New insights into lineage restriction of mammary gland epithelium using parity-identified mammary epithelial cells
- 3.6k Downloads
Parity-identified mammary epithelial cells (PI-MECs) are an interesting cellular subset because they survive involution and are a presumptive target for transformation by human epidermal growth factor receptor 2 (HER2)/neu in mammary tumors. Depending on the type of assay, PI-MECs have been designated lobule-restricted progenitors or multipotent stem/progenitor cells. PI-MECs were reported to be part of the basal population of mammary epithelium based on flow cytometry. We investigated the cellular identity and lineage potential of PI-MECs in intact mammary glands.
We performed a quantitative and qualitative analysis of the contribution of PI-MECs to mammary epithelial cell lineages in pregnant and involuted mammary glands by immunohistochemistry, fluorescence-activated cells sorting (FACS), and quantitative polymerase chain reaction. PI-MECs were labeled by the activation of Whey Acidic Protein (WAP)-Cre during pregnancy that results in permanent expression of yellow fluorescent protein.
After involution, PI-MECs are present exclusively in the luminal layer of mammary ducts. During pregnancy, PI-MECs contribute to the luminal layer but not the basal layer of alveolar lobules. Strikingly, whereas all luminal estrogen receptor (ER)-negative cells in an alveolus can be derived from PI-MECs, the alveolar ER-positive cells are unlabeled and reminiscent of Notch2-traced L cells. Notably, we observed a significant population of unlabeled alveolar progenitors that resemble PI-MECs based on transcriptional and histological analysis.
Our demonstration that PI-MECs are luminal cells underscores that not only basal cells display multi-lineage potential in transplantation assays. However, the lineage potential of PI-MECs in unperturbed mammary glands is remarkably restricted to luminal ER-negative cells of the secretory alveolar lineage. The identification of an unlabeled but functionally similar population of luminal alveolar progenitor cells raises the question of whether PI-MECs are a unique population or the result of stochastic labeling. Interestingly, even when all luminal ER-negative cells of an alveolus are PI-MEC-derived, the basal cells and hormone-sensing cells are derived from a different source, indicating that cooperative outgrowth of cells from different lineages is common in alveologenesis.
KeywordsMammary Gland Mammary Epithelial Cell Luminal Cell Mammary Stem Cell Whey Acidic Protein
E74-like factor 5
fluorescence-activated cell sorting
green fluorescent protein
human epidermal growth factor receptor 2
mammary epithelial cell
no reverse transcriptase
parity-identified mammary epithelial cell
quantitative polymerase chain reaction
sodium dodecyl sulfate
smooth muscle actin
yellow fluorescent protein
yellow fluorescent protein-negative
yellow fluorescent protein-positive.
Mammary epithelium is maintained by a pool of stem or progenitor cells that upon asymmetric cell division undergo a process of lineage restriction to generate the different epithelial cell types therein [1, 2]. The existence of lineage-restricted cell populations in mammary epithelium has long been recognized [3, 4], and because the molecular portraits of breast tumors revealed a clear separation of breast cancer subtypes based on lineage characteristics , there has been a concerted effort in recent years to map the mammary stem cell hierarchy . However, the extent of lineage restriction and parent-progeny relationships in this hierarchy are still unclear because most of the data showing multi- or bi-lineage potential has been obtained by isolating single mammary epithelial cells and evaluating their progeny either in vitro or by transplantation into de-epithelialized mammary fat pads. For example, several groups have shown that cells belonging to the basal population (based on cell surface markers) have the potential to generate all mammary epithelial cell types when transplanted by themselves in cleared mammary fat pads, and these cells are referred to as mammary stem cells [7, 8, 9]. However, lineage-tracing techniques using a basal cell-specific promoter to permanently label basal cells in intact mammary glands showed that these cells gave rise only to basal progeny but not to cells belonging to the luminal layer . Therefore, lineage potential appears to be more restricted in unperturbed tissue.
Another subpopulation of mammary epithelial cells, parity-identified mammary epithelial cells (PI-MECs), has also been shown to have multi-lineage potential in transplantation assays . In the intact mammary gland, however, it is unclear where PI-MECs are positioned in the lineage hierarchy. PI-MECs are an interesting cell population because they are long-lived and have been suggested to be the cancer cell of origin in Her2/neu-driven tumorigenesis [12, 13, 14]. PI-MECs are identified by a reporter which is irreversibly activated through Cre-mediated recombination . The Cre recombinase is expressed under control of the whey acidic protein (WAP) promoter, which is strongly induced during pregnancy and throughout lactation [16, 17]. After weaning, the secretory milk-producing cells are removed during a process termed involution  and WAP expression becomes undetectable by Western blot . Unexpectedly, initial studies using WAP-Cre and LacZ-reporter mice showed that not all cells that expressed WAP during lactation are removed, but in fact a significant portion of LacZ+ cells survived the involution process . These LacZ+ cells were found to be a major source for the newly developing structures required for milk production in subsequent pregnancies  and are now referred to as PI-MECs .
