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First published online 19 September 2007
doi: 10.1242/dev.006585
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1 Department of Cell Biology, NYU School of Medicine, MSB 618, 550 1st Avenue,
New York, NY 10016, USA.
2 Department of Molecular and Cellular Biology, M533A, Baylor College of
Medicine, One Baylor Plaza, Houston, TX 77030, USA.
3 Department of Dermatology, NYU School of Medicine, MSB 618, 550 1st Avenue,
New York, NY 10016, USA.
* Author for correspondence (e-mail: cowinp01{at}med.nyu.edu)
Accepted 27 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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N89ß-catenin. Cells at
ductal tips are inherently ß-catenin-responsive and form alveoli in the
absence of PR. However, PR activity confers ß-catenin responsiveness to
progenitor cells along the lateral ductal borders in the virgin gland. Once
activated by ß-catenin, responding cells switch on an alveolar
differentiation program that is indistinguishable from that observed in
pregnancy and is curtailed by PR signaling.
Key words: Beta-catenin, Breast, Progesterone receptor, Mammary gland, Wnt, Mouse, Stem cells
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Postnatal mammary development is temporally regulated by hormonal signaling
(Lyons et al., 1958;
Nandi, 1958). Puberty induces
ductal elongation and bifurcation by stimulating cell proliferation within
terminal end buds (TEBs) found at the ductal tips
(Daniel and Silberstein, 1987;
Bocchinfuso and Korach, 1997;
Kleinberg et al., 2000). As
puberty wanes, TEBs vanish and the ductal tree undergoes progressive
estrus-induced maturation (Purnell and
Saggers, 1974; Haslam,
1987). During early pregnancy, tertiary side-branches sprout from
the ductal tree and alveolar buds form at their tips
(Russo and Russo, 1978;
Brisken, 2002). Alveolar
differentiation proceeds in two stages termed lactogenesis I and II.
Lactogenesis I occurs during mid-pregnancy and involves the expression of
genes encoding the milk proteins ß-casein and WDNM1 and accumulation of
lipid droplets inside alveolar luminal cells. During late pregnancy, a
lactogenic switch occurs that permits differentiation to proceed to
lactogenesis II (Neville et al.,
2002; Brisken and Rajaram,
2006). This stage involves expression of whey acidic protein
(Wap) and 
-lactalbumin genes, closure of tight junctions and
secretion of milk and lipid droplets into the alveolar lumen
(Robinson et al., 1995;
Mather and Keenan, 1998;
Nguyen et al., 2001).
Lactation is followed by involution, which restores a pre-pregnant morphology
to the parous gland (Lund et al.,
1996; Wagner et al.,
2002; Boulanger et al.,
2005).
Experiments involving hormonal supplementation of oophorectomized and
hypophysectomized mice first established the contributions of individual
hormones to specific stages of mammary development. Growth hormone and
estrogen (E) promote ductal elongation. Progesterone (P) induces
side-branching and alveologenesis and prolactin (PRL), in combination with P
promotes alveolar development (Lyons et
al., 1958; Nandi,
1958; Plaut et al.,
1999; Shyamala,
1999; Atwood et al.,
2000; Hovey et al.,
2002; Aupperlee and Haslam,
2007). Recent studies on hormone receptor-null mice have confirmed
and extended these findings. For example, mammary glands with targeted
disruption of the estrogen receptor fail to undergo ductal extension
(Bocchinfuso et al., 2000;
Mallepell et al., 2006).
Progesterone receptor (PR; Pgr - Mouse Genome
Informatics)-null glands lack side-branches and alveoli
(Lydon et al., 1995;
Brisken et al., 1998). The
PR-A isoform is dispensable to mammary development but
PR-B-/- mice have fewer side-branches and alveoli
resulting in impaired lactation, despite complete alveolar differentiation
(Mulac-Jericevic et al., 2000;
Shyamala et al., 2000;
Mulac-Jericevic et al., 2003).
Prolactin receptor (PRLR) is also essential for alveologenesis and is required
for complete alveolar differentiation. Prlr-/- glands fail
form alveoli when transplanted into pregnant hosts and
Prlr+/- glands show arrested alveolar development at day
15.5 of pregnancy (Ormandy et al.,
1997a; Brisken et al.,
1999). The hormonal ablation and supplementation studies taken
together with the genetic studies show that PR and PRLR co-operate in early
pregnancy to promote alveologenesis. However, in late pregnancy, PR activity
restrains alveolar differentiation as demonstrated by the observation that
declining P levels, in the presence of high PRL, trigger the lactogenic switch
(Deis and Delouis, 1983).
