GATA-3 Expression in the Mammary Gland
We devised a microarray strategy to identify novel regulators of mammary development. β-actin-GFP reporter mice were used to visualize the mammary epithelium in vivo (Hadjantonakis et al., 1998
). TEBs, mature ducts, and epithelium-free stromal microenvironments were surgically microdissected from 5-week-old β-actin-GFP mice, and RNA was immediately isolated for analysis (). The RNA expression profiles of the TEB and mature-duct microenvironments were compared to the epithelium-free stroma by using long-oligonucleotide spotted microarrays with 19,500 features. An analysis of the most highly enriched genes in the mammary epithelium revealed that members of the keratin family (keratins 8
) were the most highly enriched genes in the TEB microenvironment, whereas members of the casein family (casein k
and casein α
) were the most highly enriched genes in the mature-duct microenvironment (). In this analysis, we identified GATA-3
as the most highly enriched transcription factor in both the TEB (13.9-fold change) and mature-duct (10.6-fold change) microenvironments. A full analysis of the microarray data can be found elsewhere (Kouros-Mehr and Werb, 2006
Expression of GATA-3 in the Mammary Gland
The Most Highly Enriched Genes in the Mammary Epithelium
We further investigated the localization of the GATA-3 protein by immunofluorescence. GATA-3 was found in all luminal cells of mature ducts, both within pubertal (5-week-old) and adult (12-week-old) virgin mice (). Costaining with α-smooth muscle actin (SMA) revealed that SMA-positive myoepithelial cells were GATA-3 negative, suggesting that GATA-3 was restricted to the luminal cells of the mammary gland (). GATA-3 was also expressed in the body cells of TEBs; however, the intensity of GATA-3 staining was lower in the distal end of the TEB (i.e., nearest to the invading front) compared to the proximal end (). GATA-3 was not found in the cap cells of the TEB (, arrowhead).
GATA-3 Is Necessary for Mammary Development
We used a loss-of-function strategy to study the role of GATA-3 in the mammary gland. GATA-3
null mice display embryonic lethality between days 11 and 12 postcoitum, so we were unable to use these mice to study mammary development (Pandolfi et al., 1995
). Instead, we used the Cre/loxP
recombination system to generate mice that were conditionally deficient in GATA-3
. Mice with a floxed GATA-3
locus were obtained, as described elsewhere (Pai et al., 2003
). In the floxed construct, exons 4 and 5 of the native GATA-3
locus were replaced by a cassette containing exons 4, 5, and 6, flanked by loxP
sites. The loxP
sites were further flanked by a phosphoglycerokinase (PGK
) promoter and an enhanced green fluorescent protein (GFP
) cassette, such that Cre-mediated recombination led to the placement of the GFP
immediately downstream of the PGK
promoter. We thus used GFP as a marker of Cre-mediated recombination to assess the efficiency of GATA-3 deletion.
We first crossed the floxed GATA-3
mouse with the constitutively active MMTV-Cre line, where Cre
expression has been reported in the mammary gland, skin, salivary glands, and other sites (Wagner et al., 2001
). Homozygous floxed mice carrying the MMTV-Cre transgene (MMTV-Cre; GATA-3flox/flox
) were born in normal Mendelian ratios compared to their littermates (data not shown). This suggested that the conditional deletion approach rescued the embryonic lethality that had been observed in GATA-3
null mice. Whereas heterozygous floxed mice expressing MMTV-Cre (MMTV-Cre; GATA-3flox/+
) appeared in all respects normal compared to non-Cre littermate controls (Non-Tg; GATA-3flox/x
), MMTV-Cre; GATA-3flox/flox
exhibited severe defects in their skin and mammary glands beginning at puberty. MMTV-Cre; GATA-3flox/flox
mice began to lose hair shortly after puberty, and by adulthood, these mice displayed total alopecia. The mice also exhibited other major skin defects, including epidermal hyperplasia, dermal fibrosis, and hyperkeratosis (data not shown).
