Previous analysis of mammary glands from an MMTV-SmoM2 transgenic mouse showed increased proliferation, and ductal hyperplasia in virgin mice. In this model, SmoM2 expression was driven by the MMTV-LTR in a small percentage of mammary epithelial cells. To ensure that similar changes also occurred in the Cre-dependent conditional model in which SmoM2 was knocked into the ROSA locus, we harvested mammary glands from SmoM2;+ (wild type) and SmoM2;Cre (SmoM2 expressing) animals at 5, 7, 9 and 10 weeks of age and analyzed for overall morphology. Consistent with previous results using MMTV-SmoM2 mice, at 5 weeks of age there was a significant increase in the number of terminal end buds (TEBs), the highly proliferative structures that drive ductal morphogenesis in SmoM2;Cre mice relative to Cre-negative controls (). Accelerated ductal outgrowth was apparent at 7 weeks of age when the ductal epithelia from SmoM2;Cre animals reached the end of the fat pad, in contrast to the ducts from Cre-negative littermates (SmoM2;+) (). At 9 weeks of age, ductal epithelia from both Cre-positive (SmoM2;Cre) and Cre-negative (SmoM2;+) animals completely filled the fat pad, and were morphologically indistinguishable from one another (). By flow cytometric analysis, approximately 45% (44.7 ± 12.78%) of MECs from Cre-positive animals expressed the SmoM2-YFP fusion protein ().
SmoM2 expression leads to increased number of TEBs and hyper-branching of the mammary gland
Whereas glands in SmoM2;Cre and SmoM2;+ mice were indistinguishable at 9 weeks of age, a dramatic phenotypic transition occurred at 10 weeks of age. Glands from SmoM2;Cre animals showed a significant increase (p<0.01) in the number of branches along the mammary ductal tree compared to Cre-negative littermates (). Additionally, alveolar bud-like structures were observed throughout the Cre-positive mammary glands that were reminiscent of alveolar development normally observed during pregnancy. To ensure that this phenotypic change was not simply due to a change in the percentage of cells expressing SmoM2-YFP, we repeated the FACS analysis. Again, approximately 50% (52.2 ± 2.3%) of the MECs from SmoM2;Cre mice expressed the SmoM2-YFP protein (Fig. S2
To determine if the morphological and developmental changes observed were associated with increased proliferation, immunostaining for BrdU incorporation was used as an indicator for cells in S phase. At 5 weeks of age, we observed a significant increase in BrdU incorporation in the TEBs of Cre-positive mammary glands relative to Cre-negative glands (). At 9 weeks of age, the Cre-negative animals exhibit low levels of proliferation in the mammary gland, characteristic of the mature virgin state (), whereas Cre-positive animals showed a significant increase in the number of proliferating cells (). Unexpectedly, at 10 weeks of age, there was no difference in proliferation between SmoM2;Cre and SmoM2;+ mice (), with ductal epithelium showing approximately 2% BrdU positive cells.
SmoM2 expression leads to increased proliferation in the developing mammary gland
Epithelial SmoM2 expression disrupts histoarchitecture, increases periductal collagen deposition, and increases macrophage recruitment
Histological examination of mammary gland sections from 10-week-old mice revealed the presence of alveolar-like structures in the Cre-positive mammary glands as compared to controls (). Cre-positive mammary glands also exhibited dissociation of the luminal and myoepithelial cell layers, and luminal cells had an enlarged, clear cytoplasm as compared to Cre-negative littermates. Additionally, the periductal stroma surrounding the hyper- branched Cre-positive 10-week old mammary glands was thickened with an increase in stromal cellularity. In order to characterize the thickened stroma, Masson’s Trichrome staining was performed, which revealed an increase in collagen deposition surrounding the ducts of the Cre-positive glands (blue staining) (). To help determine the identity of the cells in the periductal stroma, we performed immunohistochemical staining for F4/80, a macrophage marker. This revealed a marked increase in macrophages in the periductal stroma in approximately 50% of the SmoM2;Cre glands () as compared to SmoM2;+ controls (). Taken together, these data indicate that SmoM2 expression in the mammary epithelium results in alterations in the mammary gland stromal microenvironment.
