Estrogen modulates mesenchymal-epidermal interactions in the adult nipple

Maintenance of specialized epidermis requires signals from the underlying mesenchyme; however, specific pathways involved remain to be identified. By recombining cells from ventral skin of the K14-PTHrP transgenic mice with those from wild-type, we show that transgenic stroma is sufficient to reprogram wild-type keratinocytes into nipple-like epidermis. To identify candidate nipple-specific signaling factors, we compared gene expression signatures of sorted Pdgfrα-positive ventral K14-PTHrP and wild-type fibroblasts, identifying differentially-expressed transcripts of the WNT, HGF, TGFβ, IGF, BMP, FGF and estrogen signaling. Considering that some of the growth factor pathways are targets for estrogen regulation, we examined the hormone’s upstream role in maintaining the nipple. Ablation of estrogen signaling by ovariectomy produced nipples with abnormally thin epidermis, and we identified TGFβ as a negatively regulated target of estrogen signaling. Estrogen treatment represses Tgfβ1 at the transcript and protein levels in K14-PTHrP fibroblasts in vitro, while ovariectomy increased Tgfβ1 in K14-PTHrP ventral skin. Moreover, ectopic delivery of Tgfβ1 protein into nipple connective tissue reduced epidermal proliferation. Taken together, specialized nipple epidermis is maintained by estrogen-induced repression of TGFβ signaling in the local fibroblasts. D ev el o pm en t • A dv an ce a rt ic le Introduction Vertebrates interact and manipulate their environment using regions of specialized skin. Epidermis in such specialized skin sites often has distinct stratification patterns, expresses unique differentiation markers, and features novel appendages (Billingham and Silvers, 1967). In humans, such sites include nipples, lips, palms, soles, anal and genital skin (Schweizer et al., 1984). Mice share these sites and also feature distinct tail, muzzle and ear skin (Schweizer, 1993). Among these, the nipple stands out for its pivotal role in the mammalian life cycle as a key site for milk removal from mother to offspring. Thickened and highly proliferative, nipple epidermis is uniquely adapted to withstand high mechanical and frictional forces associated with milk delivery (Eastwood et al., 2007; Koyama et al., 2013; Mahler et al., 2004), and it expresses a unique set of keratins and intermediate filament associated proteins, largely absent in the trunk epidermis (Eastwood et al., 2007; Mahler et al., 2004). Despite its important function and unique characteristics, little is known about how this epidermal differentiation state is acquired and maintained. Generally, epithelial specialization is thought to require inductive signals from the underlying connective tissue (Dhouailly et al., 1998). For example, seminal experiments conducted by the Billingham group found that grafts of sole dermis recombined with ear or trunk epidermis produced a thickened and expanded cornified layer, indicating that the mesenchyme specifies the phenotype of the grafted epithelium (Billingham and Silvers, 1967). In other classic experiments, recombination of the palatal epithelium with the cheek connective tissue or vice versa resulted in the de novo keratin expression pattern normally associated with the connective tissue type (Mackenzie and Hill, 1981, 1984; Schweizer et al., 1984). D ev el o pm en t • A dv an ce a rt ic le Additionally, grafting of the non-palmoplantar epidermal cells onto human injured soles led to epidermis adopting palmoplantar phenotype, complete with the expression of the site specific keratin K9 (Yamaguchi, 1999). The lead role of fibroblasts in determining palmoplantar characteristics of the associated epidermis was further confirmed in follow-up experiments (Yamaguchi, 1999). These classic studies suggest a central role for fibroblasts in determinng the anatomical specificity of epidermal cell fate and characteristics. The inductive and maintenance growth factors produced by site-specific and appendage-specific skin fibroblasts are just starting to be defined (Driskell et al., 2013; Driskell and Watt, 