A series of studies have indicated that PR activities during pregnancy are
mediated by several paracrine factors, including Wnts
(Brisken et al., 2000;
Brisken et al., 2002;
Mulac-Jericevic et al., 2003).
Seven Wnt proteins are expressed during mammary development
(Gavin and McMahon, 1992;
Weber-Hall et al., 1994).
However, their contributions to specific mammary developmental processes
remain unclear, and whether they signal through canonical
ß-catenin-mediated or non-canonical pathways has not been determined.
ß-catenin has been strongly implicated in pregnancy-induced mammary
development by both gain- and loss-of-function experiments.
MMTV-N89ß-catenin induces precocious development in virgin
females, whereas mice expressing suppressors of ß-catenin signaling, such
as axin or ß-eng (in which the ß-catenin C-terminal domain is
replaced by the Drosophila Engrailed repressor domain), show impaired
alveolar development in pregnancy (Hsu et
al., 2001; Imbert et al.,
2001; Hatsell et al.,
2003; Rowlands et al.,
2003; Tepera et al.,
2003). These phenotypes suggest that hormonal and ß-catenin
pathways intersect to regulate postnatal mammary development.
To explore the relationship between PR and ß-catenin signaling during
mammary development, we tested the ability of MMTV-N89ß-catenin to
rescue the PR-/- phenotype. Our results show that
stabilized ß-catenin rescues alveologenesis at ductal tips in
PR-/- mammary glands. By contrast, PR is essential for the
emergence or priming of a ß-catenin-responsive subset of cells along the
lateral borders of mammary ducts. In the presence of PR,
N89ß-catenin-responding cells differentiate to lactogenesis I. In
the absence of PR, N89ß-catenin-responding cell differentiation
proceeds to lactogenesis II.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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N89ß-catenin mice cannot feed their pups after the third
pregnancy owing to progressive alveolar hyperplasia, the following breeding
strategy was taken to generate mice for analyses.
MMTV-N89ß-catenin females
(Imbert et al., 2001) on an
FVB/n strain background were mated to PR-/- males on a
129Sv X C57BL/6 strain background (Ismail
et al., 2002), and the resulting F1 progeny were intercrossed. F2
PR-/- males were crossed to wild-type FVB/n to generate
large numbers of F3 PR+/- female offspring, which were
crossed to PR-/-;N89ß-catenin males. All
analyses were performed on the resulting F4 females of this cross. Mice were
genotyped by PCR for the PR-lacZ allele using primers lacZF
(5'-CATCCACGCGCGCGTACATC-3') and lacZR
(5'-CCGAACCATCCGCTGTGGTAC-3') and for the wild-type allele using
primers P1F (5'-TAGACAGTGTCTTAGACTCGTTGTTG-3') and P2R
(5'-GATGGGCACATGGATGAAATC-3'). Mice were screened for expression
of stabilized ß-catenin by Southern blot analysis
(Imbert et al., 2001).
Axin2dEGFP and conductin-lacZ mice were kind gifts of Dr Walter
Birchmeier (Max Delbrueck Center, Berlin, Germany) and Dr Frank Costantini
(Columbia University Medical Center, New York, NY). Conductin-lacZ
mice were generated by homologous recombination using a bacterial
lacZ gene, with a nuclear localization signal, to replace the second
exon of the conductin (Axin2) gene as described
(Lustig et al., 2002). These
mice were genotyped by PCR for the conductin-lacZ allele using the
lacZF and lacZR primers described above.
Carmine and X-Gal staining of mammary gland wholemounts
Inguinal mammary glands were fixed in Carnoy's solution and stained in
carmine alum overnight. Alternatively, glands were fixed in 4%
paraformaldehyde (PFA) in PBS for 1 hour, washed three times in rinse buffer
(2 mM MgCl2, 0.2% sodium deoxycholate, 0.2% NP40 in PBS) and
stained overnight at room temperature in
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal)
staining solution (1 mg/ml X-Gal, 2 mM MgCl2, 0.2% sodium
deoxycholate, 0.2% NP40, 5 mM potassium ferrocyanide, 5 mM potassium
ferricyanide). X-Gal-stained glands were post-fixed for 4 hours in 4% PFA and
counterstained with carmine. Glands were then dehydrated in ethanol, cleared
in Citrisolv (Fisher Scientific, Suwanne, GA) and mounted in Cytoseal (VWR,
West Chester, PA).
