplanted into multiple hosts for
detailed analysis. In order to investigate the e€ects of
PAX2 ablation on mammary growth and gene
expression, fragments of PAX2 null and homotypic
wild-type mammary tissue were simultaneously transplanted into contralateral mammary glands of individual hosts. This bilateral transplant experimental
design ensures that mutant and control tissues are
exposed to identical endocrinological and physiological
backgrounds. Transplants were allowed 12 weeks to
establish and grow, after which animals were treated
with progesterone and estrogen for 7 days prior to
sacri®ce.
Progesterone, but not estrogen, stimulates the
growth of the higher order, lateral branching that is
commonly associated with pregnancy in the mouse
(Atwood et al., 2000). When PAX2 null mammary
glands were stimulated with a mixture of progesterone
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PAX2 mammary expression and function
GB Silberstein et al
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Table 2
PAX2
Positive
Negative
PAX2 expression in breast tumors relative to tumor grade, steroid hormone receptor status, and WT1 expression
Pathological Grade a
1±2
3
57% (12/21)
43 (9/21)
14 (1/7)
86 (6/7)
+
ER/PR Status b
47% (7/15)
53 (8/15)
7
+
25 (2/8)
75 (6/8)
72% (18/25)
28 (7/23)
WT1 c
7
15 (2/13)
85 (11/13)
a
28 tumors were screened for pathological status; the criteria for immuno-positive and -negative status is de®ned in Table 1, footnote. bER,
estrogen receptor; PR, progesterone receptor: 23 tumors were screened for estrogen and progesterone receptor status. Concordance of Grade 3 and
receptor-negative status was 100% (®ve out of ®ve of the tumors that comprised an overlapping set, e.g. were scored for each condition.) cWT1
immuno-stained tumors. Tissue sections were cut from the same paran-embedded specimens that were used for the PAX2 study (Table 1)
and estrogen the loss of PAX2 function resulted in
stunted to non-existent lateral branching and poor
lobular development, evidenced by the open architecture of the mutant gland (Figure 2a) compared to its
wild-type counterpart (Figure 2b). In contrast to e€ects
on lateral branching and lobular development, growth
of the primary ductal framework was una€ected;
mutant and wild-type ducts grew the full length and
breadth of the fat mammary fat pad. This point was
con®rmed by examination of transplants after 7 weeks,
at which time it was noted that mutant and wild-type
ducts had extended an identical distance from their
origin and had similar numbers of end buds (ductal
growth points) of normal histo-morphology (not
shown).
Examination of a€ected glands under higher magni®cation revealed that duct tips in the mutant tissue had
failed to branch into the fatty stroma (compare boxed
zones in Figures 2c,d). In addition, the larger ducts had
numerous, compact outgrowths (Figure 2c, large
arrows) comprised of epithelial cells lining a lumen
(Figure 2e). While not overtly dysplastic, these
structures were unusual. The tips of PAX2 null ducts
failed to undergo normal branching development
(compare Figure 2c and d, small arrows). This was
con®rmed at the histological level, where extensive
normal branching at duct termini (Figure 2g, top)
contrasted with simpli®ed termini in the mutant tissue
(Figure 2f). The accretion of ®brous stroma around the
mutant ducts and smaller branches was similar to that
seen in the wild-type tissue. It should be noted that
some normal-appearing lobules were found in mutant
tissue, usually in the vicinity of the transplant origin.
This probably re¯ects known regional di€erences in
ductal sensitivity to hormones, in which the most
di€erentiated ducts (in the vicinity of the nipple in
intact glands) require the least complex mixture of
hormonal and growth factor stimulation (unpublished
observations and DuBois and Elias, 1984). In a second
host strain (Balb/c, nu/nu), the PAX2 null phenotype
was sometimes less pronounced indicating that stromal
and physiological background play a role.