SmoM2 expression leads to histological changes in the mammary gland epithelium and stroma
SmoM2 expression is required in the luminal epithelium, but not in the myoepithelium for the observed mammary gland defects
In order to study the effect of SmoM2 expression in different cell types in the mammary gland, we employed adenoviral-Cre (Ad-Cre) infection in vitro on freshly isolated MECs from SmoM2;+ mice followed by subsequent transplantation. First SmoM2 mice were crossed to the R26R line, a lacZ reporter for Cre expression. MECs from SmoM2; R26R animals and R26R animals as a control were obtained and transduced with Ad-Cre to induce recombination. Ad-Cre elicits recombination in multiple cell types including stem and lineage-restricted progenitor cells. As a result, chimeric outgrowths containing recombined and un-recombined areas, as well as outgrowths in which only luminal or myoepithelial cell-types underwent recombination were obtained. Every outgrowth from the Ad-Cre treated SmoM2 MECs (8/8) was either hyper-branched and budded, or displayed aberrant TEB structures, or in some cases both phenotypes. A representative example of the outgrowths obtained is shown in . Control R26R outgrowths appeared normal and exhibited radial outgrowth as well as a moderate level of side branching (). In contrast, SmoM2;R26R outgrowths exhibited hyper-branching and budding and also appeared disorganized (). A chimeric SmoM2;R26R outgrowth is depicted in . Recombined areas of the outgrowth (illustrated by the blue X-gal staining) showed hyper-branching and budding as compared to the un-recombined (dashed box) confirming that only areas containing SmoM2 expressing cells exhibit the hyper-branched phenotype observed. Significantly, areas of the outgrowths consisting of only a recombined myoepithelial cell layer with un-recombined luminal epithelium (observed in multiple ducts from three independent outgrowths) did not display the hyper-branched and budded phenotype (). Thus, SmoM2 expression in the luminal epithelium is required for the formation of the hyper-branched and budded mammary ductal trees and the changes in luminal, myoepithelial and periductal histology as illustrated in .
Mammary gland reconstitution experiments after Ad-Cre treatment reveal a requirement for SmoM2 in luminal epithelium
The ductal hyperplasia phenotype requires interactions between SmoM2-expressing and wild type cells
In order to examine the consequences of activated Smo in SmoM2-expressing cells, non-expressing cells, and the surrounding environment more effectively, we crossed the SmoM2 animals to mice carrying a dual fluorescent reporter for Cre activity targeted to the Rosa26 locus (mT-mG). Cells in which SmoM2 is activated by Cre-mediated recombination express Cre-dependent EGFP, while cells in which SmoM2 is not activated by recombination remain Tomato Red-positive. Fluorescent imaging of whole mammary glands indicated that neither Cre-negative control mice () nor Cre-positive mice lacking the conditional SmoM2 allele () showed evidence of hyperplasia. In contrast, SmoM2;Cre mice exhibiting recombination (EGFP expression) showed hyper-branching and budding of the mammary ductal tree (). However, areas of the mammary glands that exhibited 100% recombination did not display the hyperplastic phenotype (). The hyper-branched and budded phenotype was only present in chimeric outgrowths containing both recombined (SmoM2-expressing) and un-recombined cells (). These results demonstrate that wild type cells are required in order for the hyper-branching to occur, and further suggest that this phenotype is dependent upon either a paracrine or juxtacrine cell-cell interaction between the SmoM2 expressing cells and nearby wild type cells.
