The epithelium of the mammary gland is composed of luminal and
basal/myoepithelial cell lineages (Richert et al., 2000). Luminal
cells line the ductal lumen and secrete milk upon terminal
differentiation into lobulo-alveolar cells. Basal/myoepithelial cells
reside between the luminal cells and the basement membrane and
are necessary for ductal contractility. Breast cancer subtypes
(luminal versus basal) have been defined by patterns of gene
expression that reflect these lineages (Sorlie et al., 2003). Luminal
subtype tumors maintain a more differentiated state and are less
aggressive than basal subtype cancers. Processes of normal
postnatal mammary gland development directly mirror those of
tumorigenesis (e.g. invasion, proliferation, angiogenic remodeling
and apoptotic resistance) (Wiseman and Werb, 2002). Hence,
determining how cell fate is regulated during normal mammary
gland development should facilitate identifying the mechanistic
basis for phenotypic differences between luminal and basal breast
cancers, and should advance the development of subtype-specific
Expression of the transcription factors ERa (ESR1 – Mouse
Genome Informatics), GATA3 and FOXA1 strongly correlates with
the luminal subtype of breast cancer and favorable patient
prognosis (Badve et al., 2007; Habashy et al., 2008; Mehra et al.,
2005; Sorlie et al., 2003). Estrogenic signaling through ERa, a
member of the nuclear receptor superfamily, is a primary
determinant of luminal tumor biology, and patients with luminal
tumors have a better prognosis, owing partly to estrogen-targeted
therapies (EBCTCG, 2005). The consistent concomitant expression
of FOXA1, ERa and GATA3 in this subtype is suggestive of a co-
modulatory loop that may be responsible for maintaining the
luminal phenotype. In breast cancer cells, FOXA1 facilitates
estrogen responsiveness by modulating ERa binding to a subset of
target gene promoters (Carroll et al., 2005; Laganiere et al., 2005).
For example, FOXA1 is specifically required for ERa-induced
transcription of CCND1 (cyclin D1) (Eeckhoute et al., 2006), an
established oncogene in breast cancer (Buckley et al., 1993;
Eeckhoute et al., 2006). In contrast to the role of FOXA1 in ERa
activity, ERa and GATA3 have been suggested to function in a
positive feedback loop, in which expression of ERa is required for
the transcription of GATA3, and vice versa (Eeckhoute et al., 2007).
These data imply an interdependence of FOXA1, ERa and GATA3
in the maintenance of luminal breast cancer. Further defining this
collaboration should provide insight into how ERa-positive tumors
become resistant to anti-hormone therapies as well as reveal the
function of FOXA1 that occurs in tumors in the absence of ERa
(Habashy et al., 2008) (R.A.K., unpublished).
The ability of FOXA1, ERa and GATA3 to form a regulatory
network in luminal breast cancer cells suggests that they may also
co-modulate normal mammary gland morphogenesis, a process
Development 137, 2045-2054 (2010) doi:10.1242/dev.043299
© 2010. Published by The Company of Biologists Ltd
1Department of Pharmacology, Case Western Reserve University School of Medicine,
10900 Euclid Avenue, Cleveland, OH 44106, USA. 2Department of Pathology,
University Hospitals-Case Medical Center, Cleveland, OH, 44106, USA. 3VBCRC
Laboratory, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria
3050, Australia. 4Laboratory of Reproductive and Developmental Toxicology,
National Institute of Environmental Health Sciences, Research Triangle Park,
NC 27709, USA. 5Department of Internal Medicine, Vanderbilt University, Nashville,
TN 37235, USA. 6Department of Medical Oncology, Fox Chase Cancer Center,
Philadelphia, PA 19111, USA. 7Department of Genetics, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104, USA. 8Department of Pathology, Case
Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
9Division of General Medical Sciences-Oncology, Case Western Reserve University
School of Medicine, Cleveland, OH 44106, USA. 10Department of Genetics, Case
Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
*Author for correspondence (email@example.com)
Accepted 13 April 2010
FOXA1, estrogen receptor a (ERa) and GATA3 independently predict favorable outcome in breast cancer patients, and their
expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis
of various organs, with ERa and GATA3 being established regulators of mammary gland development. Interdependency between
these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is
not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated
with a loss of terminal end bud formation and ERa expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike
ERa and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of
the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines,
we also demonstrate that FOXA1 regulates ERa expression, but not GATA3. These data reveal that FOXA1 is necessary for
hormonal responsiveness in the developing mammary gland and ERa-positive breast cancers, at least in part, through its control
of ERa expression.
KEY WORDS: FOXA1, ERa a, GATA3, Mammary gland, Breast cancer, Mouse
FOXA1 is an essential determinant of ERa expression and
mammary ductal morphogenesis
Gina M. Bernardo1, Kristen L. Lozada1, John D. Miedler2, Gwyndolen Harburg3, Sylvia C. Hewitt4,
Jonathan D. Mosley5, Andrew K. Godwin6, Kenneth S. Korach4, Jane E. Visvader3, Klaus H. Kaestner7,
Fadi W. Abdul-Karim2,8, Monica M. Montano1and Ruth A. Keri1,9,10,*
that requires ERa and GATA3. ERa is expressed within a subset
of the normal luminal epithelial and stromal populations of the
mammary gland (Haslam and Nummy, 1992). Disruption of Esr1
blocks development at a rudimentary ductal structure, and signaling
from estradiol through ERa during puberty is required for
mammary epithelial proliferation, ductal elongation, bifurcation
and invasion throughout the mammary fat pad (Feng et al., 2007;
Mallepell et al., 2006; Mueller et al., 2002). GATA3 is also
necessary for mammary gland development (Asselin-Labat et al.,
2007; Kouros-Mehr et al., 2006). Specifically, Gata3 deficiency
leads to expansion of the luminal progenitor population positive for
CD61 (ITGB3 – Mouse Genome Informatics), indicating that
GATA3 is necessary for terminal differentiation of the luminal
lineage (Asselin-Labat et al., 2007). Further investigation revealed
that forced expression of GATA3 induces tumor differentiation and
inhibits metastatic progression (Kouros-Mehr et al., 2008). In
support of a transcriptional interdependence between ERa, GATA3
and FOXA1, loss of Gata3 in the normal mammary gland
decreases the ERa-expressing luminal population (Asselin-Labat
et al., 2007; Kouros-Mehr et al., 2006), and overexpression of
GATA3 in murine mammary tumors (Kouros-Mehr et al., 2008)
and a human embryonal kidney epithelial cell line increases
FOXA1 mRNA (Usary et al., 2004). Moreover, chromatin
immunoprecipitation (ChIP) in primary mammary cells revealed
that GATA3 binds, and can potentially regulate, transcription of
Foxa1 (Kouros-Mehr et al., 2006), although this has not been
Although FOXA1, GATA3 and ERa are positively correlated in
breast tumors and form a co-regulatory network in breast cancer
cell lines, the functional relationship between these three factors
during development and tumor initiation has not been fully
explored. In particular, the role of FOXA1 in mammary
morphogenesis remains unknown. Herein, we report that Foxa1
deficiency in the mammary gland results in loss of ERa, a block in
terminal end bud formation and an inability of the ducts to properly
invade the mammary fat pad in response to pubertal or pregnancy
hormones. By contrast, Gata3 expression and formation of lobulo-
alveoli are independent of FOXA1. These data provide the first
direct evidence that FOXA1 is crucial for mammary gland
morphogenesis and maintenance of ERa expression.