The development of the mammary gland was severely disrupted in MMTV-Cre; GATA-3flox/flox mice. Prior to puberty at day 19 postpartum, MMTV-Cre; GATA-3flox/flox mice contained a rudimentary mammary gland that appeared to be similar to littermate controls by whole-mount inspection (). However, with the onset of puberty, the mammary glands of MMTV-Cre; GATA-3flox/flox mice failed to develop TEBs (). As a result, by 5 weeks postpartum, the ductal epithelium failed to invade into the fatty stroma and remained in a compact and highly defective state (). At 8 weeks postpartum, a few sporadic TEBs emerged in MMTV-Cre; GATA-3flox/flox mammary glands, but these TEBs were incapable of filling the mammary stroma by 30 weeks postpartum, suggesting that GATA-3 played an essential role for normal TEB function (). Moreover, the 8-week-old null outgrowths displayed gross structural defects, including irregular luminal diameters and deficiencies in side branching ().
Constitutive Loss of GATA-3 in the Mammary Gland Causes Defect in Ductal Invasion
Histologic analysis of the MMTV-Cre; GATA-3flox/flox outgrowths further demonstrated an essential role for GATA-3 in mammary development. Hematoxylin and eosin (H&E) staining showed profound defects in the luminal epithelium of 8-week-old MMTV-Cre; GATA-3flox/flox outgrowths (). Whereas MMTV-Cre; GATA-3flox/+ ductal epithelium contained a single layer of luminal cells, the MMTV-Cre; GATA-3flox/flox ductal epithelium contained regions that lacked luminal cells and regions that contained a multilayered luminal epithelium (, arrowheads). Interestingly, immunostaining revealed that these defective outgrowths remained GATA-3 positive despite having undergone recombination (GFP+) (). The multilayered epithelium in the null outgrowths contained a basally located population of GATA-3/GFP double-positive luminal cells and a distinct population of GFP-positive, GATA-3-negative cells in the ductal lumen (, yellow arrowhead). A similar histologic profile was observed in 19-day-old MMTV-Cre; GATA-3flox/flox outgrowths (data not shown).
GATA-3 Is Essential for Mammary Development
To determine the mechanism for the expression of GATA-3 in the 8-week-old MMTV-Cre; GATA-3flox/flox outgrowths, we purified genomic DNA from the recombined (GFP+) cells of MMTV-Cre; GATA-3flox/+ and MMTV-Cre; GATA-3flox/flox mammary glands. PCR analysis using primers specific to wild-type, floxed, and deleted GATA-3 loci revealed that the null outgrowths contained a nondeleted GATA-3 allele despite being GFP+ (). Thus, there was a selective pressure to retain a functional GATA-3 allele in the surviving outgrowths, which further suggested that GATA-3 is essential for mammary development.
GATA-3 Is Necessary for the Maintenance of the Luminal Epithelium in the Adult Mammary Gland
To further analyze the molecular function of GATA-3 in the mammary gland, we crossed the floxed GATA-3
mouse with the mammary-specific, doxycycline-inducible Cre line WAP-rtTA-Cre. Cre
expression in the latter is both highly specific to the mammary gland and tightly regulated by doxycycline administration (Utomo et al., 1999
). This enabled us to study the role of GATA-3 in the mammary gland while minimizing possible secondary effects from GATA-3
deletion in other organs. Furthermore, this approach enabled us to delete GATA-3
in the adult mammary gland after development had taken place.