In the virgin (nulliparous) mouse or in a mouse that has completed the involution process after weaning (parous), the mammary epithelium consists mainly of bi-layered milk ducts . The outer basal layer contains contractile myoepithelial cells as well as the mammary stem cells, and the luminal layer contains both steroid receptor-positive and -negative cells. The steroid receptor-positive cells, or hormone-sensing cells, relay systemic growth signals such as estrogen, progesterone, and prolactin via paracrine signals to neighboring cells, thereby orchestrating dynamic changes in mammary gland morphology [20, 21]. Under the influence of pregnancy-induced hormone surges, side branches sprout from the epithelial ducts. On these side branches, lobules of alveoli are formed to become the sites of milk production . LacZ-staining demonstrated a clear contribution of PI-MECs to these alveolar lobules and PI-MECs are therefore also referred to as lobule-limited progenitors . In these analyses based on intact mammary glands, PI-MECs are presumed to generate both the basal and luminal cells that make up the alveoli . Interestingly, transplantation studies of unsorted mammary epithelial cells showed that PI-MECs can generate all mammary epithelial cell types. However, they had a limited capacity for self-renewal when outgrowths consisted of PI-MECs only and therefore they were designated stem/progenitor cells . Fluorescence-activated cell sorting (FACS) studies on cultured primary cells suggested that PI-MECs are part of the basal population , in line with the position of the mammary stem cells .
In the study presented here, we used a fluorescent reporter gene to analyze the cellular identity and lineage contribution of PI-MECs in unperturbed mammary glands. We demonstrate by both FACS and histology that PI-MECs are steroid receptor-negative luminal cells of the alveolar lineage. Their characteristics are indistinguishable from those of unlabeled alveolar progenitor cells of the same mammary gland. During pregnancy, PI-MECs contribute steroid receptor-negative luminal cells, but not hormone-sensing or basal cells, to developing alveoli.
WAP-Cre mice (Jax#008735)  and Rosa26-lox-Stop-lox-YFP mice  were backcrossed up to four generations on an FVB background. All of the mice used in this study contain one allele each of the WAP-Cre and Rosa-lsl-YFP transgenes. For timed matings, female mice were placed in the cage of a male after 5 p.m. and checked for vaginal plugs at 9 a.m. the following morning (day 0). Mice were euthanized by carbon dioxide inhalation and immediately dissected for thoracic (#3), abdominal (#4), and inguinal (#5) mammary glands at the indicated time points. All animal protocols were approved by the SingHealth Institute for Animal Care and Use Committee.
Imaging and cryosectioning of whole-mounted mammary glands
Thoracic glands were whole mount-photographed on an Olympus SZX-12 fluorescent stereoscopic microscope with green fluorescent protein (GFP) filter (Olympus, Tokyo, Japan), and images were acquired by DP2-BSW software through an Olympus DP72 CCD detector. One #3 gland was subsequently fixed overnight in 4% buffered formaldehyde (ICM Pharma, Singapore) for paraffin embedding. The other #3 gland was fixed 1.5 hours in 2% formaldehyde in phosphate-buffered saline, prior to embedding in Tissue Tek OCT (Sakura Finetek, Tokyo, Japan) for cryopreservation. Cryosections (15 to 30 μm) were cut on a Leica CM1950 cryostat onto SuperFrost Plus-coated slides (Menzel-Gläser, Braunschweig, Germany), and stained with Rhodamine-conjugated phalloidin (Molecular Probes, now part of Invitrogen Corporation, Carlsbad, CA, USA) in accordance with the instructions of the manufacturer. Sections were mounted in Vectashield fluorescence mounting media (Vector Laboratories, Burlingame, CA, USA), and images were acquired on a Zeiss 710 confocal microscope (Carl Zeiss, Jena, Germany) with a pinhole aperture of 1 airy unit.