MATERIALS AND METHODS
Immunohistochemistry and immunofluorescence
For analysis of proliferation, mice were injected i.p. with 10 mg/g BrdU
(Sigma) 2 hours before sacrifice. Glands were fixed in 4%
paraformaldehyde for 4 hours, transferred to 1?PBS, paraffin embedded
and sectioned (5 m). Sections were re-hydrated, and antigen retrieval
performed using 10 mM sodium citrate (pH 6) in a pressure cooker (125°C
for 10 minutes; 90°C for 2 minutes) or for milk antibody, incubated in 10
g/ml pepsin in 0.01 N HCl for 15 minutes at room temperature. Sections
were blocked with peroxidase blocking reagent (DAKO) and incubated
with primary antibody overnight at 4°C [FOXA1, Santa Cruz; ERa, Santa
Cruz; E-cadherin, Cell Signaling; CK8 (TROMA-1), Developmental
Studies Hybridoma Bank, University of Iowa; a-SMA, Sigma; BrdU,
Becton-Dickinson) or at room temperature for 1 hour (milk, Nordic
Immunology; PR, DAKO)]. Secondary detection of FOXA1, ERa, E-
cadherin, milk and PR was performed using the appropriate Vectastain
Elite ABC Kit (Vector Laboratories) as per the manufacturer’s
recommendations. CK8, a-SMA and BrdU were detected using the
EnVision+System-HRP for mouse antibodies (DAKO) as recommended.
Secondary conjugates were detected using 3,3?-diaminobenzidine
(DAKO). Sections were counterstained with Gill’s #3 Hematoxylin
(Fisher), dehydrated and mounted. TUNEL was performed as per the
manufacturer’s recommendations except using Gill’s #3 Hematoxylin as a
counterstain (ApopTag Peroxidase In Situ Apoptosis Detection Kit,
Chemicon). Alexa fluor 596 (anti-goat) and Alexa fluor 488 (anti-rabbit)
secondary antibodies (Invitrogen) were used for detection of FOXA1 and
ERa by immunofluorescence (IF). IF and immunohistochemistry (IHC)
were quantified by counting the percentage of positive epithelium in at
least two to five fields per section per mouse. Hematoxylin and Eosin
staining was performed by the Case Western Reserve University Tissue
Procurement and Histology Core Facility.
All animal procedures, except production of Ex3aERKO and MMTV-
cre;Gata3f/fmice, were approved by the Case Western Reserve University
IACUC. Ex3aERKO mice were generated under an approved protocol at
the National Institute of Environmental Health Sciences/NIH, and a
contract to Xenogen (Caliper Life Sciences) using a strategy similar to that
described previously (Dupont et al., 2000). This resulted in an Esr1 gene
with exon 3 flanked by loxP sites. Exon 3 was deleted by crossing mice
carrying the floxed exon 3 Esr1 to a global Sox2-cre mouse line [Tg(Sox2-
cre)1Amc/J; Jackson Labs]. DNA was evaluated by PCR using P1 and P3
primers as described (Dupont et al., 2000). The MMTV-cre;Gata3f/fmice
were generated at the Walter and Eliza Hall Institute of Medical Research
as described (Asselin-Labat et al., 2007). Foxa1+/–males (Kaestner et al.,
1999) were bred with wild-type C57BL/6 females generating Foxa1+/–
progeny that were intercrossed to generate Foxa1–/–progeny. Transgenic
mice were identified by PCR using primers as described (Kaestner et al.,
Mammary anlagen transplantation
Transplantation of mammary anlagen into recipient mice has been
described (Robinson et al., 2000). Briefly, the mammary anlagen of
embryonic day 14 (E14) female mouse embryos were dissected and
cultured at 37°C/5% CO2in DMEM/F12 (supplemented with 10% FBS,
1% Pen-Strep, 2 mM L-Glutamine and 0.75 g/ml Fungizone) until the
genotypes were determined. Three-week-old recipient C57BL/6 females
were anesthetized with 2.5% avertin, and inguinal fat pads were cleared of
endogenous epithelium. The cleared fat pad was examined by whole mount
to verify successful clearing. A Foxa1+/+anlage was inserted into the
cleared fat pad, and a Foxa1–/–anlage was inserted into the contralateral
cleared fat pad of the same mouse. The incision was sutured and infiltrated
with marcaine (0.25%). Recipient mice were aged 5 or 8 weeks, and the
transplanted glands were collected and whole mounts examined.
Alternatively, recipient mice were aged 8 weeks, mated with C57BL/6
males, and the transplanted glands collected and whole mounts assessed at
18.5 days postcoitum (dpc).
Renal capsule grafting
Tissue grafting into the renal capsule has been described (Cunha et al.,
2000). Briefly, inguinal fat pads of postnatal day 1 female pups were
removed, and incubated at 4°C in DMEM/F12 culture media as described
above until the genotypes were determined. Recipient C57BL/6 females
were anesthetized with 2.5% avertin, and a kidney exteriorized. A small
incision was made to separate the kidney capsule from the parenchyma. A
polished glass pipette was used to create a pocket between the kidney
capsule and parenchyma. The inguinal fat pad from a Foxa1+/+pup was
grafted into the pocket, and the kidney placed back into the body cavity.