2015; Sriram et al., 2015). Perhaps, the most well-studied in this respect are the dermal papilla fibroblasts of the hair follicle, that induce both the formation (Jahoda et al., 1984; Jahoda et al., 1993; Plikus, 2014; Yang and Cotsarelis, 2010) and regenerative cycling of hair follicles (Chi et al., 2013; Clavel et al., 2012; Enshell-Seijffers et al., 2010; Morgan, 2014; Rendl et al., 2005; Sennett and Rendl, 2012). Dermal papilla fibroblast-specific factors are fibroblast growth factors Fgf7 and Fgf10 (Chi et al., 2013; Clavel et al., 2012; Enshell-Seijffers et al., 2010; Morgan, 2014; Rendl et al., 2005; Sennett and Rendl, 2012), bone morphogenetic proteins Bmp4 and Bmp6 (Clavel et al., 2012; Rendl et al., 2005; Rendl et al., 2008), BMP antagonists noggin (Botchkarev et al., 1999; Botchkarev et al., 2002; Rendl et al., 2005), transforming growth factor TGFβ2 (Oshimori and Fuchs, 2012), and many others (Morgan, 2014; Rendl et al., 2005; Sennett and Rendl, 2012). Outside of the hair follicle, skin fibroblasts also feature significant specialization and heterogeneity; however, our understanding of their molecular profiles is still rudimental (Driskell et al., 2013; Driskell and Watt, 2015; Sriram et al., 2015). For instance, recent studies show that papillary (upper) dermis fibroblasts D ev el o pm en t • A dv an ce a rt ic le express higher levels of growth factors that control epidermal proliferation and differentiation, whereas those from the reticular (lower) dermis produce high levels of signaling molecules associated with matrix production (Driskell et al., 2013; Driskell and Watt, 2015; Sriram et al., 2015). These findings imply that transcriptional heterogeneity may be an intrinsic property related to the developmental history of specific fibroblasts. The notion is further supported by the transcriptional profiling studies on skin fibroblasts from different anatomical locations (Chang et al., 2002; Rinn et al., 2006; Rinn et al., 2008). Signature profiles of regionally specific skin fibroblasts are in part maintained by their unique homeobox (HOX) gene activities. For example, in human skin the HOXB genes are expressed in the trunk and nondermal fibroblasts, whereas HOXD4 and HOXD8 are found exclusively in trunk and proximal leg fibroblasts. Intriguingly, HOXA13 is limited in expression to fibroblasts in distal body sites including hands, feet and foreskin, and its activity is required for the expression of the distal-specific Wingless-Int family growth factor WNT5A (Chang et al., 2002; Rinn et al., 2006; Rinn et al., 2008). In mice, differential expression of the HOX gene Tbx15 between dorsal and ventral skin fibroblasts contributes to the differential skin pigmentation along the sagittal body axis (Candille et al., 2004). Taken together, these findings imply that the developmental history of fibroblasts profoundly impacts their inductive and maintenance interactions with the epidermis. The specific developmental origin of the nipple connective fibroblasts that underlie the structure has not been completely established. The mammary gland develops at the interface of what will become dorsal and ventral skin. The mesenchymal cells that underlie the mammary line at E10.5, may be derived from the hypaxial mesoderm (Dhouailly and Oftedal, 2016; Oftedal and Dhouailly, 2013). However, it is not clear whether these or other cells condense around the bud as the D ev el o pm en t • A dv an ce a rt ic le primary mammary mesenchyme at E11 to E12.5 (Sakakura, 1987; Sakakura et al., 1987). The differentiation of the mammary mesenchyme is driven in part by PTHrP signaling from the developing epithelial cells via PTH/PTHrP receptor expessed on the stroma. In turn, inductive signaling from these differentiated fibroblasts is required to produce nipple sheath, which is of distinct evolutionary origin from the gland (Oftedal and Dhouailly, 2013) at E17 (Dunbar et al., 1999; Dunbar and Wysolmerski, 1999; Wysolmerski et al., 1998). Intriguingly, ectopic expression of PTHrP driven by the human Keratin 14 promotor in K14-PTHrP mice (aka KrP mice) dramatically expands the differentiation