Fixed #3 mammary glands were processed and embedded in paraffin wax. Paraffin sections of 5 μm were prepared and subjected to 1 mM disodium-ethylenediaminetetraacetic acid (EDTA) antigen retrieval as described previously . Primary antibodies used for immunofluorescence are the following: cytokeratin-8 (CK8) (TROMA-I, rat, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), estrogen receptor (NCL-ER-6 F11, mouse, 1:100; Novocastra, which is part of Leica, Wetzlar, Germany), GFP (600-401-215, rabbit, 1:100; Rockland Immunochemicals Inc., Gilbertsville, PA, USA), GFP (600-141-215, goat coupled to Dylight-488, 1:300; Rockland Immunochemicals Inc.), progesterone receptor (MAB9785, rabbit, 1:400; Abnova, Taipei, Taiwan), and smooth muscle actin (SMA) (A2547, mouse, 1:1,000; Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies used at 1:400 dilution are from Invitrogen Corporation: Alexa488-coupled goat anti-mouse (A11029), Alexa488-coupled goat anti-rabbit (A11034), Alexa568-coupled goat anti-mouse (A11031), and Alexa568-coupled goat anti-rabbit (A11036). Additionally, CF633nm-coupled donkey anti-rat (20137–1; Biotium, Hayward, CA, USA) was used at 1:400 dilution.
Isolation of primary mammary epithelial cells for fluorescence-activated cell sorting, RNA, and genomic DNA
Abdominal and inguinal glands were pooled and processed either for single mammary epithelial cell isolation for FACS analysis as previously described  or else processed just to the organoid stage for purposes of harvesting RNA and genomic DNA. At this point, three quarters of the organoid suspension was pelleted for lysis in PureZOL RNA isolation reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and total RNA then was prepared in accordance with the instructions of the manufacturer. The remaining quarter was pelleted and resuspended in 0.1 M Tris-Cl pH 8/0.2 M NaCl/5 mM EDTA/0.2% SDS/0.2 mg/mL Proteinase K for overnight digestion at 50°C. Genomic DNA was precipitated out from the digest with 1 volume isopranol, washed in 70% ethanol, and resuspended in 100 μL of water. A hundred nanograms of genomic DNA was amplified per sample, 35 cycles with Taq polymerase (Fermentas, Vilnius, Lithuania).
Cell labeling, flow cytometric analysis, and fluorescence-activated cell sorting
Fluorochrome-conjugated antibodies were titrated on primary mammary epithelial cells to ensure maximal positive signal-to-background fluorescence ratio. Anti-mouse or anti-rat compensation beads (BD 552843 and 552845, respectively) or both were used for single-stain antibody controls. Compensation controls also included two cellular samples: unstained cells and cells stained with 4′,6-diamidino-2-phenylindole (DAPI) (D8417; Sigma-Aldrich). Cells were incubated with antibodies on ice for 45 minutes with agitation every 15 minutes. Samples were then washed with twice the sample volume and resuspended in L15 (Gibco-Life Technologies, now part of Invitrogen Corporation) with 6% fetal calf serum (FCS) (HyClone, Logan, UT, USA) and 200 ng/mL of DAPI, except non-DAPI compensation controls. All multiple-labeled samples were gated on FSC-A versus SSC-A and doublet discrimination (FSC-H versus FSC-W and SSC-H versus SSC-W) and DAPI negativity. Samples contained anti-CD45 to exclude lymphocytes from analysis. Cells were analyzed and sorted on a BD FACS-Aria II containing 355 nm UV, 488 nm blue, 561 nm yellow-green, and 633 nm red lasers (BD Biosciences, San Jose, CA, USA). Specific antibodies used and gating strategy are detailed in Additional file 1.