The same procedure was used to graft the inguinal fat pad from a Foxa1–/–
pup on to the contralateral kidney. The incisions were closed using wound
clips, and the wound infiltrated with marcaine (0.25%). Recipient mice
were aged either 2 or 4-5 weeks, and the glands harvested for further
analysis. Alternatively, recipients were aged 4-5 weeks, mated, and the
glands were harvested at 18.5 dpc.
Mammary gland whole mounts
Glands were fixed in Kahle’s fixative for at least 4 hours, washed in 70%
ethanol, gradually rehydrated to 100% water, stained with carmine alum,
dehydrated, cleared in xylenes and mounted as previously described
(Rasmussen et al., 2000). Ductal area was obtained by taking the area of a
box drawn around the gland, including the nipple. Ductal length was
obtained by measuring from the farthest edge of the lymph node from the
Development 137 (12)
nipple to the end of the longest duct. When the duct did not reach the
lymph node, the distance from the end of the duct to the farthest edge of
the lymph node from the nipple was measured and given a negative value.
Mammary cell preparation and FACS
Inguinal mammary glands from ten (per experiment) wild-type FVB/N
virgin females (~8 weeks of age) were isolated and prepared as described
(Shackleton et al., 2006). Experiments #1 and #2 were sorted by
fluorescence-activated cell sorting (FACS) using antibodies against CD24,
CD29 (ITGB1 – Mouse Genome Informatics) and CD61 as described
(Asselin-Labat et al., 2007). Experiment #2 was sorted twice to enhance
Quantitative real-time PCR
Total RNA was isolated using TRIzol Reagent (Invitrogen), treated with
DNAse I (DNA-free, Ambion), and cDNA produced using SuperScript II
Reverse Transcriptase (Invitrogen). Real-time PCR was performed using
Applied Biosystems TaqMan Gene Expression Assays (Assay IDsFoxa1,
Mm00484713_m1; Pgr, Mm00435625_m1; Gata3 Mm00484683_m1;
Krt8, Mm00835759_m1; ESR1
Hs99999905_m1) or SYBR-green primers (Foxa1-Foward: GGATCCC-
Cell culture and RNA interference
All cell lines were obtained from ATCC. MCF7 cells were grown in
DMEM (Mediatech); T47D cells in RPMI 1640 (Gibco). Media was
supplemented with 10% FBS and 1% Penicillin-Streptamycin (Invitrogen).
Cells were seeded in 100 mm dishes to be 30-50% confluent upon
transfection. siRNA targeting firefly luciferase mRNA (siCONTROL Non-
targeting siRNA #2, Dharmacon) or human FOXA1 mRNA (siGENOME
M-010319-01 and -04, Dharmacon) were transfected in OPTI-MEM media
(Invitrogen) using Lipfectamine 2000 (Invitrogen) to a final concentration
of 100 nM. Culture media was changed to complete growth medium after
16-24 (MCF7) or 24 (T47D) hours. Cells were harvested 36 (MCF7) or 72
(T47D) hours post-transfection.
Cells were lysed [50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 1 mM EDTA;
1 mM EGTA; 1 mM NaF; 0.1% SDS; 0.5% Sodium Deoxycholate; 1%
Triton-X-100; 10% Glycerol; 2 mM Sodium Orthovanadate; Protease
Inhibitor Cocktail (Sigma)], and protein levels quantified (Bradford Assay,
Biorad). Protein lysate was resolved using SDS-PAGE, and transferred to
PVDF membrane (BioRad). Blots were blocked (5%-milk-1XPBST) and
incubated overnight at 4°C with primary antibody (FOXA1, Santa Cruz;
ERa, Santa Cruz; GATA3, Santa Cruz; b-actin, Sigma) diluted in 5%-
BSA-1XPBST. Blots were incubated with an HRP-conjugated secondary
antibody (Santa Cruz) diluted in 5%-milk-1XPBST and developed using
ECL reagent (Amersham). Quantification of protein levels was determined
using Image J (Abramoff et al., 2004).
ChIP was performed as previously described (Wittmann et al., 2005). For
the FOXA1 ChIP analysis, MCF7 cells were treated with vehicle or 17b-
estradiol (10–7M) for 45 minutes. Cleared lysate was incubated with either
normal goat IgG or FOXA1 antibody (Abcam, Ab5089). For RNA
polymerase II ChIP analysis, MCF7 cells were transfected with a non-
targeting siRNA or FOXA1 siRNA as described above. Cells were
harvested 36 hours post-transfection. Cleared lysate was incubated with
either normal mouse IgG or RNA polymerase II antibody (Covance,
8WG16). Binding of FOXA1 and RNA polymerase II to the ESR1
proximal promoter was detected using the following primers: 5?-AG-
GCTG-3?. Quantification of precipitated DNA relative to input was
accomplished using Image J (Abramoff et al., 2004).
Significance was determined by Student’s t-test assuming a two-tailed
distribution and equal variance among sample populations.
FOXA1 is expressed in the developing mammary
gland in conjunction with ERa a
The consistent expression of FOXA1 in luminal breast cancers led
us to postulate that FOXA1 may also regulate luminal epithelial
cells in the normal breast. To begin to address this possibility, we
examined the pattern of FOXA1 expression throughout various
stages of murine mammary gland development. Given the ability
of FOXA1 to regulate ERa activity at numerous target genes in
breast cancer, we also assessed the pattern of ERa expression.
FOXA1 is expressed in the majority of body cells (i.e. luminal
progenitors), but is absent from cap cells (i.e. myoepithelial
progenitors) within the terminal end bud (TEB) (Fig. 1A). TEBs
appear at the leading edge of the duct during puberty, and are the
highly proliferative structures required for ductal elongation and
branching of the mammary epithelium throughout its associated fat
pad (Richert et al., 2000). The expression pattern for FOXA1 was
similar to that observed for ERa in the pubertal gland (Haslam and
FOXA1 regulates mammary morphogenesis
Fig. 1. FOXA1 is expressed in the developing mammary gland in
conjunction with ERa a. (A)Representative images of FOXA1 and ERa
IHC in virgin terminal end buds (TEBs) (5 weeks), virgin ductal
epithelium (8 weeks), virgin alveoli (20 weeks), pregnant alveoli (day
18), lactating alveoli (day 2) and an involuting gland (day 5). Within the
TEB, arrows mark the luminal progenitor cells and arrowheads mark the
basal/myoepithelial progenitors. Unfilled arrowheads indicate
expressing cells in the pregnant alveoli and involuting gland. FOXA1
and ERa expression (brown nuclei) is counterstained with Hematoxylin.