Histology and immunohistochemistry
Mammary glands were dissected, fixed in 10% phosphate-buffered formalin
overnight, processed and embedded in paraffin. Histological analyses were
performed on Hematoxylin and Eosin-stained sections of paraffin-embedded
tissues. Alveolar luminal area was quantified by thresholding the image and
computing the luminal area and the number of alveoli in each field using
ImageJ software
(http://rsb.info.nih.gov/ij/;
National Institutes of Health, Bethesda, Maryland, USA). The graph represents
an average of five fields that were examined in three
PR+/-;N89ß-catenin and five
PR-/-;N89ß-catenin mice. Immunofluorescence
was performed on formalin-fixed tissues and immunohistochemistry on
X-Gal-stained, PFA-fixed, paraffin-embedded tissues after antigen retrieval.
Antigen retrieval was performed by microwaving sections in 6.52 mM sodium
citrate (pH 6.0) for 30 minutes. Primary rabbit antibodies directed against
NKCC1 (gift of Dr Jim Turner, NIH, Bethesda, MD; 1:2000), PR (A0098, DAKO,
Carpinteria, CA; which recognizes both PR-A and PR-B isoforms; 1:500), casein
(gift of Dr Margaret Neville, University of Colorado, Denver, CO; 1:4000) and
Ki67 (Novocastra, Newcastle, UK, NCL-Ki67p; gift of Dr Susan Logan; 1:1000),
and mouse antibodies recognizing the Myc epitope (9E10, 1:100; a gift of Dr
Harold Varmus, Memorial Sloan Kettering, New York, NY; 1:500) and PCNA (M0879,
DAKO; 1:1000) were used for these analyses. For histochemical analyses,
biotinylated secondary antibodies (anti-rabbit or anti-mouse IgG, Vector
Laboratories) were used in conjunction with streptavidin peroxidase (Fisher
Scientific) that was colorimetrically detected using diaminobenzidine (K3466,
DAKO). Alternatively, FITC-conjugated goat anti-mouse and Cy3-conjugated
donkey anti-rabbit secondary antibodies (Cappel, Solon, OH; 1:100 and 1:200,
respectively) were used for immunofluorescence.
Oil Red O staining
Frozen sections (10 µm) were air-dried, fixed in 3.7% formaldehyde for
10 minutes and rinsed in deionized water and 60% triethylphosphate (TEP)
(Fluka Chemie, Buchs, Switzerland). A working dilution of Oil Red O (Fluka
Chemie, Buchs, Switzerland) was prepared fresh each time by diluting the stock
solution (5 mg/ml in 60% TEP) to 36% with deionized water. Sections were
stained for 25 minutes in the working solution of Oil Red O, counterstained
with Hematoxylin 2 (Richard-Allan Scientific, Kalamazoo, MI), differentiated
in running tap water, mounted in Gelmount (Fisher Scientific) and photographed
immediately.
RNA isolation, northern blot analysis and real-time RT-PCR
Total RNA was extracted from frozen mammary gland samples, stored in liquid
nitrogen, using the TotallyRNA Isolation Kit (Ambion, Austin, TX). Total RNA
(20 µg) from all samples was separated by electrophoresis on a 1%
phosphate-glyoxal agarose gel using the NorthernMax-Gly Kit (Ambion). 28S rRNA
was used as a loading control for the total amount of RNA in the sample.