Synthesis of cDNA and quantitative polymerase chain reaction analysis
For analysis of transcript levels by quantitative polymerase chain reaction (qPCR) in FACS-sorted populations, cells were sorted directly into lysis buffer (10 IU RNase inhibitor (Invitrogen Corporation), 2 mM dithiothreitol (DTT), 0.15% Tween-20 (Bio-Rad Laboratories, Inc.) in 12 μL of nuclease-free water) in PCR tubes by using a direct reverse transcription (RT) method described by Ho and colleagues . Five hundred cells were sorted into each tube, and RT was performed by using Superscript VILO (Invitrogen Corporation) in accordance with the protocol of the manufacturer. In the case of PureZOL-purified total RNA harvested from epithelial organoids, cDNA was prepared by using the Bio-Rad iSelect kit (Bio-Rad Laboratories, Inc.) and a pool of gene specific RT primers (each at 0.42 mM final concentration), comprising the reverse primers for each of the amplified genes: WAP, Cre transgene, YFP transgene, Elf5, and hypoxanthine-guanine phosphoribosyltransferase (HPRT). Primers were designed that span introns to exclude the detection of genomic DNA and selected for optimum melt curve and amplification profiles. The primers to detect the WAP-Cre transgene expression were designed from the Cre sequence and rabbit β-globin polyA sequence which spans the β-globin intron that is spliced out in MECs (data not shown). All primer sequences are listed in Additional file 2. In the case of the intronless YFP transcript, a no-reverse-transcriptase (NRT) control reaction was run in parallel with the normal RT reaction, and qPCR background signal from the NRT reaction (due to genomic DNA contamination in those samples with low RNA yields) was subtracted from the RT reaction quantification for YFP in the reported results. qPCR was performed by using Sso Fast Evagreen supermix reagent (Bio-Rad Laboratories, Inc.) in accordance with the protocol of the manufacturer. To assess genomic recombination of the Rosa-lsl-YFP locus, primer sequences flanking the loxP sites were designed such that the recombined locus could be amplified from organoid genomic DNA as a 578-bp band (Additional file 3), whereas the unrecombined locus would generate a 3,244-bp band (data not shown).
Genetic labeling of parity-identified mammary epithelial cells
At 3 days post-partum, virtually every alveolus contained only YFP-expressing cells (Figure 2C, see also Additional file 5C), consistent with complete recombination of the Rosa-lsl-YFP locus at this stage and underscoring the efficiency of the WAP-Cre/YFP labeling system. Larger ducts of glands at 3 days lactation often remained negative for YFP, in contrast to secondary ducts or side branches that contain multiple YFPpos cells (Figure 2C, top panel and Additional file 7). Six weeks after weaning, the alveolar structures had been cleared by involution. Only the ductal network and regressed terminal branches remained in the post-involution glands, and YFPpos cells were readily detectable in involuted mammary epithelium (Figure 2D). These YFPpos cells are referred to as PI-MECs, cells present in parous tissue that survived involution. In contrast, a large proportion of cells that were initially labeled during pregnancy terminally differentiated and were removed. Thus, not all YFPpos cells that are present during pregnancy and lactation will become PI-MECs. In parous epithelium, WAP and Cre expression dropped by several orders of magnitude, consistent with loss of detectable WAP protein at this stage . Notably, the baseline WAP mRNA expression remains higher after involution than in virgin mice (Figure 2G), consistent with a recent study from the Bentires-Alj laboratory . Overall, these results were concordant with observations with previously published LacZ and GFP reporters [11, 15, 22, 23, 28].
At day 7 of the second pregnancy, alveoli emerged from the involuted ductal network. In contrast to the first pregnancy, some of these contained mostly YFPpos cells, whereas others were unlabeled (Figure 2E). The majority of these YFPpos cells are likely progeny generated by PI-MECs (this will be addressed later). By day 14 of the second pregnancy, most alveoli were completely labeled by YFP (Figure 2F) but with a marked proportion of alveoli that were still partially labeled or even unlabeled. This suggests that, in addition to PI-MEC-derived alveoli which are already YFP-labeled, some alveoli were derived from unlabeled progenitors that progressively undergo YFP recombination and activation, perhaps at a somewhat accelerated pace compared with the first pregnancy (compare Figure 2B and F). The increase in cells with a de novo recombined YFP reporter coincides with the renewed induction of WAP (and in parallel Cre) during the second pregnancy (Figure 2G, note the logarithmic scale).
Parity-identified mammary epithelial cells are luminal cells that express markers of alveolar progenitor cells
FACS analysis of MECs isolated from involuted mammary glands and stained for Sca1 and CD49b showed that about a quarter of luminal cells fell within the gate for hormone-sensing cells, whereas the remaining three quarters were found within the alveolar progenitor gate (Figure 4B). YFP-negative (YFPneg) cells belonged to both cell types, but the vast majority of YFPpos cells were part of the alveolar progenitor population (Figure 4C). Only around 6% of YFPpos cells were part of the hormone-sensing cell population (Figure 4D). This rare population expresses ERα mRNA to the same extent as the collective hormone-sensing cell population (Additional file 8), suggesting that their FACS profile truly reflects a hormone-sensing identity of these cells. Notably, even though almost all YFPpos cells belonged to the alveolar progenitor population, about 40% of the alveolar progenitor population in the involuted mammary gland lacked YFP expression (Figure 4D). Since secretory alveoli were close to 100% YFPpos at lactation (Figure 2C), this YFPneg population may represent alveolar progenitors that resided in the primary ducts, where most cells are YFPneg even during lactation (Additional file 7). In summary, there appear to be three major populations of luminal cells in involuted mammary epithelium: (a) YFPneg hormone-sensing cells, (b) YFPneg alveolar progenitor cells, and (c) YFPpos alveolar progenitor cells (PI-MECs). Immunofluorescence analysis on tissue sections confirmed the presence of all of these three types of cells and showed their juxtaposition within the luminal layer of ducts in involuted mammary glands (Figure 4E).