(B)Representative image of dual IF for FOXA1 and ERa in virgin ductal
epithelium (n4). The luminal epithelium consists of four populations:
cells co-expressing FOXA1 and ERa (31.8±4.4%) (yellow cells in
‘Merge’), expressing FOXA1 (12.1±5.0%) or ERa (3.8±0.6%) alone
(arrowheads), or expressing neither (52.3±6.8%). Scale bars: 20m.
Nummy, 1992). Both FOXA1 and ERa are maintained in the
ductal epithelium of post-pubertal virgin mammary glands, but the
subset of cells that are positive for either protein decreases within
the virgin alveolar population and is further reduced during
pregnancy, when only a few positive cells are present per field.
Importantly, lobulo-alveoli do not express either FOXA1 or ERa.
Detection of the cell population that expresses FOXA1 and ERa is
restored as the mammary gland undergoes involution. These data
indicate that FOXA1 is present within the structures that are
necessary for puberty-associated mammary morphogenesis (i.e.
TEBs) and in the same developmental stages as ERa. To define
whether FOXA1 and ERa are co-expressed within the same cells,
we performed dual IF within the adult virgin gland (Fig. 1B). At
this stage, approximately 30% of luminal epithelial cells express
both FOXA1 and ERa, whereas a subset of cells express FOXA1
alone, or to a lesser degree, ERa alone. In addition, whereas ERa
is present within the stroma, FOXA1 expression is undetectable
(data not shown).
FOXA1 is essential for mammary ductal invasion
The pattern of FOXA1 expression in the TEB (Fig. 1A) suggests
that it may contribute to mammary morphogenesis. To determine
whether loss of Foxa1 disrupts embryonic development of the
mammary primordium, we analyzed mammary rudiments from
Foxa1+/+(wild-type) and Foxa1–/–(null) mice (Kaestner et al.,
1999) on postnatal day 1 (see Fig. S1A in the supplementary
material). Using whole mount analysis, we observed no difference
between Foxa1+/+and Foxa1–/–rudiments in the number of
terminal ducts or area occupied by ducts (see Fig. S1B in the
supplementary material). These results indicate that FOXA1
expression is not required for embryonic development of the
mammary ductal rudiment.
Other than early rudiment formation, most mammary gland
development occurs postnatally with the onset of puberty. Thus,
we next examined the impact of FOXA1 on postnatal mammary
morphogenesis in Foxa1–/–mice. These mice exhibit postnatal
lethality as a result of severe hypoglycemia and dehydration
(Kaestner et al., 1999). Thus, we implemented two well-
established rescue strategies to investigate postnatal mammary
gland development: orthotopic transplantation and renal capsule
grafting (Cunha et al., 2000; Robinson et al., 2000). The
orthotopic transplantation paradigm examines whether epithelia
are capable of growing and invading the wild-type stroma in
response to the pubertal and post-pubertal hormonal milieu of
the recipient. Mammary anlagen were collected from E14
Foxa1+/+or Foxa1–/–embryos and inserted into the cleared fat
pads of wild-type syngeneic recipient females (Fig. 2).
Transplanted glands were retrieved from recipient mice 5 and 8
weeks later (Fig. 2A,B). In addition, a subset of the 8-week
recipients was mated, and transplanted glands were collected
from mice with a verified pregnancy (Fig. 2C). At 5 weeks post-
transplant, 50% (3/6) of the wild-type anlagen formed mammary
ductal outgrowths. This take-rate is consistent with previous
reports using this approach (Chakravarty et al., 2003; Robinson
et al., 2001). In contrast to the wild-type donor glands, no
detectable outgrowths occurred from Foxa1–/–anlagen (0/5). At
8 weeks post-transplant, 59% (10/17) of the wild-type vs 0%
(0/9) of the Foxa1–/–anlagen formed mammary ductal
outgrowths. Even when exposed to pregnancy-associated
hormones, all of the Foxa1–/–mammary anlagen failed (0/9) to
develop, whereas 71% (5/7) of the wild-type anlagen formed
outgrowths with extensive alveoli. Although FOXA1 is not
necessary for formation of the primordial ductal tree at postnatal
day 1 (see Fig. S1 in the supplementary material), these results
reveal that the presence of FOXA1 in the mammary epithelium
is essential for the ductal outgrowth that occurs with puberty.
The complete absence of ductal outgrowth in transplanted
glands from Foxa1–/–mice could be a result of either an invasion
defect of the developing gland, or a loss of epithelial cells from
the transplanted embryonic anlagen. To test this directly, we
utilized renal capsule grafting of postnatal day 1 glands (Cunha
et al., 2000). This approach maintains the endogenous epithelial-
stromal architecture, rather than requiring the formation of a
tissue recombinant. In the case of Foxa1–/–glands, the intact
mammary rudiment/fat pad serves as an ideal source of donor
tissue because the glands have a normal rudimentary ductal
structure postnatally (see Fig. S1 in the supplementary material).
The grafts were harvested 2 weeks after transplantation, and
whole-mount analysis revealed numerous TEBs actively
invading the fat pad within wild-type glands (Fig. 3A). By
contrast, TEBs are not present in Foxa1 null glands. Rather, the
rudimentary ducts have not extended into the fat pad. Hence,
loss of Foxa1 leads to failed development of TEBs and a
subsequent inability to invade the mammary fat pad in response
to an adult milieu of mammogenic hormones. Whole mount
analysis at 4-5 weeks post-transplant demonstrates that even
after wild-type glands have completed invasion of the fat pad,
Foxa1–/–glands remain severely dysmorphic (Fig. 3B).