PAX2 and WT1 expression are coordinated and
co-localize in human mammary cells
The PAX2 null status of mammary tissue derived from
a rescued anlage was con®rmed by RT ± PCR using
RNA isolated from bilaterally transplanted mutant and
wild-type mammary glands (Figure 3a). Since mutant
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epithelium grew into wild-type stroma the absence of
an ampli®cation product also means that the bulk of
PAX2 mRNA is epithelial and not stromal in origin. In
the PAX2 null glands, there was a signi®cant reduction
in WT1 mRNA apparently con®rming the predicted
positive regulatory relationship between the two genes
(Figure 3a). PCR primers spanning the alternative
splice site in the WT1 regulatory domain (exon 5) were
used and demonstrated that expression of both the plus
and minus exon 5 isoforms were a€ected by PAX2.
Similar results were seen with primers spanning a splice
donor site (KTS) in the DNA binding domain (data
not shown). Starting RNA concentrations were
approximately equivalent as indicated by b-actin
expression (Figure 3c).
The relationship between PAX2 and WT1 was
further investigated by double-antibody immuno-¯uorescence. In the normal duct, PAX2 and WT1 coexpression was detected in luminal and myoepithelial
cells (Figure 3c,d), and in tumors, the nuclei of
numerous in®ltrating cells contained both proteins
(Figure 3e,f). As noted earlier (Table 2), PAX2 and
WT1 expression were correlated in a high percentage of
breast tumors. While suggestive, this positive correlation does not comment on same-cell expression because
for the tumor survey immunostaining for each protein
was examined in separate tissue sections.
Discussion
A function for PAX2 in the mammary gland had not
been surmised in the previous investigations of gene
expression patterns or mutant analyses. The early
death of PAX2 mutant mouse newborns precludes
any phenotypic characterization of mammary development directly. Once expression of PAX2 in the normal
human mammary gland and in a high proportion of
human breast cancers had been established (Figure 1,
Table 1), we investigated the PAX2 mammary
phenotype by grafting PAX2 null embryonic glands
into normal hosts. Whereas PAX2 mutant mammary
epithelium underwent normal ductal development, as
indicated by elongation and branching that was
comparable to the wild-type, the injection of progesterone with estrogen failed to stimulate typical secondary
branching and lobular growth (Figure 2). The mutant
phenotype is consistent with higher levels of PAX2
expression in and around the branch points (Figure
1b), and indicates an important function for PAX2 in
PAX2 mammary expression and function
GB Silberstein et al
1013
Figure 2 E€ects of the PAX2 null mutation on mouse mammary ductal and lobular growth. Contralaterally transplanted mutant
and wild-type glands were taken from a single, bilaterally transplanted RAG1 mouse that had been treated with progesterone and
estrogen for 7 days prior to sacri®ce. Whole-mount images of PAX2 null (a) and wild-type (b) mammary glands. Lateral (secondary)
branching and lobular growth was considerably reduced in the mutant gland. Note that both the mutant and wild-type primary
ducts ®lled the available fat pad. The mutant gland exhibited regional variation in secondary growth with some lobular growth
nearest the site of transplantation (left side of image out of the picture; the direction of growth is from left to right as pictured).
(Bar=2.5 mm). Details of branching and lobular growth in mutant (c) and wild-type (d) glands. The in-®lling of available fatty
stroma by lateral branches and lobules was attenuated in the mutant (boxes highlight reduced lateral in-®lling in similar spaces in
the mutant versus the wild-type gland. The ends of mutant ducts exhibited truncated branching compared with equivalent wild-type
structures (small arrows) and unusual, compact outgrowths appeared on mutant ducts (c, large arrows). (Bar=625 mm). Histomorphology of mutant (e, f) and wild-type (g) ducts. Mutant glands exhibited numerous compact outgrowths that arose from larger
ducts (large arrows). The ends of wild-type ducts typically exhibit extensive tertiary branching (g, top half of image) that was absent
in the mutant (f, small arrow). Stromal investment of mutant outgrowths (e, small arrow) compared to wild-type (g, small arrow).
(Bar=200 mm)
mediating the progesterone response in the mature
mammary gland.
The inhibition of side branching in PAX2 mutant
mammary glands was similar to that seen in mammary
glands in which the progesterone receptor or wnt-4 had
been genetically ablated (Brisken et al., 1998, 2000).