Northern blot analyses were performed using end-labeled oligonucleotide probes
for ß-casein (5'-GTCTCTCTTGCAAGAGCAAGGGCC-3'), Wap
(5'-CAACGCATGGTACCGGTGTCA-3') and WDNM1
(5'-CAGAGCCCAGGCAGTAGTCATTGTC-3')
(http://mammary.nih.gov/tools/markers/molecular/MMD.html)
and random-prime-labeled cDNA probes (Roche, Indianapolis, IN) for
-lactalbumin (GenBank BC069916) and K18 (Krt18)
(Imbert et al., 2001) using
the NorthernMax-Gly Kit (Ambion). All analyses were performed on a single
membrane. RNA (2 µg) was reverse transcribed using Superscript III reverse
transcriptase (18080-044, Invitrogen, Carlsbad, CA) and 1 µl of the cDNA
thus obtained was used in the real-time PCR reaction. Real-time reverse
transcriptase (RT) PCR was performed according to the instructions provided
with the SYBR Green Quantitative RT-PCR Kit (Sigma Aldrich, St Louis, MO)
using a Light Cycler (Roche). The following primers were used: ß-casein,
5'-GCCTTGCCAGTCTTGCTAAT-3' and
5'-GGAATGTTGTGGAGTGGCAG-3'; WDNM1,
5'-ACTGCCTGGGCTCTGTCTAA-3' and
5'-TCTCCTGTGCATCGTTCATC-3'; -lactalbumin
5'-CATAGCGTGTGCCAAGAAGA-3' and
5'-CACATGGGCTTGTAGGCTTT-3'; Wap,
5'-GTAGGACCCGCAAAACTCCT-3' and
5'-TAGATTCCAAGGGCAGAAGC-3'; and 28S rRNA,
5'-AAACTCTGGTGGAGGTCCGT-3' and
5'-CTTACCAAAAGTGGCCCACTA-3'
(Teuliere et al., 2005;
Oxelmark et al., 2006).
Analyses were performed in duplicate and values were normalized to those
obtained for 28S rRNA. Fold changes were calculated relative to mRNA
expression levels in a 12-week-old virgin mouse.
Mammary gland transplants
Three-week-old Rag1-/- recipient mice were anesthetized
by intraperitoneal injection of ketamine (1 mg/g body weight) and xylazine
(0.5 mg/g body weight). A vertical, midline, abdominal incision was made to
expose both inguinal mammary glands. The mammary fat pad was cleared of
epithelium by resecting the gland lateral to the lymph node. Each recipient
received 1 mm3 epithelial fragments either from 6- to 10-week-old
donor mice (PR-/- and
PR-/-;N89ß-catenin or
PR+/- and
PR+/-;N89ß-catenin) in contra-lateral cleared
fat pads. The abdominal incision was closed with 4.0 Ethilon interrupted
sutures
(http://mammary.nih.gov/tools/mousework/index.html).
Transplanted fragments were allowed to grow for 6 weeks to reconstitute the
mammary ductal system. Recipient mice were then mated and sacrificed at days
8.5 and 14.5 of pregnancy. Transplanted glands were stained with X-Gal
(Applichem, Cheshire, CT) to differentiate between ductal systems generated
from transplanted fragments and those arising from incompletely removed
endogenous epithelium. Endogenous glands from the Rag1-/-
mice were used to determine the extent of pregnancy-induced normal alveolar
development and as negative controls for X-Gal staining.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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N89ß-catenin expression rescues alveologenesis but not side-branching in PR-/- glands;
Brisken et al., 1998). To
investigate the intersections between ß-catenin and PR signaling, we
asked whether expression of N89ß-catenin could restore
pregnancy-like morphogenesis to PR-/- glands. As
PR-/- mice are infertile, these studies were performed in
mammary glands transplanted into immunocompromised pregnant hosts.
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N89ß-catenin glands
(Fig. 1B,F) that resembled
structures that formed in control transplanted PR+/-
(Fig. 1C,G) and endogenous
Rag1-/- glands (Fig.
1D,H) but which were consistently more distended. Wholemount
analyses showed a significant increase in these structures between 8.5 and
14.5 days of pregnancy (compare Fig. 1B-D
with F-H). Histological analysis identified the presence of
alveolar features, such as casein expression, in these protrusions
(Fig. 2A,B). To determine
whether N89ß-catenin also rescued ductal side-branching, we
analyzed the expression of the Na-K-Cl co-transporter, NKCC1, in transplanted
mammary glands during pregnancy
(Shillingford et al., 2003).
In wild-type glands, NKCC1 is highly expressed along the basolateral membrane
of ductal cells of virgin animals (Fig.
2C), but is reduced within alveoli during pregnancy
(Shillingford et al., 2002).