of the lateral plate mesoderm derivatives to the mammary mesenchyme fate. However, this has minimal impact on cells of the dorsal dermis (Foley et al., 2001). In part, this appears to be the result of increased Bmp4 signaling in the developing ventral skin of the embryo, driving mammary mesenchyme differentiation (Hens et al., 2007). However, other factors such as timing of trangene expression or sensitivity of somitic versus lateral plate-derived mesenchymal cells to PTHrP may be involved (Foley et al., 2001). In the adult female mouse, the ectopic expression of PTHrP results in a hairless skin with a thickened epidermis and complex connective tissue consistent with the nipple (Abdalkhani et al., 2002; Foley et al., 2001; Foley et al., 1998). Whether the fibroblasts that underlie the nipple are simply derived from mammary mesenchyme cells remains to be determined. Nevertheless during skin development these cells appear to have the capacity to induce and maintain the specialized epidermis that characterizes the structure (Foley et al., 2001; Wu et al., 2015). In this study, we set out to define the inductive and signaling properties of the specialized fibroblasts from the nipple skin. We established that KrP fibroblasts robustly induce reprograming of the trunk epidermis toward nipple fate. We also D ev el o pm en t • A dv an ce a rt ic le established the unique transcriptional signature of the KrP fibroblasts and evaluated the impact of key pathways identified by that analysis. Results Fibroblast-induced reprogramming of trunk keratinocytes into nipple-like epidermis The instructive capacity of highly specialized nipple fibroblasts, however, has not been established and this has been limited by the very small size of the appendage in mice. To determine if nipple fibroblasts have the capacity to reprogram the fate of trunk epidermis, a grafting experiment was carried out using purified cells from ventral skin of neonatal female KrP mice (Wysolmerski et al., 1994), in which close to 1/4 of the entire body’s skin is nipple-like (Foley et al., 2001). KrP fibroblasts were recombined with wild-type (WT) neonatal trunk keratinocytes from both dorsal and ventral skin. The resulting grafts were compared to those reconstituted from WT epidermal and dermal cells, as well as the ventral skin of KrP mice (Fig. 1A) (Lichti et al., 2008). When WT keratinocytes and WT fibroblasts were grafted, hairy skin was produced, as previously reported (n=3) (Fig. 1B, left) (Lee et al., 2011; Lichti et al., 2008; Lichti et al., 1995; Lichti et al., 1993). As expected, reconstitution of KrP neonatal keratinocytes with KrP fibroblasts produced pigmented nipple-like skin with a thickened epidermis and without hair follicles (n=3) (Fig. 1B, 1C, right panels). Importantly, grafts that recombined WT neonatal keratinocytes with KrP fibroblasts also resulted in pigmented nipple-like skin (n=3) with similar histology to those reconstituted from KrP cells only (Fig. 1B, third panel and 1C, middle panel). As shown on Fig. 1D and 1E, grafts that contained KrP ventral fibroblasts expressed D ev el o pm en t • A dv an ce a rt ic le nipple epidermis-specific markers, keratin K2e, and expanded filaggrin, whereas WT cell only grafts did not. In normal haired mouse skin melanocytes localize exclusively to hair follicles (Quevedo and Fleischmann, 1980). In contrast, connective tissue of grafts based upon ventral KrP dermal cells contained pigmented cells that we have previously determined to be melanocytes (Abdalkhani et al., 2002) (Fig. 1C, arrows). Thus, dermal cells from KrP ventral skin produce a signaling environment sufficient to induce reprogramming of neonatal trunk keratinocytes toward nipple-like epidermis, and formation of pigmented connective tissue. Pdgfrα is a marker for nipple fibroblasts Platelet-derived growth factor receptor α (Pdgfrα) was previously reported to be a useful marker for isolating dermal fibroblasts from haired skin (Collins et al., 2011). Here, using immunofluorescence, we show that Pdgfrα is expressed in nipple fibroblasts, but not in basal keratinocytes or fibroblasts in WT