We administered doxycycline to 12-week-old WAP-rtTA-Cre; GATA-3flox/flox and WAP-rtTA-Cre; GATA-3flox/+ mice and analyzed GFP expression to determine the timing and efficiency of recombination. GFP was not detected in the absence of doxycycline administration, indicating that there was no leakiness of Cre expression. Although GFP was not detectable after a 3 day course of doxycycline, by 5 days of doxycycline, high levels of GFP were observed throughout the ductal epithelium of both WAP-rtTA-Cre; GATA-3flox/flox and WAP-rtTA-Cre; GATA-3flox/+ mammary glands (). GFP was induced in a large fraction of luminal cells but was absent in myoepithelial and stromal cells, suggesting that WAP-rtTA-Cre was highly specific to the luminal epithelium (). Interestingly, the WAP-rtTA-Cre; GATA-3flox/flox mammary glands showed greatly reduced GFP expression after a 14 day course of doxycycline, whereas the WAP-rtTA-Cre; GATA-3flox/+ glands maintained a high level of GFP expression (). Both the relative intensity and the distribution of GFP along the ductal epithelium were reduced (), suggesting that these cells had been lost.
Disruption of Epithelial Architecture in Adult Mammary Glands after Induction of GATA-3 Recombination
On histologic examination, the 5 and 14 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mammary glands showed severe cellular defects in the luminal epithelium. After 5 days of doxycycline treatment, WAP-rtTA-Cre; GATA-3flox/flox mammary glands developed a disorganized multilayered epithelium (). There appeared to be a substantial increase in cell number and heterogeneity of nuclear size and orientation. Detachment of single cells into the ductal lumen was observed, but only sporadically (see below). After 14 days of doxycycline treatment, WAP-rtTA-Cre; GATA-3flox/flox mammary glands showed additional defects in the luminal epithelium, including cell detachment into the ductal lumen, disruption of the ductal architecture, and widespread cell death (). In some areas, individual luminal cells detached from the basement membrane, whereas in other areas, large portions of ductal epithelium detached (). No phenotype was observed in 5 or 14 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/+ mammary glands ().
Immunostaining of WAP-rtTA-Cre; GATA-3flox/flox mammary glands verified that GATA-3 was lost after doxycycline administration. In 5 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mammary glands, we identified two populations of luminal cells: a GATA-3-positive population lining the basement membrane and a GATA-3-negative population that had detached from the basement membrane (, white arrow). Both populations expressed similar levels of GFP, suggesting that only the subset of recombined luminal cells that lost the GATA-3 protein had detached from the basement membrane at this time point. In 14 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mammary glands, we detected a single population of GATA-3-positive, GFP-negative cells. We did not detect GFP-positive cells in histologic analysis, suggesting that recombined cells had undergone negative selection by this time point (). Taken together, these data suggest that GATA-3 is necessary in adult mammary glands to maintain the integrity of the luminal epithelium.
Acute Loss of GATA-3 Leads to the Expansion of Undifferentiated Luminal Cells
To characterize the immediate consequences of GATA-3 deletion in the adult mammary gland, we further immuno-stained the 5 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mammary glands with structural and differentiation markers. Coimmunostaining with the luminal marker keratin 18 and the myoepithelial marker keratin 14 revealed that the multilayered epithelium in 5 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mammary glands was exclusively keratin 18 positive, suggesting that they retained luminal character and had not trans-differentiated into myoepithelial cells ().
Acute Loss of GATA-3 in Adult Mammary Glands Leads to the Expansion of an Undifferentiated Luminal Population
Despite being a luminal cell population, the 5 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox epithelium expressed significantly reduced levels of luminal differentiation markers (). There was a substantial reduction of β-casein immunostaining in the null mammary glands compared to littermate controls (). There was also a reduction of E-cadherin and an absence of ERα in the null cells that exhibited detachment from the basement membrane (). To verify these findings, we compared the RNA expression profiles of 5 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox and WAP-rtTA-Cre; GATA-3flox/+ whole mammary glands by microarray (). The data confirmed that there was a relative increase in the expression levels of the luminal keratins 18, 19, and 8 and a decrease in various luminal differentiation markers (including members of the casein, cadherin, and estrogen receptor families) in the null mammary glands compared to heterozygous controls.
These data suggested that the acute loss of GATA-3 led to an expansion of a luminal cell population that lacked markers of differentiation. To confirm that this phenomenon indeed represented cellular proliferation, we analyzed BrdU and PCNA staining in the 5 day doxycycline-treated mammary glands. WAP-rtTA-Cre; GATA-3flox/flox mammary glands showed a significant increase of PCNA and BrdU-positive luminal cells compared to WAP-rtTA-Cre; GATA-3flox/+ controls, suggesting that the acute loss of GATA-3 led to cell-cycle progression ().