High levels of NKCC1 expression, indicative of ductal differentiation, were
maintained in PR-/- glands
(Fig. 2D). However, NKCC1
expression was reduced in the protrusions that develop in
PR-/-;N89ß-catenin glands
(Fig. 2E) and in endogenous
Rag1-/- alveoli (Fig.
2F). These data suggest that that N89ß-catenin rescues
alveologenesis, but not side-branching, in PR-/- glands
during pregnancy. Wholemounts of transplanted glands at 8.5 days of pregnancy
showed no statistically significant difference in the number of secondary
branches among the four genotypes of mice examined
(Fig. 2G,H). Consistent with
previous observations, PR-/- mice showed significantly
fewer side-branches than PR+/- glands (t-test,
P=0.008; Fig. 2H)
(Lydon et al., 1995). Although
on average N89ß-catenin glands contained more side-branches than
their non-transgenic counterparts, these differences were not statistically
significant (t-test, P=0.057).
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N89ß-catenin expression induces precocious mammary development in the absence of PR signalingN89ß-catenin expression could induce
precocious alveolar development in virgin mice in the absence of PR.
Wholemounts of virgin mammary glands from 12-week-old adolescent
(Fig. 3A-D) and 26-week-old
mature adult (Fig. 3E-H) female
PR+/- and PR-/- mice were compared
with those from mice transgenically expressing N89ß-catenin.
Pubertal ductal extension was completed normally in virgin
PR+/- and PR-/- glands and their
transgenic counterparts. In adolescents, PR+/-
(Fig. 3A) and
PR-/- (Fig.
3C) ducts remained smooth but 5/6
PR+/-;N89ß-catenin
(Fig. 3B) and 2/6
PR-/-;N89ß-catenin
(Fig. 3D) glands showed
precocious mammary development. By 26 weeks of age, PR+/-
(Fig. 3E) and
PR-/- (Fig.
3G) ducts remained smooth but all transgenic glands displayed
precocious development regardless of PR status
(PR+/-;N89ß-catenin, n=7,
Fig. 3F; and
PR-/-;N89ß-catenin, n=7,
Fig. 3H). These data show that
ß-catenin signaling induces precocious mammary development in the absence
of PR activity.
PR signaling alters the pattern of ß-catenin response within the mammary gland
Further examination of wholemounts from 26-week-old virgin
PR+/-;N89ß-catenin and
PR-/-;N89ß-catenin mammary glands revealed
striking differences in the pattern of development
(Fig. 3F,H and
Fig. 4A,B). In
PR+/-;N89ß-catenin glands, development
occurred at regular intervals along the lateral borders of the secondary
ductal branches as well as at ductal tips
(Fig. 4A). By contrast,
PR-/-;N89ß-catenin mammary development
occurred exclusively at the ends of ductal branches
(Fig. 4B). Thus, a
N89ß-catenin-responsive subset of cells exists at ductal tips
regardless of PR activity, but the emergence of a second subset of
N89ß-catenin-responsive cells along the lateral borders of ducts
is strictly dependent upon PR activity within the virgin gland. As the
MMTV-LTR is hormonally responsive, we examined whether PR produced these
effects by altering N89ß-catenin expression
(Shyamala and Dickson, 1976;
Truss et al., 1992;
Witty et al., 1995). However,
indirect immunofluorescence detected Myc-tagged N89ß-catenin in
all luminal cells regardless of PR expression
(Fig. 4C,D). These data show
that P/PR signaling has no effect on transgene expression but critically
determines the pattern of response to N89ß-catenin in the adult
mammary tree.
In other tissues it is known that only a subset of cells are capable of
mounting a response to a uniformly expressed ß-catenin transgene
(DasGupta and Fuchs, 1999). To
examine whether a similar phenomenon operates in the mammary gland, we
investigated the expression of conductin-lacZ, a reporter of
ß-catenin signaling. Conductin (Axin2) is a target gene and
negative-feedback regulator of the canonical Wnt signaling pathway. Expression
of the conductin-lacZ knock-in allele provides a context-independent,
reliable reporter of ß-catenin signaling
(Jho et al., 2002;
Lustig et al., 2002;
Yu et al., 2005). Analyses of
PR+/+;conductin+/lacZ;N89ß-catenin
mammary glands showed that, despite uniform N89ß-catenin transgene
expression in all luminal cells (Fig.
5A,B), only a subset of cells expressed conductin-lacZ
(Fig. 5B).