ventral skin. It is also expressed in ventral dermis of KrP mice (Fig. 2A). Next we evaluated Pdgfrα staining along with staining for fibroblast marker vimentin and found substantial overlap (Fig. 2B). Dual staining for smooth muscle marker Acta2 and Pdgfrα revealed co-expression in blood vessels, arrector pili muscles, smooth muscle-like cells along the lactiferous duct of the nipple, and nests of contractile cells within KrP ventral dermis. This data indicates that Pdgfrα can be used as a marker for isolating specialized fibroblasts from KrP ventral skin, however the Pdgfrα-positive fraction will also include some, albeit few Acta2-positive vascular and muscle cells. Next, fibroblasts were isolated on cell sorting (FACS) from the nipple-like skin of virgin adult female KrP mice and ventral skin of their WT littermates, respectively using anti-Pdgfrα (aka anti-CD140) antibody (Fig. 3A). To purify CD140 population, D ev el o pm en t • A dv an ce a rt ic le we set up a dump channel to exclude cells expressing CD31 (endothelial cells), CD117 (melanocytes), CD45 (hematopoietic cells) or CD49f markers (keratinocytes) (Collins et al., 2011). Next, we harvested both WT and KrP RNAs from three distinct cell populations: CD140/dump cells, CD140/dump cells and viable cells expressing none of the markers (Fig. 3B). There was also almost a two-fold increase of Pdgfrα and an elevated expression of Vim and Col1a2 in KrP comparing to WT CD140 sorted cells. Next, sorted CD140 or dump cell populations were transiently cultured for 48 hours. While cultured CD140 cells had branched cytoplasm and elongated, spindle-like shape, morphologically similar to a typical fibroblast, dump cells, were mostly epithelial in appearance (Fig. 3C vs. 3D). (Kalluri and Zeisberg, 2006). Thus, we confirmed that Pdgfrα is expressed highly on dermal fibroblasts from the ventral skin of both WT and KrP female mice and that it can be used as a marker for their isolation by sorting. Microarray profiling reveals a unique gene expression signature of KrP fibroblasts To identify candidate inductive and maintenance factors present in nipple fibroblasts, we performed microarray-based transcriptome profiling on FACS-sorted CD140 fibroblasts from KrP and WT ventral skin. 123 genes were upregulated and 118 were downregulated in KrP as compared to ventral WT fibroblasts. To identify signaling pathways altered in KrP fibroblasts, differentially expressed genes were subjected to Gene Ontology (GO) analysis and altered signaling of WNT, HGF, TGFβ, IGF, BMP and FGF pathway elements were identified (Fig. 4 Fig. S1). Interestingly, selected extracellular matrix transcripts, including Col1a, as well as metalloproteinase inhibitors Timps were upregulated, while several metalloproteinase genes were downregulated in the KrP fibroblasts. D ev el o pm en t • A dv an ce a rt ic le The differential expression of 30 transcripts (20 increased, 6 decreased and 4 unchanged in KrP relative to WT fibroblasts), many of which are part of the pathways identified by the Ingenuity and GO term analysis, was largely confirmed by qRT-PCR using sorted fibroblasts as well as micro-dissected virgin WT nipples, WT and intact KrP ventral skin. (Fig. S2 and S3). We grew both WT and KrP ventral fibroblasts in vitro for up to 5 passages. and found signature genes remained differentially regulated in KrP relative to WT ventral fibroblasts (Fig. S4). Overall, differential expression of most genes that comprise the KrP fibroblast signature are consistent in the nipple and stable in primary culture (Rinn et al., 2006; Rinn et al., 2008). Estrogen receptor signaling in fibroblasts maintains nipple structure Fibroblasts associated with the mammary gland in mice have long been recognized to be responsive to estrogen (Hiremath et al., 2012) (Cunha et al., 1997). (Wu et al., 2015). Not surprisingly, pregnancy and lactation related hormone receptors, including progesterone receptor (Pgr), oxytocin receptor (Oxtr), and relaxin receptor (Rxsp1) were strongly upregulated in the nipple-like KrP fibroblasts on the microarray analysis (Fig. 5A). Since both oxytocin and