Tagged SmoM2;Cre mammary glands exhibit hyper-branching and budding and reveal a possible requirement for SmoM2-wildtype cell interactions
SmoM2-expressing cells stimulate the proliferation of neighboring wild type cells in a paracrine or juxtacrine manner
To demonstrate whether SmoM2 expressing cells stimulate proliferation of wild type cells in a paracrine or juxtacrine manner, we employed intraductal Ad-Cre delivery to activate SmoM2 and EGFP expression, with contralateral injection of adenoviral-LacZ as a negative control. This technique allowed us to achieve SmoM2 expression in situ
in only a subset of cells, and to examine the effects on surrounding cells at specific times after SmoM2 expression. Two weeks after injections, the glands were harvested following a two hr BrdU pulse and processed for staining. Co-staining for EGFP and BrdU revealed a significant increase in proliferation in cells surrounding SmoM2 expressing (EGFP positive) cells (). Quantification of the staining () demonstrated that 14% (± 4.1%) of the cells exhibited recombination (as determined by EGFP expression) in Ad-Cre treated ducts and Ad-lacZ treated ducts exhibited no recombination. Ad-Cre treated ducts showed a significant increase in BrdU incorporation (2.91 ± 0.8%)as compared to Ad-lacZ treated ducts (0.21 ± 0.1%) (p=0.01). Significantly, in Ad-Cre treated ducts, the majority of proliferation occurred in un-recombined cells(2.67 ± 0.73%), but not in EGFP expressing cells (0.24 ± 0.05% ) (p< 0.01). These results indicate that SmoM2 cells are stimulating the proliferation of the surrounding wildtype cells while not significantly increasing proliferation in the SmoM2 expressing cells themselves. It is important to note that within Ad-Cre treated ducts, the increase in proliferative un-recombined (Tomato Red+) cells was only observed in those ducts containing at least one recombined (EGFP+) cell (Fig. S3
), indicating that the proliferative stimulus from the SmoM2 cells requires either a close proximity or direct contact with wildtype cells.
SmoM2 expression stimulates proliferation of neighboring wildtype cells
SmoM2-expressing cells exhibit increased Hh signaling activity and possible downregulation of Notch pathway activity
In previously published models of Hh signaling activation in the mammary gland, the hallmark transcriptional targets of high level Hh signaling were not detected (Moraes et al., 2009
; Moraes et al., 2007
). Several studies have suggested that canonical Hh signaling must be kept in a repressed state for normal virgin mammary gland development to occur (Hatsell and Cowin, 2006
). In order to determine if SmoM2 expression could elicit a canonical Hh transcriptional response, we took advantage of our tagged mouse model to sort recombined (EGFP) and un-recombined (Tomato Red) MEC populations from SmoM2;Cre mice and wildtype MECs from SmoM2;+ littermates (). Total RNA was isolated from epithelial-enriched cell populations and qPCR was performed. As expected, SmoM2 transgene expression was increased 13-fold (± 4.8) in EGFP-positive SmoM2:Cre MECs compared to the un-recombined Tomato Red-positive population (). illustrates the gene expression of the Hh signal pathway components in all three sorted cell populations. An induction of Gli2 (18 ± 7.2 fold change p=0.03) expression as well as a marked increase of Gli1 (71 ± 24.2 fold change p=0.04) and Ptch2 (55 ± 20.3 fold change p=0.04) was observed in EGFP+ (recombined) SmoM2;Cre MECs as compared to SmoM2;+ cells. An induction (5 ± 0.9 fold) in Gli2 was observed in Tomato Red+ (un-recombined) cells. Additionally, and induction of Smo expression was observed in both recombined (EGFP+) and un-recombined (Tomato Red+) cells from SmoM2;Cre mice as compared to cells from Cre negative litter-mates. These data indicate that activation of canonical Hh signaling occurred in the SmoM2 expressing cells.
SmoM2 expressing cells have active Hh signaling and downregulation of the Notch pathway
Since SmoM2-expressing cells were stimulating proliferation of wildtype cells adjacent or in-close proximity, we next investigated whether expression of members of the Notch signaling pathway, which is known to function in a juxtacrine manner, was altered. illustrates the gene expression patterns of several Notch pathway members in all three sorted cell populations. A marked upregulation of the Notch ligands delta-like 1 (Dll-1), (21 ± 6.8 fold change p=0.02) and Jagged-2 (Jag2) (5 ± 1.0 fold change p<0.01) was detected in EGFP+ (recombined) cells from SmoM2;Cre mice, as compared to wildtype MECs from SmoM2;+ mice. Additionally, the Notch 4 receptor (18 ± 11.9 fold change p<0.01) along with the Hes6 target gene (7 ± 3.7 fold change p=0.03) and the Delta-like 4 (Dll4) ligand (14 ± 9.1 fold change p<0.01) were found to be downregulated in the EGFP-positive cell population. The Notch1 receptor was found to be slightly upregulated (less than 1 fold) in both EGFP and TomatoRed populations. No significant changes were observed in other pathway members tested.