We have optimized a direct lysis method for qPCR analysis of limited numbers of cells  and used this method to validate the molecular identity of the cell populations identified by FACS. Estrogen receptor alpha (ERα) and progesterone receptor (PR) expression was largely limited to hormone-sensing cells (Figure 4F), whereas both YFPpos and YFPneg alveolar progenitor populations exclusively expressed the alveolar marker genes Elf5 and β-Casein  (Figure 4G). PI-MECs thus clearly belong to the luminal alveolar lineage within the involuted mammary epithelium.
Initially, we performed a microarray on YFPpos and YFPneg luminal cells in order to identify unique cell surface markers of PI-MECs to facilitate future studies of this cell population without the need for generating parous double transgenic mice. However, the presence of a significant proportion of YFPneg alveolar progenitor cells that express Elf5 and β-casein to the same extent as the YFPpos cells explains why we were unable to identify unique markers for PI-MECs. Apart from YFP, which was enriched approximately 30-fold and validates the sorting procedure, no other transcripts were significantly enriched in the YFPpos population (data not shown). This suggests that the YFPpos and YFPneg alveolar progenitor populations share their transcriptome profile beyond Elf5 and β-Casein.
An unlabeled pool of alveolar progenitor cells is equipotent to parity-identified mammary epithelial cells
To evaluate whether PI-MECs or the unlabeled alveolar progenitor cells were the main source for the development of new alveoli, we analyzed WAP-Cre;Rosa-lsl-YFP females at day 7 of their second pregnancy. In typical lineage-tracing techniques, Cre is activated for a short time period following administration of a chemical inducer . In contrast in PI-MEC labeling, Cre expression is controlled by WAP promoter activity and therefore the progeny of PI-MECs can be traced only during developmental time periods when WAP is not expressed. We chose the 7-day second pregnancy time point because alveologenesis is already apparent but WAP expression is not significantly induced yet ([15, 16] and Figure 2G). Thus, YFPpos alveoli at this time point likely originate from PI-MECs and are not the result of de novo activation of the reporter, which occurs again later in the second pregnancy (Figure 2F).
Overall, the relative contribution of PI-MECs to alveologenesis, as measured by the fraction of total YFPpos alveoli, varied from 4% to 79%. A similar variation was found when four independent animals were analyzed by FACS (27% to 77%, Figure 6H). It is currently unclear whether this variation is stochastic or whether certain conditions favor one population over the other. It should be noted, however, that the females in Figure 5A and B, which show the most divergence in PI-MEC contribution, are littermates who shared nearly identical histories. The proportion of YFPpos alveoli was roughly similar in the right and left mammary glands of each individual mouse (data not shown). This suggests that the relative contribution of PI-MECs and unlabeled alveolar progenitors to alveologenesis is due to systemic regulation rather than independent gland-autonomous effects.
In summary, even though PI-MECs contribute significantly to the generation of new alveoli, a substantial proportion of newly developing alveoli are unlabeled. Therefore, the unlabeled alveolar progenitor cells that are present after involution not only are very similar to PI-MECs in their transcriptome and cell surface markers but also have the ability to contribute to alveologenesis to the same extent as PI-MECs.
Basal and hormone-sensing cells of developing alveoli are not generated by parity-identified mammary epithelial cells
Since PI-MECs can give rise to all mammary epithelial cell types in transplantation experiments [11, 15, 22], we further evaluated their lineage potential in vivo during a normal pregnancy by FACS analysis (Figure 6C). Separating the mammary epithelial population of WAP-Cre;Rosa-lsl-YFP mice at 7 days of the second pregnancy into YFPneg and YFPpos subpopulations showed that the majority of YFPpos cells remained restricted to the luminal cell population (Figure 6D). Only 0.29% ± 0.03% of PI-MEC-derived cells were found in the basal cell compartment, indicating that PI-MECs cannot be the source of the expansion of the basal layer during pregnancy. A closer examination of the luminal subpopulations showed that during pregnancy the hormone-sensing cells lose their CD49b and especially their Sca1 expression (Figure 6E versus Figure 4A), consistent with a previous report . The cellular identity of the FACS populations was again validated by qPCR. Even during pregnancy, more than 98% of PI-MECs remained firmly within the luminal alveolar progenitor population (Figure 6F and G). These data show that, as a rule, PI-MECs remain restricted to the luminal alveolar lineage during pregnancy and do not contribute significantly to the other lineages.