progesterone receptors are regulated by estrogen (Lapidus et al., 1998; Zingg et al., 1998), we next performed qRT-PCR analysis on these hormone receptor-regulated genes using intact ventral skin, as well as sorted fibroblasts. Indeed, transcripts for Esr1, Oxtr and Pgr were significantly increased in the WT nipple and KrP nipple-like ventral skin relative to WT non-nipple skin (Fig. 5B). Similar results were also observed for the CD140 sorted KrP fibroblasts (Fig. 5B). Focusing on Esr1, its differential expression between primary cultured fibroblasts from WT vs. KrP virgin female ventral skin was confirmed at the protein level on the Western blot (Fig. 5C) and in the intact WT D ev el o pm en t • A dv an ce a rt ic le nipple and by immunofluorescence (Fig. 5D). At the cellular level, nipple fibroblast had robust nuclear Esr1 expression (Fig. 5D) (McCormack and Greenwald, 1974). These findings suggest that nipple fibroblasts, including these from the KrP mouse model, maintain stable Esr1 expression and are the target sites for estrogen action. To further investigate the role of ovarian hormones in the nipple, we ovariectomized (ovex) pre-pubertal WT and KrP mice and then harvested nipples and KrP ventral skin between 1.5 to 3 months later. We now show that the nipple epidermis in ovexed mice is much thinner than in controls, and the basal layer is less markedly invaginated (Fig. 5E). These changes were accompanied by the diminished expression of the nipple epidermal markers, K2e and filaggrin (Fig. 5F Fig S5). Moreover, nipple size, largely controlled by extracellular matrix (Wu et al., 2015), was ~30% smaller in the ovexed WT mice as compared to age-matched controls, and specialized nipple connective tissue, typically composed of small tightly packed collagen bundles, was markedly diminished in the ovexed KrP mice (Fig. 5E). Thus, the removal of ovarian hormones, including estrogens, impacts both the epidermis and the connective tissue of the nipple. TGFβ signaling is downregulated in KrP fibroblasts In reviewing the growth factor pathways differentially represented in the KrP fibroblasts by GO analysis, we found that several of them can be regulated by estrogen in non-nipple tissues (Hewitt et al., 2010; Knabbe et al., 1987; Yokota et al., 2008). Among these is the TGF pathway, which has been shown to be inhibited by estrogen signaling components at multiple levels (Cherlet and Murphy, 2007; Colletta et al., 1990), including repression of ligand secretion (Knabbe et al., 1987). This prompted us to evaluate Tgfb1 production and signaling in the context of nipple D ev el o pm en t • A dv an ce a rt ic le tissue further. Indeed, Tgfb1 transcript was decreased by 50% in sorted KrP fibroblasts as compared to WT ventral skin fibroblasts (Fig. 6A). Similar decrease, by ~70%, was found in intact WT nipple and KrP skin as compared to ventral WT skin of virgin mice (Fig. 6A). To further validate reduced TGF signaling, we examined the expression levels of TGF-specific phospho-Smad2/3 (pSmad2/3). We show that while nuclear pSmad2/3 expression was observed in most of the ventral skin fibroblasts, but less nipple fibroblasts were positive (Fig. 6B). Secreted Tgfb1 protein (as measured by ELISA) was reduced by ~50% at 48 or 72 hours of culture of primary KrP vs. WT ventral skin fibroblasts (Fig. 6C), while Tgfb1 transcript expression remained stably reduced in cultured primary KrP fibroblasts (Fig. 6C) and this was maintained for 4 passages (not shown). On Western blot, pSmad2/3, but not total Smad2/3 protein was reduced in KrP vs. WT cultured fibroblasts (Fig. 6D). Taken together, these data suggest TGFβ signaling is downregulated in nipple fibroblast. Estrogen regulates Tgfb1 transcript levels in KrP fibroblasts To further establish a functional relationship between estrogen and TGFβ signaling in nipple fibroblast, we employed primary fibroblast culture in stripped serum phenol-red free media. WT or KrP fibroblasts were treated with 0.5 or 10 nM estradiol for 48 or 72 hours, followed by qRT-PCR analysis. For KrP fibroblasts culture, removal of phenol-red, a known weak estrogen mimetic, resulted