Wnt-4 is a secreted glycoprotein that was recently
shown to act under progesterone stimulation in a
paracrine mode driving ductal cells into secretory
di€erentiation (Brisken et al., 1998, 2000). In the
developing kidney, PAX2 appears to act upstream of
wnt-4 and low levels of PAX2 can be detected in wnt-4
mutants, indicating a regulatory relationship (Dressler,
2002; Stark et al., 1994). Alternative explanations for
the mutant phenotype include possible e€ects on
mammary stem cell survival and faulty induction of
the progesterone receptor. In other developing systems
the loss of PAX2 leads to stem cell death and
subsequent agenesis of the developing tissue (Brunner
et al., 1999; Ostrom et al., 2000; Porteous et al., 2000).
If some PAX2 positive ductal cells are undi€erentiated,
secretory progenitors (Figure 1a), their absence in the
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PAX2 mammary expression and function
GB Silberstein et al
1014
Figure 3 Expression of PAX2 and WT1 in normal or tumorous mammary tissue. RT ± PCR analysis of PAX2, WT1, and b actin
gene expression in PAX2 null and wild-type mouse mammary glands. (a) PAX2 and WT1. No ampli®cation signal was detected for
PAX2 in the mutant mammary gland (KO) after two rounds of PCR with nested primers whereas a signal of predicted molecular
size was detected in the wild-type (Cont). Ampli®cation of WT1 was reduced in KO compared with wild-type preparations for each
of the exon 5 alternative splice isoforms. (b) b-actin. Ampli®cation products of the predicted size for the b-actin primers were
detected mutant and wild-type preparations in approximately equivalent concentrations over a 10-fold dilution range. (c ± f) Double
immuno-¯uorescence detection of PAX2 and WT1 expression in normal and tumorous human breast tissue: PAX2, (c and e); WT1,
(d and f), (c and d) normal duct with myoepithelial cells, to the right of dotted line, and luminal cells expressing both PAX2 and
WT1 (arrows); cell expressing PAX2 only (arrowhead, c); L, lumen. (e and f) in®ltrating ductal carcinoma cells strongly expressing
both PAX2 and WT1 (arrows). (Bar=20 mm)
mutant could be expected to impede progesteronedependent development of lateral branching. If PAX2
is necessary for normal mammary tissue estrogen
responsiveness, then it is also possible that the loss of
PAX2 could result in a failure of estrogen to properly
induce the progesterone receptor thereby accounting
for the PAX2 null phenotype.
A role for PAX2 in the pathogenesis and possibly
the ontogeny of breast cancer is also suggested by our
studies. As seen in polycystic kidney disease, the
persistent (deregulated) expression of PAX2 contributes to pathogenesis by the combined mechanisms of
preventing di€erentiation and interfering with apoptosis (Ostrom et al., 2000). Apoptosis is an important
aspect of breast cancer pathogenesis that presents a
complex and somewhat counter-intuitive picture. The
highest rates are seen in the most aggressive, highly
proliferative tumors, which were mainly PAX2 negative, while lower grade tumors, which are frequently
PAX2 positive (Table 2), have relatively low rates
(Lipponen, 1999). If PAX2 does a€ect mammary
apoptosis, its expression could contribute to the
pathogenesis of some breast tumors by blocking
apoptosis early in progression or, by its absence during
later stages, it could favor the selection of apoptosisresistant cells (Zhang et al., 1998). Thus, PAX2
expression in breast tumors may contribute not only
to proliferation, but also to cell survival by preventing
di€erentiation and apoptosis. Given its persistent
expression in a high percentage of tumors, a role for
PAX2 in tumor resistance to certain chemotherapies
that depend for their e€ect on the stimulation of
apoptosis is certainly worth investigating.