Out of all the hormone-sensing cells analyzed by FACS at the 7-day second pregnancy time point, only 1.7% ± 1.2% were YFPpos (Figure 6H). Even though the percentage of YFPpos cells in the hormone-sensing cell gate was small, it is not noise in the FACS data, because rare cells that were positive for both ER and YFP were detectable by confocal microscopy of tissue sections from pregnant mice (Figure 6I).
Taking these findings as a whole, we conclude that, in contrast to the clear multi-lineage potential of PI-MECs in transplantation assays, the lineage potential of PI-MECs in vivo is almost entirely restricted to luminal ER-negative cells, even during pregnancy.
Parity-identified mammary epithelial cells are restricted luminal estrogen receptor-negative progenitors
We show that PI-MECs are a strictly luminal population, both by FACS analysis (including qPCR validation of the different populations) and by confocal microscopy on tissue sections from primiparous and pregnant mice. Matulka and colleagues  reported that PI-MECs were located at the tip of the basal cloud, the same position as the mammary stem cells. They used a GFP reporter strain to label the PI-MECs and made use of the same cell surface markers (CD24 and CD49f), but an important difference is that the cells were cultured for 3 days before FACS analysis. Based on the reproducibility of our results in all animals examined and the agreement between different methods, we conclude that PI-MECs are a luminal cell type.
In line with the labeling based on expression of one of the milk genes (WAP), we found that PI-MECs have an alveolar identity, based on cell surface expression (Sca1loCD49fhi) and gene expression (Elf5, β-casein). Consistent with previous reports, PI-MECs contribute significantly to alveolar development in pregnancy, and we therefore designate them luminal alveolar progenitor cells.
Unexpectedly, we noted that, at least in primiparous mice, there is a population of YFPneg alveolar progenitor cells that displays characteristics similar to those of PI-MECs. Possibly, these cells resided in the larger ducts during lactation where hardly any cells were YFP-labeled, in contrast to the almost complete labeling of the secretory cells in the alveoli (Additional file 7). Interestingly, there seemed to be a gradient of reporter activation in the ducts because we detected quite a number of YFPpos cells in the smaller ducts of lactating glands. This raises the question of whether PI-MECs might be part of an alveolar progenitor reservoir that is located in the ducts rather than representing fully committed secretory cells that dedifferentiated and reintegrated into the ducts during involution, but a different experimental system is required to address this.
The use of the WAP promoter to label PI-MECs creates a technical challenge to investigate PI-MEC progeny, because new YFPpos cells could be either daughter cells of PI-MECs or the result of activation of the WAP promoter. Even though we found a general increase in baseline activity of the WAP promoter in parous MECs (similar to ) and in early pregnancy, our data indicated that de novo floxing activity was low at day 7 of the second pregnancy and we used this time point to analyze the lineage potential of PI-MECs. We used both FACS and confocal microscopy to analyze the progeny generated by the YFP-labeled alveolar progenitors in early pregnancy and found that PI-MECs can produce all the steroid receptor-negative luminal cells of an alveolus; however, as a rule, they do not generate basal or hormone-sensing cells of alveoli (Figure 7A). The more restricted lineage potential observed in recent lineage-tracing strategies based on intact mammary glands compared with previous transplantation studies has prompted the revision of current models of the mammary stem cell hierarchy [10, 32]. Previously, PI-MECs have been designated (limited) stem cells based on their contribution to multiple lineages in cleared fat pad transplantations [11, 14, 32]. The data presented here warrant a revision of the place of PI-MECs in the hierarchy: strictly into the luminal lineage (Figure 7B). Note that our observation that PI-MECs generate luminal but not basal cells is consistent with a study showing that in intact mammary glands basal cells maintain the basal layer and luminal cells maintain the luminal layer .