Conductin-lacZ-positive cells were distributed at intervals along the
ducts of
PR+/+;conductin+/lacZ;N89ß-catenin
glands (Fig. 5B,C) and were
spatially distinct from cells expressing PR
(Fig. 5C).
Conductin-lacZ expression was prominent in all regions undergoing
development, i.e. at ductal tips and along the ductal borders of
PR+/+;conductin+/lacZ;N89ß-catenin
glands (Fig. 5D). These results
show that in PR+/+ glands, the subset of cells capable of
responding to ß-catenin signaling is evenly patterned with respect to
PR.
To determine which cell-types are proliferating in
PR+/+;conductin+/lacZ;N89ß-catenin
glands, we first analyzed the expression of PCNA and PR by double
immunofluorescence. PR and PCNA were found in mutually exclusive expression
patterns (Fig. 5E), reminiscent
of previous reports in human breast and mouse mammary glands
(Clarke et al., 1997;
Seagroves et al., 2000). Thus,
PR cells are quiescent in MMTV-N89ß-catenin virgin glands. By
contrast, we observed that 34.9+11.3% of conductin-lacZ-positive
cells express Ki67 (Fig. 5F,G).
We conclude that the segregation between PR and proliferating cells is
maintained in N89ß-catenin-expressing mice and that
conductin-lacZ-positive cells are proliferating.
N89ß-catenin-induced alveolar differentiation is accentuated in the absence of PR signaling
ß-catenin-induced structures faithfully recapitulate many features of
normal alveolar development. For example, they are composed of bilayered
structures (Fig. 6A,B) that
contain eosinophilic secretions, and they express casein
(Fig. 6C,D) and lipid droplets
(Fig. 6E,F). In addition to
having alveolar morphology, ß-catenin-induced structures showed
downregulation of PR-lacZ in a manner similar to that observed in
pregnancy-induced alveoli (data not shown). We observed that alveoli from
PR-/-;N89ß-catenin glands were consistently
larger than their counterparts in
PR+/-;N89ß-catenin glands
(Fig. 6A-G), suggesting that
they have progressed to a more advanced stage of lactogenic differentiation.
To determine the extent to which N89ß-catenin induces alveolar
differentiation, we analyzed milk protein gene expression by northern blotting
(Fig. 7) and real-time RT-PCR
(Table 1) performed on RNA
isolated from virgin glands. Early milk protein genes including WDNM1
and ß-casein were expressed in all
PR+/-;N89ß-catenin (n=3) and
PR-/-;N89ß-catenin (n=3) mice at
levels above those detected in their non-transgenic virgin littermates.
However, transcripts for late milk protein genes, including
-lactalbumin and Wap, which were present in barely detectable
amounts in PR+/-;N89ß-catenin glands
(n=3), were expressed at higher levels in 2/3
PR-/-;N89ß-catenin glands
(Fig. 7 and
Table 1). We conclude that
N89ß-catenin-induced alveoli undergo secretory differentiation but
are restrained by PR activity from undergoing the lactogenic switch.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Imbert et al.,
2001; Tepera et al.,
2003). However, connections between hormonal and ß-catenin
signaling have not been investigated. Here, we show that two distinct subsets
of mammary cells are responsive to ß-catenin. One subset, located at
ductal tips, is intrinsically ß-catenin-responsive. A second subset,
distributed at intervals along the lateral ductal borders, requires PR
signaling to become ß-catenin-responsive.
N89ß-catenin-induced alveoli commence secretory differentiation
but are restrained by PR from undergoing the lactogenic switch
(Fig. 8).
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|
N89ß-catenin specifically restores alveologenesis to PR-/- mammary glandsN89ß-catenin specifically restores alveologenesis to ductal tips,
it does not rescue side-branching in PR-/- mice.