Oncogene
The expression patterns of PAX2 and WT1 showed
a high degree of coincidence in the normal mammary
gland as well as in tumors, consistent with, but not
proving, a regulatory relationship between the two
genes. PAX2 expression was seen in a relatively high
percentage of tumors (Table 1) and of these, over 70%
also expressed WT1 (Table 2). If these tumors
represent clonal expansions of PAX2 or WT1-positive
ductal cells, this would indicate the special vulnerability
of these cells to transformation. PAX2 null glands
showed a marked reduction of WT1 mRNA expression
that is consistent with a relationship that was
previously established in the kidney (Figure 3a). Twoantibody immunostaining demonstrated co-localization
of PAX2 and WT1 in the nuclei of luminal and
myoepithelial cells of the normal duct as well as in cells
of an in®ltrating breast tumor supporting a potential
direct regulatory relationship between the two genes in
the mammary gland (Figure 3b). Alternatively, lower
WT1 mRNA levels (Figure 3a) could also be due to
selective death of PAX2/WT1 double-positive cells,
leaving only the WT1 single-positive cells remaining in
the mutant gland.
The e€ect of progesterone on normal and neoplastic
mammary epithelial cells is paradoxical. During
pregnancy, for example, the hormone simultaneously
stimulates cell proliferation and lobulo-alveolar di€erentiation and, depending on dosage and cell type, it
can either stimulate or inhibit mammary tumor cell
proliferation. Recent models now suggest that progesterone modulates a switching mechanism(s) that acts,
in part, through nuclear transcription factors to control
the activity of key genes involved in breast cell fate
PAX2 mammary expression and function
GB Silberstein et al
(Lange et al., 1999). While the elements of this system
are coming into focus, the transcription factor(s) that
mediate the switching are unknown. In light of its role
in cell fate determination in other systems and its
dramatic e€ect on progesterone responsiveness, we
suggest that PAX2 is a candidate to modulate fate
switching in progesterone-responsive mammary cells
and thus merits further investigation.
Materials and methods
Human mammary tissue
Specimens of ®xed, sectioned breast tumors were provided by
the University of Michigan Breast Cell/Tissue Bank, where
histological grading and steroid hormone receptor status were
determined. Additional specimens used for immunohistochemical or in situ hybridization analysis were obtained
directly after surgical excision, transferred immediately to
chilled (48C) 4% paraformaldehyde in phosphate-bu€ered
saline (PBS) and ®xed for 3 h. Histological and steroid
receptor typing of some of these specimens was performed by
Dr KR O'Keefe, Dominican Hospital, Santa Cruz, CA,
USA. Pathological grading and steroid hormone receptor
status were available only for a subset of samples.
Rescue and clonal expansion of PAX2-homozygous recessive
(null) mouse mammary anlagen
E17 embryos from PAX2 heterozygote crosses in mice were
removed from the uterus and PAX2 homozygous recessive
mutants identi®ed by the characteristic exencephaly of the
hindbrain and con®rmed by genotyping (Torres et al., 1996).
Dead mutant and companion embryos were then ¯own
overnight in ice-cold DMEM from the Dressler laboratory to
Santa Cruz. PAX2 null and wild-type mammary anlage were
surgically removed from the embryos and, without culturing,
transplanted contralaterally into the inguinal fat pads of
immune-ablated, Balb/C nu/nu mice, whose endogenous
mammary parenchyma had previously been surgically excised
(Robinson et al., 2000). To guarantee complete `clearing' of
the host parenchyma, each of the excised fragments was
stained and examined in whole-mount to ensure that it
contained the host parenchyma in its entirety. To determine
transplant success, anlage were given approximately 4 weeks
to grow, after which fragments of each gland were removed
without sacri®cing the host and examined in whole-mount.
Three independent sets of mutant and control transplants
were established, and one set was selected for clonal
expansion by transplantation into RAG1 immune-ablated
mice.
Induction of ductal side-branching and lobular development
Transplants were allowed to grow for at least 6 weeks. To
stimulate branching and lobular development animals were
then treated with estrogen and progesterone (5 mg Depoestradiol in cottonseed oil plus 5 mg Depo-provera;
Pharmacia/Upjohn, Kalamazoo, MI, USA) for 7 days via
a long-acting, subcutaneous injection. To investigate
morphology, glands were stained with hematoxylin for
whole-mount analysis (Silberstein and Daniel, 1982),
photographed, then sectioned at 7 mm and restained with
hematoxylin and eosin.