Epithelial SmoM2 expression disrupts luminal-myoepithelial cell interactions and alters luminal epithelial cell differentiation
In order to determine if the morphological changes observed after SmoM2 expression were due to aberrant differentiation and organization of distinct mammary epithelial cell subtypes, we examined the luminal and myoepithelial cell layers by immunostaining with antibodies that detect keratin 8 (K8), a luminal cell marker and keratin 14 (K14), a myoepithelial cell marker (). While both of these markers were readily detected in SmoM2;Cre glands, the typical bilayer of ductal epithelium was disrupted as compared to SmoM2;+ controls. Furthermore, K14-expressing cells were rounded as opposed to their characteristic elongated shape (), and appeared dissociated from the luminal cells and possibly from the basement membrane ( and insets). In this trigenic model, recombination and SmoM2 expression (as measured by EGFP detection) occurs in both luminal and myoepithelial cell layers (Fig. S4
SmoM2 expression leads dissociation between the luminal and myoepithelial cell layers
In order to determine if the dissociation of the mammary gland bilayer was due to expression of SmoM2 in the myoepithelial cell layer, we examined K8-K14 expression in SmoM2/mT-mE glands injected intraductally with Ad-Cre. Serial sections were stained for EGFP and K-14 expression () and K8 and K-14 expression () in order to examine the bilayer interaction in areas with low or no recombination as well as in areas with high levels of SmoM2 expression. Disruption of the luminal and myoepithelial cell layers was only apparent in ducts containing EGFP expression in luminal cells () as compared to ducts containing no EGFP expression (). Ad-lacZ injected glands exhibited the normal pattern of K8 and K14 expression and typical bilayer interaction and appeared indistinguishable from the Ad-Cre injected ducts with no EGFP expression (Fig. S5
). Since recombination in the Ad-Cre injected glands occurs primarily in the luminal epithelium (Fig. S6
), it is likely that SmoM2 transgene expression in only the luminal compartment is sufficient for the observed phenotypes.
To examine the proper differentiation of luminal epithelial cells further, particularly with respect to hyperplastic alveolar buds, we performed immunostaining for a specific marker for the transition from luminal to alveolar cells. The Na-K-Cl co-transporter-1 (NKCC1) is normally expressed throughout the differentiated luminal epithelium of virgin mammary glands and is absent in alveolar cells during pregnancy (Shillingford et al., 2002
). SmoM2;Cre mammary glands showed loss of NKCC1 expression in a large percentage of cells as compared to SmoM2;+ littermates (). This loss of expression occurred not only in the alveolar-like cells as occurs during pregnancy, but also throughout the ductal epithelium in virgin mice. We also examined NKCC1 expression in Ad-Cre and Ad-LacZ injected glands and found that in comparison to Ad-LacZ treated glands () this marker again was absent in Ad-Cre injected glands predominantly in SmoM2 (EGFP+) cells (). Arrows illustrate regions of the duct in which NKCC1 expression is absent.
SmoM2 expression leads to changes in luminal epithelial cell identity
Despite these changes in differentiation, no detectable changes in the expression or distribution of estrogen receptor (ER) or progesterone receptor (PR) were observed (data not shown). Therefore, these studies suggest that although SmoM2;Cre glands still maintain a distinct luminal and myoepithelial cell identity as well as normal levels of hormone receptors, SmoM2 expression in the luminal epithelium is sufficient to cause altered luminal cell differentiation and disrupted interactions between the luminal and myoepithelial cell layers.