Side-branching and alveologenesis are abolished in PR-/-
glands but can be restored by placing PR-/- cells in the
proximity of PR+/+ cells, thus indicating that PR exerts
its effects by inducing paracrine factors
(Brisken et al., 1998). Wnt
and RANKL have been implicated as downstream paracrine effectors in these
processes (Gavin and McMahon,
1992; Weber-Hall et al.,
1994; Bradbury et al.,
1995; Humphreys et al.,
1997; Brisken et al.,
2000; Fata et al.,
2000; Grimm et al.,
2002; Mulac-Jericevic et al.,
2003). ß-catenin is the central transducer of the canonical
Wnt pathway and can also synergize with RANKL signaling
(Fig. 8)
(Fantl et al., 1999;
Shtutman et al., 1999;
Tetsu and McCormick, 1999;
Cao et al., 2001;
Lamberti et al., 2001;
Rowlands et al., 2003). The
observation that N89ß-catenin rescues alveologenesis but not
side-branching in PR-/- mice is entirely consistent with
previous findings that ß-catenin signaling suppressors specifically
impair alveologenesis (Hsu et al.,
2001; Tepera et al.,
2003). From these results, we conclude that PR induction of
side-branching must be transduced through ß-catenin-independent pathways
in luminal cells, or, through effects on other cell types
(Fig. 8). Alternatively,
cell-fate choice could be determined in the mammary gland by specific
ß-catenin levels as is the case for other epidermal appendages, where
lowering the ß-catenin activity promotes sebaceous and epidermal fate and
raising ß-catenin signaling induces hair-cell fate
(Gat et al., 1998;
Niemann et al., 2002;
Niemann and Watt, 2002).
PR signaling is essential for ß-catenin responsiveness along lateral ductal borders
The most surprising finding of the current study is that PR signaling
induces dramatic changes in the pattern of ß-catenin responsiveness
within the virgin gland. Using conductin-lacZ as a reporter of
ß-catenin signaling, we identified a subset of luminal cells that are
capable of responding to ß-catenin. These proliferative responder cells
are regularly distributed with respect to PR-positive cells and appear to be
precursors of conductin-lacZ-positive precocious alveoli. Alveolar
development is found along the lateral ductal borders in
PR+/+;N89ß-catenin and
PR+/-;N89ß-catenin glands but is restricted to
the ductal tips in PR-/-;N89ß-catenin glands.
One interpretation of our findings is that PR is essential for the spatial
patterning of a ß-catenin-responsive cell type along the lateral ductal
borders (Fig. 8). Precedence
for PR mediating patterning comes from analyses of PR expression in
PR-/- mice. PR-/- ducts fail to
undergo the transition from a juvenile, uniform pattern of PR expression to an
adult, non-uniform PR expression pattern that is seen in wild-type and
PR+/- animals (Ismail
et al., 2002; Shyamala et al.,
2002). Our results suggest that PR activity plays a more global
role in patterning the virgin gland during the process of ductal maturation
and might regulate the positioning of multiple progenitors (bipotent, ductal
and alveolar) along lateral borders. As alveoli emerge directly from lateral
borders in PR+/-;N89ß-catenin virgin glands
(Fig. 4A), their formation is
dependent upon PR activity but not upon the prior formation of a ductal
side-branch.
An alternative interpretation of our data is that PR acts downstream of
ß-catenin. For example, temporal increases in PR activity during estrus
and pregnancy could control ß-catenin responsiveness by regulating the
availability of a limiting factor required for ß-catenin signaling
(Fig. 8). At a molecular level,
a number of factors are known to regulate ß-catenin signaling. Regulated
nuclear entry of ß-catenin has been observed in Xenopus embryos
and the ß-catenin partners, Legless (LGS; BCL9)/BCL9-2 and Pygopus
(PYGO), are required for this step in flies
(Schneider et al., 1996;
Kramps et al., 2002;
Thompson et al., 2002;
Hoffmans and Basler, 2004;
Townsley et al., 2004;
Stadeli et al., 2006). Once
inside the nucleus, assembly of ß-catenin transcription complexes is
dependent upon the expression of DNA-binding partners, such as Tcf
proteins/LEF1, which play important roles in mammary development.
Lef1-/- mice fail to develop mammary buds and
Tcf1 and Tcf4 are expressed in adult mammary glands
(van Genderen et al., 1994;
Korinek et al., 1997;
Barker et al., 1999;
Roose et al., 1999).
Intriguingly, recent studies have shown that P primes uterine cells for
ß-catenin signaling by increasing Tcf/Lef and reducing GSK3ß levels
but E is required for nuclear entry (Rider
et al., 2006).