Even though it is clear that PI-MECs are remarkably restricted to the luminal alveolar lineage, we observed a small percentage of labeled cells that belonged to the luminal ER+ population by FACS and in tissue sections of early pregnancy. It remains to be determined whether this reflects a low level of plasticity of PI-MECs in situ (Figure 7B) or whether hormone-sensing cells induce WAP expression in particular circumstances. Interestingly, the multipotency of PI-MECs in reconstitution assays suggests that the conditions of transplantation can unlock their lineage restriction. However, cells from the basal layer have the most robust long-term multipotent potential in transplantation assays [9, 33], whereas the reconstitution potential of PI-MECs or luminal populations identified by other methods is more limited [11, 33], suggesting that some lineage restriction remains even in transplantation assays. It will be interesting to investigate what factors control the lineage potential of these different cell populations both in intact mammary glands and in reconstitution assays and to what extent transplant conditions reflect circumstances relevant for tumorigenesis and metastasis.
Alveoli are formed by collaborating cell types
An alternative version of the mammary stem cell hierarchy has been proposed on the basis of transplantation experiments that show ductal-limited and lobule-limited mammary epithelial outgrowths . Instead of a division into a basal and a luminal lineage as discussed above , this alternative model proposes that stem cells give rise to a ductal- and lobule-restricted lineage whereby the lobule-restricted progenitors are thought to give rise to both the basal and luminal cells of alveoli [6, 13]. We did not detect the activity of a bipotent lobule-restricted progenitor, because we did not observe any YFPpos basal cells by histology, and less than 0.1% of basal cells were YFPpos by FACS analysis and could be technical noise. Consistent with our observation that the basal cells in alveoli are derived from a different source are recent lineage-tracing experiments with the Axin2-promoter whereby Cre-ER was activated briefly before puberty. In those experiments, clonal Axin2-traced offspring contributed exclusively to the basal layer of multiple clusters of alveoli . In contrast, when Cre activity was briefly induced in adult females, the authors found evidence that Axin2-traced progeny could contribute to both the luminal and basal layers of alveoli  (Figure 7B). Interestingly, after involution, Axin2-traced cells remained in the luminal layer, suggesting that PI-MECs could potentially be generated from Axin2-traced basal cells. The Axin2-lineage tracing experiments demonstrate that bipotent activity can be detected in intact mammary glands of adult mice; however, it is unclear how common this activity is. Our data show that PI-MECs make a significant, though variable, contribution to alveologenesis, but this does not include basal alveolar cells.
Moreover, PI-MECs generally do not generate the luminal ER-positive cells, even when all ER-negative luminal cells of an alveolus are derived from PI-MECs. Interestingly, lineage-tracing of cells that expressed Notch2 in puberty revealed two previously unrecognized cell types—small string cells and large L cells—and these cell types do not fit in current models of the stem cell hierarchy . The cells from the Notch2-lineage are functionally required for alveologenesis, and the authors showed that each alveolus contains a small proportion of L cells. An inverse picture of what we have observed was found in Notch2-lineage tracing experiments in early pregnancy: alveoli in which only a single L cell is labeled and the remaining luminal alveolar cells are unlabeled and thus derived from a different source . In addition, hormone-sensing cells play an important role in the early stages of alveologenesis due to their secretion of paracrine factors such as receptor activator of nuclear factor kappa-B ligand (RANKL) and insulin-like growth factor 2 (IGF-2) [21, 36, 37, 38]. Together, these observations warrant the investigation of steroid-receptor status of Notch2-lineage traced L cells, and if they prove to be ER-positive, they could be incorporated into the hormone-sensing lineage of the stem cell hierarchy.
Overall, the Notch2-lineage tracing data and the experiments presented here highlight that, as a rule, alveoli develop through collaboration of different cell types (Figure 7B-1) rather than being generated as clonal units from a single progenitor. In the case of cooperative growth, basal cells generate the basal cells of alveoli and luminal alveolar progenitors such as PI-MECs produce luminal ER-negative cells. Hormone-sensing cells may be recruited in small numbers from the ducts and are the likely instigators of alveolar outgrowth [20, 21]. The Axin2-lineage tracing data suggest that under certain circumstances alveolar luminal and basal cells can derive from the same source (Figure 7B-2). It will be interesting to determine whether this includes ER-positive cells and how these different modes for alveologenesis, collaboration versus clonal origin, are regulated.
In intact mammary glands, PI-MECs are strictly luminal and express markers of the secretory alveolar lineage. Notably, an equipotent population of unlabeled alveolar progenitors exists in primiparous glands, raising the question of whether PI-MECs are a unique population or the result of stochastic reporter activation. In line with other studies that showed a more restricted lineage potential of other stem/progenitor populations in intact mammary glands compared with transplantation assays, we found that the potential of PI-MECs in vivo is restricted to luminal steroid receptor-negative cells. Our data underscore that individual alveoli are not necessarily clonal and are frequently formed by cooperative outgrowth of cells from different lineages, including at least one basal cell, one ER-positive hormone-sensing cell, and one restricted luminal alveolar progenitor cell such as a PI-MEC.