Long-Term Loss of GATA-3 Leads to Luminal-Cell Death and Lactational Insufficiency
We determined the long-term effects of GATA-3 loss by further characterizing the 14 day doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mammary glands. In contrast to the 5 day doxycycline treatment, the 14 day doxycycline treatment led to a relative reduction in the number of keratin 18-positive cells in WAP-rtTA-Cre; GATA-3flox/flox mammary glands compared to controls (). There was also a reduction of E-cadherin and ERα-positive cells after 14 days of doxycycline (). To determine if the reduction in cell number was due to cell death, we immunostained with the apoptosis marker M30, which recognizes a caspase-cleaved epitope of keratin 18. We observed widespread M30-positive luminal cells in 14 day, but not 5 day, doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mammary glands (). This suggested that cell death was not a primary event after GATA-3 loss but was rather a long-term consequence of GATA-3 loss.
Chronic Loss of GATA-3 in the Adult Mammary Glands Leads to Cell Death and Lactational Insufficiency
The lactational competence of doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mice was assessed to determine the functional consequence of GATA-3 loss. WAP-rtTA-Cre; GATA-3flox/flox mice that had received a 14 day course of doxycycline were bred and sacrificed at day 10 postpartum. The mammary glands of WAP-rtTA-Cre; GATA-3flox/flox mothers exhibited severe defects in lactation as a functional consequence of GATA-3 loss, including a reduction in the number and size of milk-producing alveolar units (). As a consequence of the lactational defect, the pups of null mothers displayed a significant reduction in weight at day 10 post-partum (). Doxycycline-treated WAP-rtTA-Cre; GATA-3flox/+ mothers showed normal lactation and exhibited a heterogeneous pattern of GFP expression, indicating mosaicism of recombination in the WAP-rtTA-Cre line (). In contrast, the ductal structures in doxycycline-treated WAP-rtTA-Cre; GATA-3flox/flox mothers were GFP negative, which suggested there was a negative selection for recombined cells in the WAP-rtTA-Cre; GATA-3flox/flox mammary glands ().
GATA-3 Binds to the FOXA1 Promoter in the Mammary Gland
To identify potential downstream effectors of GATA-3 signaling, we performed bioinformatic analysis of our mammary microarray data. We identified 22 transcription factors that were highly enriched in TEBs and mature ducts of the mammary gland, a pattern that mirrors GATA-3
expression (Figure S1A in the Supplemental Data
available with this article online). These genes included FOXA1
, as well as several members of the Id
, and TCFAP-2
transcription factor families. We then analyzed four independent microarray datasets to identify whether any of these genes were consistently correlated with GATA-3
expression in breast cancer samples. The transcription factor FOXA1
emerged as one of the best predictors of GATA-3
expression in these datasets. Of ~24,000 genes, FOXA1
emerged as the most highly correlated gene with GATA-3
in two offour datasets, and it was the second and sixth most highly correlated gene in the other two datasets (Bonferroni-adjusted p < 0.0001 in all datasets, (Figure S1B
). The strong positive correlation between GATA-3
suggested a potential interaction between these transcription factors.
To explore the possible relationship between GATA-3 and FOXA1, we analyzed the 50 kb promoter sequences of mouse and human FOXA1 and identified three conserved GATA binding sites lying 0.8, 1.7, and 3.6 kb upstream of the first exon (rVista 2.0). We performed chromatin immunoprecipitation (ChIP) of primary mammary epithelial cultures with a GATA-3 antibody and then PCR amplified the regions containing these GATA binding sites. The GATA binding site lying 0.8 kb upstream of the FOXA1 first exon coimmunoprecipitated with the GATA-3 antibody, suggesting that GATA-3 binds to the FOXA1 promoter in this region ().