The TEB contains a high frequency of luminal progenitors
(Smalley and Ashworth, 2003), and FOXA1 is expressed in a
subset of the body cells in this structure (Fig. 1A). To determine
whether Foxa1 is expressed specifically within luminal progenitors,
we compared expression levels between sorted epithelial cell
populations enriched for normal mammary stem cell (MaSC),
CD61-positive luminal progenitor and mature luminal epithelial
populations (Fig. 3C) (Asselin-Labat et al., 2007). When compared
with the MaSC-enriched population, Foxa1 mRNA expression is
increased in the luminal progenitor population, and is further
Development 137 (12)
Fig. 2. FOXA1 is required for mammary ductal outgrowth in an
orthotopic transplantation model. (A-C)Representative whole
mounts of ductal outgrowths arising from mammary anlagen collected
from E14 Foxa1+/+and Foxa1–/–mice and transplanted into cleared fat
pads of 3- to 4-week-old syngeneic C57BL/6 recipients. (A)Recipients
aged 5 weeks post-transplant. (B)Recipients aged 8 weeks post-
transplant. (C)Recipients aged 8 weeks post-transplant with
subsequent pregnancy (18.5 dpc). Epidermal cysts form as a result of
co-transplantation of hair follicles along with the mammary gland. The
number and percentage of mammary outgrowths for each donor
genotype is indicated. Scale bars: 2 mm. *, epidermal cysts.
increased in mature luminal cells. These results, in combination
with the pattern of expression of FOXA1 in the TEB, suggest that
FOXA1 may contribute to specification of the luminal lineage. To
test this directly, we analyzed the expression of proteins that
distinguish luminal [E-cadherin (CDH1 – Mouse Genome
Informatics) and cytokeratin-8 (CK8; KRT8 – Mouse Genome
Informatics)] and basal/myoepithelial [a-smooth muscle actin (a-
SMA; ACTA2 – Mouse Genome Informatics)] lineages (see Fig.
S2 in the supplementary material). Expression and localization of
CK8, E-cadherin and a-SMA are unaltered in renal grafts of
Foxa1–/–glands harvested at 4-5 weeks post-transplantation. Both
lineages were also observed in Foxa1–/–renal transplanted glands
harvested at 2 weeks post-transplant and during pregnancy (data
not shown). Hence, FOXA1 is not necessary for lineage
specification, but is essential for expansion and invasion of ductal
The blockade of ductal invasion observed in the complete
absence of Foxa1 led us to investigate whether Foxa1 displays
haploinsufficiency. Unlike Foxa1–/–mice, Foxa1+/–mice are
viable, precluding the need for transplantation. Mammary gland
development was analyzed at both mid-puberty (5 weeks) and late-
puberty (7 weeks), and a significant decrease in ductal invasion
was observed at both time points (see Fig. S3A-D in the
supplementary material). Ovariectomy and estradiol plus
progesterone (E+P) replacement did not rescue this defect (see Fig.
S3E,F in the supplementary material), indicating that it is not the
result of an ovarian steroid deficiency. Foxa1+/–mice are capable
of lactating (data not shown); thus loss of a single allele delays, but
does not prevent, mammary gland development. Growth inhibition
was associated with an increase in epithelial apoptosis without a
change in proliferation (see Fig. S3G-J in the supplementary
material). These data suggest that the expression level of FOXA1
may be crucial for ductal expansion and invasion as a consequence
of its regulation of ductal cell survival.
Alveologenesis is independent of FOXA1
The mammary luminal lineage terminally differentiates into
secretory lobulo-alveolar cells during pregnancy and lactation. To
determine whether FOXA1 is necessary for alveolar differentiation,
recipients of renal capsule grafts were mated at 4-5 weeks post-
transplantation, and transplanted glands were harvested at 18.5 dpc.
Alveoli fill the fat pads of wild-type glands, whereas Foxa1–/–
glands remain severely hampered from invading the surrounding
stroma (Fig. 4A). However, histological evaluation of epithelium
in Foxa1–/–glands revealed the presence of alveoli immediately
surrounding the truncated ducts that contained lipid droplets
indistinguishable from wild-type controls (Fig. 4A). Expression of
milk protein was confirmed in both wild-type and Foxa1–/–glands,
indicating that the stunted, non-invaded Foxa1–/–glands can
undergo terminal differentiation (Fig. 4B). Consistent with these
data, the E+P-treated Foxa1+/–mammary glands have increased
alveoli compared with Foxa1+/+controls (see Fig. S3E,F in the
supplementary material), suggesting that suppression of FOXA1
may promote alveologenesis.
FOXA1 regulates mammary morphogenesis
Fig. 3. FOXA1 is required for TEB formation and ductal invasion.
(A,B)Representative whole mounts of renal grafts of Foxa1+/+and
Foxa1–/–mammary glands (into wild-type C57BL/6 recipients) harvested
at (A) 2 weeks (+/+, n4; –/–, n3) and (B) 4-5 weeks post-
transplantation (+/+, n4; –/–, n4). Broken lines outline the mammary
fat pad. (C)Quantitative real-time PCR of Foxa1 mRNA levels in the
MaSC-enriched population (CD24+/CD29hi), the luminal progenitor
population (CD24+/CD29lo/CD61+), and the mature luminal
population (CD24+/CD29lo/CD61-) isolated from wild-type FVB/N
inguinal mammary glands (n10 per independent experiment). The
results of two independent cell-sorting experiments are shown. Values
were normalized to 18S rRNA (Exp#1) or Gapdh mRNA (Exp#2) and
then expressed relative to the values obtained with the mature luminal
population. Scale bars: 1 mm.
Fig. 4. FOXA1 is not required for alveolar differentiation during
pregnancy. (A)Representative whole mounts and Hematoxylin and
Eosin-stained sections (+/+, n5; –/–, n3) and (B) images of milk protein
IHC (brown) (+/+, n3; –/–, n3) in renal grafts from Foxa1+/+and
Foxa1–/–mammary glands harvested 4-5 weeks after transplantation
and during late pregnancy (18.5 dpc). Sections were counterstained
with Hematoxylin. Scale bars: 0.5 mm in A; 20m in B.