1015
Immunohistochemistry
Using standard procedures, formalin-®xed, paran-embedded tissue was sectioned at 7 mm, deparanized, and
sections incubated with a rabbit anti-PAX2 polyclonal
antibody (PRB-276P, Covance/BAbCo, Berkeley, CA, USA)
at a 1 : 50 ± 75 dilution in PBS. WT1 single-antibody
immunohistochemistry has been described (Silberstein et al.,
1997). A biotin ± avidin secondary antibody system (Vector
Laboratories, Burlingame, CA, USA) was used to produce
the brown detection product.
For double-antibody immunostaining, sections were incubated overnight in a mixture of the PAX2 polyclonal
antibody and a WT1 monoclonal antibody, the latter at a
1 : 25 dilution (PharMingen, San Diego, CA, USA; clone
2C12). Fluorescein-conjugated avidin was used in conjunction
with biotinylated goat anti-mouse secondary antibody to
detect WT1. Vector red dye (Vector Labs, Burlingame, CA,
USA) was used according to the manufacturer's protocol in
conjunction with biotinylated goat-anti rabbit secondary
antibody to detect PAX2. A Leitz Aristoplan microscope
interfaced with a CCD-array camera was used to capture
¯uorescent images.
In situ hybridization
A detailed protocol for in situ hybridization to mammary tissue
appears elsewhere (Silberstein et al., 1996). Digoxigenin-labeled
anti-sense and sense-strand riboprobes were generated from a
527-bp BamHI ± EcoRI fragment of cDNA PAX2 clone c31A
(Dressler et al., 1990) using 10X NTP labeling mixture
(Boehringer-Mannheim, Indianapolis, IN, USA).
RNA preparation and RT ± PCR analysis
The isolation of mammary gland RNA, reverse transcription,
and PCR analysis was as described (Silberstein et al., 1997).
RNA amounts were estimated by spectrophotometry and
concentrations were equalized by dilution before ampli®cation.
PAX2 expression was analysed with primers designed to
amplify mouse or human cDNA. To detect low message levels,
nested primer sets were used in two rounds of ampli®cation.
One-sixth (3 ml) of the puri®ed DNA/RNA volume was
subjected to ®rst round PCR ampli®cation (948C for 1 min,
588C for 2 min and 728C for 3 min for 30 cycles) using Taq
polymerase (Fisher Scienti®c) in a 20 ml reaction volume in a
Perkin-Elmer model 9600 thermal cycler. For the second
round, a 1 : 10 dilution of the ®rst-round reaction mixture was
made in a fresh reaction mixture containing the second set of
primers and ampli®ed for 30 cycles (948C for 1 min, 618C for
2 min and 728C for 4 min). Primers for the initial ampli®cation
were: (forward) 5'-CGACAGAACCCGACTATGTTCG-3';
(reverse) 5'-TGAATCTCCAAGCCTCRTTGTAG-3'. The internal primers ¯anked the exon 10 alternative splice site and
were (forward): 5'-GACC/TGGTCGTGAC/TATGA/GCGA3'; (reverse): 5'-TGTAC/TGGGTTGCCG/TGAGAACT-3'.
For b-actin ampli®cation (948C for 1 min followed by 35
cycles of 948C, 558C and 728C for 1 min each concluding with a
10 min extension at 728C), primers were (forward): 5'GCTGGTCGTCGACAACGGCT-3'; (reverse): 5'-ATGACCTGGCCGTCAGGC-3'.
Acknowledgments
We thank Drs J Snyder and KR O'Keefe (Dominican
Hospital, Santa Cruz); Mr D Albritton, Ms P Huerta and
Oncogene
PAX2 mammary expression and function
GB Silberstein et al
1016
Ms E Olson (University of California, Santa Cruz); Dr S
Ethier (University of Michigan Breast Tissue Bank) for
obtaining breast tissue samples and Drs Charles W Daniel,
Daniel Medina and Charles Roberts Jr. for helpful
suggestions and review of the manuscript. This work was
supported by National Institutes of Health Grants DK48883 (to Charles W Daniel, Santa Cruz) and D-54740 (to
GR Dressler), Department of the Army Grant DAMD1794-J-4230 (Charles W Daniel) and a gift from the Milton
Meyer Foundation (to GB Silberstein).
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