in Tgfb1 transcript to increase by 50%, whereas addition of 0.5 nM estradiol lowered Tgfb1 to baseline level and 10nM estradiol reduced it by another 50% (Fig. 6E). We also validated estradiol’s effect on Tgfb1 at the protein level by ELISA measurements. While the serum free conditions reduced total Tgfb1 levels in both WT and KrP D ev el o pm en t • A dv an ce a rt ic le fibroblasts, addition of 10 nM of estradiol decreased the protein by 40% in cultures of KrP fibroblasts only (Fig. 6F). Taken together, these in vitro assays demonstrate that Tgfb1 production is specifically repressed by exogenous estradiol in the KrP fibroblast. Next, we evaluated TGFβ signaling in an in vivo system with altered estrogen signaling, the ovexed KrP mice. As shown in Fig. 6G, Tgfb1 transcript was elevated four-fold in the ventral skin of KrP mice that had been ovexed for 16 weeks, whereas the estrogen regulated Esr1 and Pr were reduced by 50% as compared to age matched samples from intact female KrP mice. On Western blot, pSmad2/3 protein was elevated in ovexed vs. non-ovexed KrP mice (Fig. 6H, S6), indicating signaling downstream of TGFβ was elevated in KrP skin under conditions of low estrogen. Dermal but not epidermal Tgfβ partially shifts nipple features towards trunk skin To initially test the impact of Tgfβ on nipple skin, slow peptide-releasing beads were implanted into 8-week old virgin female mouse skin. Beads with or without 0.2 μg of murine recombinant Tgfb1 were implanted into the nipple dermis of WT mice or ventral skin of KrP mice and their effect was analyzed after 7 days. In contrast to buffer-treated control beads, Tgfb1-treated beads showed a trend toward a thinner epidermis in WT nipple and KrP skin and a thinner papillary dermal layer in the transgenic (Fig. 7C). This was accompanied by significant decrease in epidermal BrdU labeling by ~50%. In this experiment, Tgfb1 did not have measurable impact on WT non-nipple skin (Fig. 7C). To investigate whether Tgfb1 overexpression could directly influence nipple epidermis, we used doxycycline to induce transgene expression in the K14-rTA/tetO-TGFb1 mice (Liu et al., 2001), which produces a non-latent porcine form of the ligand. As shown in Fig. S7 and S8, nipple epidermal D ev el o pm en t • A dv an ce a rt ic le thickness and BrdU incorporation were similar in transgene expressing and noninduced double transgenic mice, whereas BrdU incorporation was substantially reduced in the ventral skin of TGFb1 overexpressing mice. Taken together, it appears that overexpression of TGFb1 within the dermis rather than the epidermis has a greater impact on nipple skin. Discussion In this work we have defined key molecular and cellular attributes of the fibroblast population that underlie specialized epidermis of the murine nipple. First, we showed that fibroblasts from newborn KrP mice induce reprogramming of WT trunk skin keratinocytes toward nipple fate. Secondly, we identified unique gene expression signature of the KrP fibroblasts. Next, we showed that Esr1 signaling is among the major pathways active in KrP fibroblasts and that it signals to suppress TGFβ signaling in KrP fibroblast in vitro. We also present the evidence suggesting that reduced TGFβ pathway activity is essential for endowing nipple skin with its unique characteristics in the adult. To-date, palmoplantar fibroblasts are the most well-defined example of sitespecific skin fibroblasts. Their ability to induce reprogramming of keratinocytes into thickened epidermis with unique keratin expression pattern (Yamaguchi, 1999), was attributed to several secreted factors. One of them is Dkk1, a soluble inhibitor of canonical WNT signaling (Yamaguchi et al., 2004; Yamaguchi et al., 2009; Yamaguchi et al., 2008), which also functions to reduce pigmentation of palms and soles by inhibiting melanocyte activity (Yamaguchi et al., 2004; Yamaguchi et al., D ev el o pm en t • A dv an ce a rt ic le 2009). Another factor is the non-canonical WNT ligand Wnt5a, a putative downstream target of Hoxa13 (Rinn et al., 2006; Rinn et al., 2008). Here, we provide the first insight into the