FOXA1 is required for ERa a expression in the
The inability of the Foxa1–/–glands to properly invade the
mammary fat pad is a phenocopy of the ERa-knockout (aERKO)
mouse (Feng et al., 2007; Mallepell et al., 2006; Mueller et al.,
2002), and FOXA1 expression colocalizes with ERa in ~30% of
luminal cells, suggesting that Foxa1 may be epistatic with Esr1
within luminal cells. Analysis of ERa expression in renal
transplanted mammary glands revealed that ERa is undetectable
within the epithelium of Foxa1–/–glands (Fig. 5A). By contrast,
ERa is readily detected in the epithelium of wild-type transplanted
controls and in the stromal population of both wild-type and
Foxa1–/–glands. To confirm that ERa activity is lost in Foxa1–/–
glands, we assessed expression of the progesterone receptor (PR),
an established transcriptional target of ERa (Clarke et al., 1997).
Similar to ERa, PR expression is undetectable in Foxa1–/–
epithelium, although maintained in wild-type glands and Foxa1–/–
stroma (Fig. 5A). PR mRNA (Pgr) is similarly decreased (Fig. 5B).
These data indicate that FOXA1 is necessary for ERa and PR
expression and that this requirement is epithelium-specific. The
percentage of epithelial cells expressing ERa and PR was
unchanged in pubertal Foxa1+/–versus Foxa1+/+control glands,
(51±6% vs 51±2%, 48±3% vs 47±2%, respectively; n3-4 per
group), indicating that retention of one FOXA1 allele is sufficient
to maintain the percentage of cells expressing these receptors.
To verify that FOXA1 is upstream of ERa during normal
mammary gland development, we analyzed FOXA1 expression in
ERa knockout (Ex3aERKO) mice. These mice are devoid of all
ERa transcriptional activity as a result of genomic deletion of exon
3, the coding region for the DNA binding domain, in Esr1. Foxa1
mRNA and FOXA1 protein levels are maintained in Ex3aERKO
mammary glands compared with wild-type controls (Fig. 5C,D).
Combined, these results indicate that FOXA1 functions upstream
of, and is necessary for, ERa expression in the normal mammary
It has been proposed that FOXA1, ERa and GATA3 collaborate
during mammary morphogenesis (Kouros-Mehr et al., 2006); thus
we also evaluated Gata3 expression in the absence of FOXA1. We
found no significant change in Gata3 mRNA in Foxa1–/–glands
(Fig. 5B), indicating that FOXA1 is not required for Gata3
transcription in the mammary gland. In addition, the presence of
Gata3, but absence of ERa, in Foxa1–/–epithelium suggests that,
in contrast to breast cancer cells (Eeckhoute et al., 2007),
transcription of Gata3 in normal mammary epithelium may be
independent of ERa. This dichotomy was further confirmed by the
sustained expression of Gata3 mRNA in Ex3aERKO mammary
glands (Fig. 5E). We also evaluated whether GATA3 regulates
expression of FOXA1 using mammary glands deficient for Gata3
(MMTV-cre;Gata3f/f) (Asselin-Labat et al., 2007). FOXA1
expressing cells in MMTV-cre;Gata3f/f(null) versus Gata3+/f
(intact) controls were indistinguishable (Fig. 5F).
FOXA1 regulates transcription of ESR1
Loss of ERa in Foxa1 null mammary glands could be a result of
either a loss of FOXA1/ERa-expressing cells or a specific
requirement for FOXA1 to induce expression of ERa. To
determine whether FOXA1 regulates expression of ERa, we
silenced FOXA1 expression and assessed the impact on ESR1
mRNA and ERa protein expression in MCF7 (Fig. 6A-C) and
T47D (see Fig. S4 in the supplementary material) breast cancer cell
lines, both of which endogenously express FOXA1 and ERa
(Williamson et al., 2006). Transient knockdown of FOXA1 resulted
in a significant reduction in ERa protein levels in both cell lines
(Fig. 6A,B; see Fig. S4A,B in the supplementary material),
recapitulating the loss of ERa in Foxa1 null mammary glands.
ESR1 mRNA levels were also significantly decreased, suggesting
that FOXA1 may regulate its transcription (Fig. 6C; see Fig. S4C
in the supplementary material). Importantly, knockdown of FOXA1
in MCF7 cells did not affect GATA3 mRNA or GATA3 protein
levels (data not shown and Fig. 6A), providing additional evidence
that FOXA1 is not required for GATA3 expression.
Development 137 (12)
Fig. 5. FOXA1 is required for expression of ERa a in the normal
mammary gland. (A)Representative images of ERa and PR IHC
(brown nuclei) in renal grafts from Foxa1+/+and Foxa1–/–mammary
glands harvested 4-5 weeks post-transplantation (+/+, n3; –/–, n3).
ERa and PR are maintained in the stroma of Foxa1–/–glands (arrows).
(B)Foxa1, Pgr and Gata3 mRNA levels in renal transplanted Foxa1+/+
and Foxa1–/–mammary glands. Values represent the average ± s.d. and
are relative to Krt8 mRNA (+/+, n3; –/–, n3; *P<0.01). (C)Quantitation
of Foxa1 mRNA and (D) representative images of FOXA1 IHC (brown) in
wild-type and Ex3aERKO mammary glands. Values represent the
average ± s.d. and are relative to Krt8 mRNA (wild-type, n3;
Ex3aERKO, n3). (E)Gata3 mRNA levels in wild-type and Ex3aERKO
mammary glands. Values represent the average ± s.d. and are relative
to Krt8 mRNA (wild-type, n3; Ex3aERKO, n3). (F)Representative
images of FOXA1 IHC in Gata3+/fand MMTV-cre;Gata3f/fmammary
glands (+/f, n3; f/f, n3). IHC quantification is depicted in the bottom
right corner of each image. All sections were counterstained with
Hematoxylin. Scale bars: 20m. NS, not significant.