ß-catenin responsiveness of cells at ductal tips is PR-independent, as
they undergo alveologenesis in PR-/- as well as
PR+/- mice. We do not know why these cells are
intrinsically responsive to ß-catenin. It is possible that these cells
are pre-existing alveolar progenitors. However, in rats it has been proposed
that ductal tips contain a remnant of TEB stem cells
(Russo and Russo, 1978).
Previous studies have suggested that only stem cells are capable of responding
to ß-catenin signaling. For example, although
keratin-14-N87ß-catenin is expressed throughout the basal
epidermal layer, TOPGAL transcriptional response is restricted to epidermal
and hair follicle stem cells (DasGupta and
Fuchs, 1999). Similarly, despite elevation of ß-catenin in
all intestinal cells of Apcmin mice,
conductin-lacZ expression is restricted to crypt stem cells and
premalignant adenomas (Lustig et al.,
2002; Maretto et al.,
2003). These studies show that only stem cells and possibly early
progenitors are ß-catenin-responsive. Consistent with these observations,
N89ß-catenin expression has been shown to expand the mammary
stem/progenitor pool (Liu et al.,
2003; Liu et al.,
2004). Thus, the inherent ability of cells at ductal tips to
respond to ß-catenin, without prior priming by PR, is in keeping with
their suggested designation as residual TEB stem cells, as proposed by Russo
(Russo and Russo, 1978).
N89ß-catenin-induced secretory alveolar differentiation is restrained by PR
Our previous studies have shown that ß-catenin stimulation results in
the emergence of structures that are morphologically indistinguishable from
normal alveoli (Imbert et al.,
2001; Rowlands et al.,
2003). Here we report that in addition to initiating
alveologenesis and causing alveolar progenitor expansion and survival,
ß-catenin induces a significant degree of alveolar differentiation that
progresses to lactogenesis II in the absence of PR
(Fig. 8). It is not clear
whether ß-catenin initiates alveologenesis and subsequent differentiation
proceeds by default, or, whether ß-catenin acts at multiple steps to
facilitate the differentiation process. During pregnancy, P and PRL intersect
in complex ways to regulate alveologenesis, differentiation and expansion. PR
and PRLR reciprocally regulate one another's expression and both pathways
phosphorylate and activate STAT5A, an important mediator of alveolar
development (Edery et al.,
1985; Ormandy and Sutherland,
1993; Gouilleux et al.,
1995; Groner and Gouilleux,
1995; Ormandy et al.,
1997b; Richer et al.,
1998). Moreover, both PR and PRLR increase the levels of RANKL and
cyclin D1, which are essential for alveologenesis and alveolar maturation,
respectively (Brisken et al.,
2002; Brockman et al.,
2002). However, numerous studies indicate that PRLR promotes and
PR activity restrains the lactogenic switch
(Neifert et al., 1981;
Graham and Clarke, 1997;
Ormandy et al., 1997a;
Nguyen and Neville, 1998;
Buser et al., 2007). For
example, in the presence of a differentiated and secretion-competent mammary
epithelium, P withdrawal triggers the lactogenic switch
(Neville et al., 2002).
Ovariectomy or treatment with the P antagonist RU486 in late pregnancy induces
lactation (Nguyen et al.,
2001). Decreasing P levels coincide with lactogenesis in many
species and P secretion from retained placental fragments delays lactogenesis
in women (Martin et al., 1978;
Neifert et al., 1981). Some
degree of PRLR activity, either through placental lactogen (PL) or PRL, is
required for the lactogenic switch as abolishing PL, by hysterectomy, and PRL,
by bromocryptine treatment, completely prevents tight-junction closure
(Nguyen et al., 2001). The
fact that ß-catenin promotes the lactogenic switch in the absence of PR
and is restrained from doing so by the presence of PR raises the possibility
that ß-catenin additionally functions downstream of PRL
(Fig. 8). Future experiments
will focus upon elucidating the relationship between ß-catenin signaling
and the PRL-JAK-STAT axis in secretory differentiation.
In summary, our data show that although ß-catenin signaling is sufficient to induce alveologenesis at ductal tips, it is insufficient to stimulate alveolar development along lateral ductal borders. We propose that a ß-catenin-independent PR activity is required to generate and/or maintain stem/progenitor cell competence along lateral ductal borders. Once rendered competent by this mechanism, a progenitor subset is poised to undergo alveologenesis in response to ß-catenin signals.
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ACKNOWLEDGMENTS |
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