signaling network that defines site-specific properties of nipple fibroblasts. Our recombination experiments confirm the leading role of fibroblasts in endowing skin with its regional specificity. We show that KrP stromal cells reprogram WT trunk skin keratinocytes to have nipple-like features, including stratification pattern and marker profile. It is noteworthy that our KrP stromal cells grafts were pigmented, similar to WT nipple and intact KrP ventral skin (Abdalkhani et al., 2002). This suggests that fibroblasts regulate site-specific skin pigmentation pattern, consistent with the previous experiments on palmoplantar fibroblasts (Yamaguchi et al., 2004; Yamaguchi et al., 2009). Commonly, specialized skin sites in mammals undergo physiological changes in adult life. For instance, palmoplantar skin responds to weight bearing by altering epidermal proliferation dynamics to produce a thick, protective callus (Menz et al., 2007). Perineal skin in the anogenital region of the Old World monkeys can dramatically change in synchrony with the altered reproductive states (VandeBerg et al., 2009). The human nipple and areola skin increases in size and pigmentation during pregnancy and lactation (Javed and Lteif, 2013; Neifert et al., 1990). Estrogen is at moderate levels during nipple morphogenesis and Esr1 is dispensable for its formation (Bocchinfuso and Korach, 1997). Our new data from the ovexed mice indicate that estrogen signaling in fibroblasts is necessary for maintaining the thickened epidermis and some connective tissue features of the adult virgin nipple D ev el o pm en t • A dv an ce a rt ic le (Fig. 5). Nipple expands during late pregnancy, when estrogen levels peak, and this is accompanied by high levels of nuclear Esr1 in the fibroblasts (Wu et al., 2015). This prompted us to speculate that estrogen likely triggers changes in the signaling environment required for the production of adult nipple connective tissue, its expansion and maintenance of the unique thickened epidermis. Among several candidate pathways identified in our transcriptomic studies, we focused on the reduction of the TGFβ signaling because it has known effects on fibroblast biology, including regulation of extracellular matrix production, modulation of growth factor secretion, including keratinocyte growth factor and colony stimulating factor Csf1, which are crucial for the regulation of epidermal proliferation and differentiation (Mauviel, 2009; Szabowski et al., 2000). Consistent with this, we now show that short term Tgfb1 bead implantation into the WT nipple and KrP ventral dermis reduced epidermal proliferation, whereas epidermal overexpression of the growth factor failed to do so. One caveat to these Tgfb1 studies is that they both produced a simple ligand in contrast the more complex endogenous form, involving the latency associated peptide as well as latent TGFβ-binding proteins (Robertson et al., 2015). Nevertheless, these findings imply that at least some aspects of the adult nipple epidermal phenotype can be reversed by ectopic activation of the TGFβ signaling and this appears to be an indirect effect of reduced autocrine TGFβ activity in nipple fibroblasts (Mauviel, 2009). We posit that further studies on some of the signaling pathways revealed here could lead to novel regenerative and tissue engineering approaches to nipple replacement. D ev el o pm en t • A dv an ce a rt ic le Material and Methods Mice The K14-PTHrP (KrP) transgenic line (Wysolmerski et al., 1994) was maintained by continual breeding against unrelated C57BL/6 mice. WT mice used in this study were either inbred C57BL/6 or littermates of the KrP mice. All animal use was approved by the Indiana University Institutional Animal Care and Use Committee and was performed in compliance with stipulations of that body. K14-rTA/tetO-TGFb1 mice were fed 1 g/kg doxycycline chow (Bio-Serve, Flemington NJ) for 3 weeks to induce transgene expression. Nipples and skin were harvested after BrdU injection (see below). Inbred 1.5 to 3 month old Foxn1nu/nu BALB/c mice (NU/J Jackson Labs, Bar Harbor, ME) were used as recipients of grafts.