To investigate the mechanism underlying regulation of ERa by
FOXA1, we queried a publicly available dataset of genome-wide
FOXA1 binding sites in MCF7 cells (Lupien et al., 2008). This
dataset indicates that FOXA1 binds to ten distinct regions of the
ESR1 gene, with five sites in the promoter and five in intragenic
regions (Fig. 6D). We then confirmed FOXA1 binding to one of
these predicted regions within the ESR1 proximal promoter through
ChIP followed by site-directed PCR. FOXA1 binds to this region
independently of estradiol treatment (Fig. 6E). To ascertain whether
FOXA1 regulates transcription of ESR1, we examined binding of
RNA polymerase II to the ESR1 proximal promoter following
transient knockdown of FOXA1 (Fig. 6F). Silencing FOXA1
reduces RNA polymerase II binding by ~50%, which is
comparable to the reduction in ESR1 mRNA levels after FOXA1
knockdown (Fig. 6C). Combined, these data reveal a previously
unrecognized requirement for FOXA1 in regulating ERa
expression, suggesting that FOXA1 may directly regulate ESR1,
although these experiments do not rule out an indirect effect of
FOXA1 on ESR1 transcription.
FOXA1 is necessary for both ERa a expression and
Previous studies using breast cancer cells revealed that FOXA1 is
required for ERa binding to target gene promoters, and subsequent
estrogen responsiveness (Carroll et al., 2005; Laganiere et al.,
2005). We predicted that FOXA1 might function similarly during
mammary morphogenesis. We found that FOXA1 and ERa follow
identical expression patterns throughout normal development, and
are co-expressed in luminal epithelial cells. Our studies also
revealed that FOXA1 is unnecessary for embryonic development
of the mammary rudiment, but is required for mammary ductal
invasion in three different models: orthotopic and renal capsule
transplantation, and Foxa1 heterozygous null mice. The absence of
TEBs in renal transplanted Foxa1 null glands, along with the
presence of Foxa1 in the luminal progenitor population, indicates
that FOXA1 is essential for ductal lineage expansion and
morphogenesis (see model, Fig. 7). The loss of epithelial ERa in
Foxa1 null glands provides a specific mechanism for this
phenotype because ERa is also essential for TEB development and
ductal morphogenesis (Feng et al., 2007; Mallepell et al., 2006;
Mueller et al., 2002).
Depletion of epithelial ERa with deficiency of Foxa1 could
result from regulation of ERa expression by FOXA1 or a loss of
differentiated cells that can express ERa, as seen in GATA3-
depleted mammary glands (Asselin-Labat et al., 2007; Kouros-
Mehr et al., 2006). Complementing our observations in the
developing mammary gland, transient suppression of FOXA1
results in decreased transcription of ESR1 and protein expression
of ERa in breast cancer cells. Hence, FOXA1 not only mediates
ERa activity as has been described (Carroll et al., 2005; Laganiere
et al., 2005), but is also essential for sustained ERa expression.
These data reveal that FOXA1 tightly regulates ERa activity
through two distinct mechanisms, i.e. basal expression and
Previous reports examining a role for FOXA1 in mediating ERa
binding to target gene promoters did not observe a decrease in ERa
expression upon transient knockdown of FOXA1 (Carroll et al.,
2005; Eeckhoute et al., 2006; Laganiere et al., 2005). The disparity
between these results might be explained by variation in
experimental conditions. For the studies reported herein, changes in
ERa in response to transient knockdown of FOXA1 were observed
using media containing hormone-replete serum. By contrast,
previous studies in which sustained ERa occurred following FOXA1
silencing were performed under hormone deprivation. The presence
of estradiol substantially decreases the stability of ESR1 mRNA and
protein (Reid et al., 2002). Thus, the experimental paradigm used
herein likely maintains a higher turnover rate of ESR1 mRNA and
ERa protein, and thus, is permissive to detecting changes in
expression as a result of FOXA1 silencing.
Expression of Gata3 is independent of FOXA1 and
FOXA1, ERa and GATA3 are positively correlated in breast
cancer, and ERa appears necessary for GATA3 expression in breast
cancer cell lines (Eeckhoute et al., 2007). However, Gata3
FOXA1 regulates mammary morphogenesis
Fig. 6. FOXA1 regulates transcription of ESR1.
(A-C)MCF7 cells were transiently transfected with non-
targeting or two different siRNAs targeting FOXA1 (si#1 and
si#4). (A)Representative immunoblots of FOXA1, ERa and
GATA3 (I3) [*, mutant form of GATA3 (Usary et al., 2004)].
(B)Quantitation of ERa protein levels relative to b-actin. Bars
represent the mean of three experiments ± s.d. (*P<0.01;
**P<0.005). (C)Quantitation of ESR1 mRNA levels. Bars
represent the mean of three experiments ± s.d. relative to
GAPDH mRNA (*P<0.005). (D)ESR1 is comprised of eight
exons and at least seven promoters (only A and F are shown)
(Reid et al., 2002). Regions previously identified to bind
FOXA1 by ChIP-chip are indicated by black boxes (Lupien et
al., 2008). (E)Representative (n3) FOXA1 ChIP of the ESR1
promoter using primers amplifying a predicted binding site (*
in D). MCF7 cells were treated with and without 17b-
estradiol (E2). (F)MCF7 cells were transiently transfected with
NT or FOXA1 si#1. Quantification of RNA polymerase II ChIP
of the ESR1 promoter (n3). Bars represent the average fold
change relative to input and normalized relative to NT ± s.d.
(*P<0.0005). NT, non-targeting siRNA.
expression is sustained in Foxa1 null mammary glands that also
lack detectable ERa. In addition, Gata3 mRNA is maintained in
mammary glands that lack functional ERa, providing further
evidence that ERa is not necessary for Gata3 expression in normal
mammary epithelium. These data reveal that expression of Gata3
occurs independently of FOXA1 and ERa during lineage
specification. We confirmed these data by silencing FOXA1 in vitro
and found that GATA3 remains constant even with a reduction in
ERa. These results contrast with previous analyses of breast cancer
cell lines (Eeckhoute et al., 2007). To reconcile these data, we
propose that although ERa may not be required for Gata3
expression under normal conditions, it may become necessary
during tumorigenesis. It is also important to note that the FOXA1
knockdown in breast cancer cells presented herein resulted in only
a 50% reduction in ERa expression, which may be sufficient to
It has also been suggested that GATA3 regulates ERa
expression in breast cancer cell lines (Eeckhoute et al., 2007).