We thank Colin Stewart and Nancy Jenkins for providing the WAP-Cre and Rosa-lsl-YFP mice, respectively, Victor Ho for FACS analysis, and Kakaly Ghosh and Bryan Tan for assistance with the mouse experiments. This work was supported by the Singapore Ministry of Health’s National Medical Research Council under its Exploratory Development Grant (NMRC/EDG/0066/2009) scheme, a grant from the Agency for Science, Technology and Research (A*Star Singapore, SSCC/10/022) and the Duke-NUS Signature Research Program.
- 2.Regan JL, Sourisseau T, Soady K, Kendrick H, McCarthy A, Tang C, Brennan K, Linardopoulos S, White DE, Smalley MJ: Aurora a kinase regulates mammary epithelial cell fate by determining mitotic spindle orientation in a notch-dependent manner. Cell Rep. 2013, 4: 110-123. 10.1016/j.celrep.2013.05.044.CrossRefPubMedGoogle Scholar
- 5.Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lønning PE, Børresen-Dale AL, Brown PO, Botstein D: Molecular portraits of human breast tumours. Nature. 2000, 406: 747-752. 10.1038/35021093.CrossRefPubMedGoogle Scholar
- 13.Jeselsohn R, Brown NE, Arendt L, Klebba I, Hu MG, Kuperwasser C, Hinds PW: Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis. Cancer Cell. 2010, 17: 65-76. 10.1016/j.ccr.2009.11.024.CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Boxer RB, Stairs DB, Dugan KD, Notarfrancesco KL, Portocarrero CP, Keister BA, Belka GK, Cho H, Rathmell JC, Thompson CB, Birnbaum MJ, Chodosh LA: Isoform-specific requirement for Akt1 in the developmental regulation of cellular metabolism during lactation. Cell Metab. 2006, 4: 475-490. 10.1016/j.cmet.2006.10.011.CrossRefPubMedGoogle Scholar
- 19.Chapman RS, Lourenco PC, Tonner E, Flint DJ, Selbert S, Takeda K, Akira S, Clarke AR, Watson CJ: Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3. Genes Dev. 1999, 13: 2604-2616. 10.1101/gad.13.19.2604.CrossRefPubMedPubMedCentralGoogle Scholar
- 26.Oakes SR, Naylor MJ, Asselin-Labat M-L, Blazek KD, Gardiner-Garden M, Hilton HN, Kazlauskas M, Pritchard MA, Chodosh LA, Pfeffer PL, Lindeman GJ, Visvader JE, Ormandy CJ: The Ets transcription factor Elf5 specifies mammary alveolar cell fate. Genes Dev. 2008, 22: 581-586. 10.1101/gad.1614608.CrossRefPubMedPubMedCentralGoogle Scholar
- 27.Meier-Abt F, Milani E, Roloff T, Brinkhaus H, Duss S, Meyer DS, Klebba I, Balwierz PJ, van Nimwegen E, Bentires-Alj M: Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium. Breast Cancer Res. 2013, 15: R36-10.1186/bcr3419.CrossRefPubMedPubMedCentralGoogle Scholar
- 29.Shehata M, Teschendorff A, Sharp G, Novcic N, Russell A, Avril S, Prater M, Eirew P, Caldas C, Watson CJ, Stingl J: Phenotypic and functional characterization of the luminal cell hierarchy of the mammary gland. Breast Cancer Res. 2012, 14: R134-10.1186/bcr3334.CrossRefPubMedPubMedCentralGoogle Scholar
- 30.Kendrick H, Regan JL, Magnay F-A, Grigoriadis A, Mitsopoulos C, Zvelebil M, Smalley MJ: Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate. BMC Genomics. 2008, 9: 591-591. 10.1186/1471-2164-9-591.CrossRefPubMedPubMedCentralGoogle Scholar
- 37.Mukherjee A, Soyal SM, Li J, Ying Y, He B, DeMayo FJ, Lydon JP: Targeting RANKL to a specific subset of murine mammary epithelial cells induces ordered branching morphogenesis and alveologenesis in the absence of progesterone receptor expression. FASEB J. 2010, 24: 4408-4419. 10.1096/fj.10-157982.CrossRefPubMedPubMedCentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.