Although our results do not refute this conclusion, they do
indicate that GATA3 alone is insufficient to maintain ERa in the
absence of FOXA1. This conclusion stems from the loss of ERa,
but not GATA3, that occurs both in Foxa1-null glands and with
transient silencing in breast cancer cells. Lastly, GATA3 was
previously reported to bind to the Foxa1 promoter in primary
mammary cells (Kouros-Mehr et al., 2006) and induce
expression of FOXA1 in mammary tumors (Kouros-Mehr et al.,
2008) and a kidney cell line (Usary et al., 2004). We found that
FOXA1 expression is maintained in glands deficient for Gata3,
indicating that GATA3 is not necessary for Foxa1 expression
during normal development.
Development of the mammary ductal, but not
alveolar lineage is dependent on FOXA1
Both orthotopic and renal transplantation models used herein
revealed that Foxa1-null glands were unable to invade the
mammary fat pad in response to pregnancy-associated hormones.
However, the rudimentary ductal epithelium that was grafted into
the renal capsule developed differentiated alveoli in response to
pregnancy. Although these alveoli arose from a rudimentary duct
and were substantially fewer in number, they were otherwise
indistinguishable from wild-type glands. These data reveal that
Foxa1 is unnecessary for lobulo-alveolar lineage specification (see
model, Fig. 7) and provide additional evidence that ductal
expansion and alveolar lineage specification are independent
processes. A similar phenotype has been observed in murine
mammary glands lacking amphiregulin (Ciarloni et al., 2007),
ERBB3 (Jackson-Fisher et al., 2008) and FGFR2b (Parsa et al.,
2008), or in glands exposed to exogenous TGFb1 (Daniel et al.,
1989; Silberstein and Daniel, 1987). Interestingly, TEB
development is also disrupted in all of these models. Thus, it is
possible that FOXA1 participates in a signaling network that
includes one or more of these mediators of breast development and
cancer progression (Holbro et al., 2003; McBryan et al., 2008;
Wakefield et al., 2001).
Previous studies have shown that ERa and PR are
independently required for alveologenesis (Brisken et al., 1998;
Feng et al., 2007; Mallepell et al., 2006). Thus, the loss of ERa
and PR in Foxa1-null glands along with the sustained ability to
form alveoli was unanticipated. A trivial explanation for these
data is that although we cannot detect ERa and PR by IHC, low
levels still occur and are sufficiently functional. Supporting this
notion, Pgr mRNA is still present, albeit only at ~10% of normal
levels. Like FOXA1, ERa is not expressed in lobulo-alveoli.
Thus it is not clear whether ERa acts in a cell-autonomous
manner to regulate alveologenesis, or if intercellular
communication or lineage progression involving ERa silencing
is involved (broken arrow in Fig. 7). It is also possible that
FOXA1 maintains the ductal epithelium in an undifferentiated
state, thus inhibiting alveologenesis. The loss of FOXA1 could
then induce alveolar differentiation in response to pregnancy-
associated hormones even in the absence of ERa. This
hypothesis is supported by the enhanced alveologenesis observed
in Foxa1 heterozygotes when treated with pregnancy-level
hormones. Notably, both orthotopic and renal capsule
transplantation models preclude investigating lactational
differentiation in detail because the transplanted glands undergo
involution post-partum due to the lack of suckling (Li et al.,
1997). Hence, conditional knockout of Foxa1 is necessary to
directly examine the function of FOXA1 in lactation and
involution, and these studies are currently underway.
Conclusions and implications
FOXA1 is essential for development and specification of cell fate
in the prostate, liver, kidney, pancreas and lung (Behr et al., 2004;
Besnard et al., 2005; Gao et al., 2005; Kaestner et al., 1999; Shih
et al., 1999). We now describe an indispensable role for FOXA1 in
mammary ductal morphogenesis (Fig. 7). Our studies also reveal
that FOXA1 is necessary for expression of ERa in the normal
mammary epithelium, and modulates transcription of ESR1 in
vitro. Approximately 75% of breast cancers are ERa-positive,
hence these findings have implications in hormone receptor-
positive disease because FOXA1 expression occurs in most, if not
all ERa-positive breast cancers. It is likely that the positive
correlation seen between FOXA1 and the differentiated luminal
breast tumor subtype stems from this previously undefined role of
FOXA1 in regulating the differentiation of the mammary ductal
lineage and controlling ESR1 transcription. We also suggest that
FOXA1 may also modulate other well-known pathways of
tumorigenesis (e.g. amphiregulin-EGFR, heregulin-ERBB3,
TGFb1) providing a possible explanation and function for FOXA1
in breast tumors lacking ERa.
Development 137 (12)
Fig. 7. Schematic of the mammary epithelial cell hierarchy. FOXA1
is expressed in and required for ductal development. GATA3 is
expressed in and required for both ductal and alveolar development
and is independent of FOXA1 expression. ERa is required for ductal and
alveolar development, but is only expressed in ductal cells. This
supports intercellular communication and/or lineage progression from
ERa-positive ductal to ERa-negative alveolar cells (broken arrow).
We thank Kay-Uwe Wagner and Jennifer Yori for instruction in performing
orthotopic transplants, Marie-Liesse Asselin-Labat for assistance with Gata3
deficient mice and Yanduan Hu for technical support for the ChIP analyses.
Paraffin embedding and sectioning was performed by the Case Western
Reserve University Tissue Procurement and Histology Core Facility. The CK8
antibody was developed by Philippe Brulet and Rolf Kemler, and obtained from
the Developmental Studies Hybridoma Bank formed under the auspices of the
NICHD and maintained by the University of Iowa, Department of Biological
Sciences, Iowa City, IA 52242, USA. G.M.B. is supported through a pre-
doctoral fellowship from the Department of Defense (DoD) (W81XWH-06-1-
0712). This work was also supported by the Case Western Reserve University
Comprehensive Cancer Center (P30 CA043703), a University Hospitals of
Cleveland Pathology Research Associates Grant (F.W.A.K. and R.A.K.), the
Division of Intramural Research of the National Institute of Environmental
Health Sciences/NIH (S.C.H. and K.S.K.), an Ohio Innovation Incentive
Fellowship (J. D. Mosley.), the NIH (T32-GM0720, J. D. Mosley; P01
DK049210, K.H.K.; R01 CA090398, R.A.K.), a Fox Chase Cancer Center
Support Grant (P30 CA006927, A.K.G.), the National Health and Medical
Research Council of Australia (J.E.V.) and the DoD (W81XWH-08-1-0347,
R.A.K.). Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material for this article is available at
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