EGF-receptor regulates salivary gland branching morphogenesis by supporting proliferation and maturation of epithelial cells and survival of mesenchymal cells

Epidermal growth factor receptor (EGF-R) regulates epithelial morphogenesis during development and is important for the proper branching of the lung, mammary gland, and pancreas. We analyzed the salivary gland phenotype of EGF-R-deficient mice and showed impaired growth, branching, and maturation of the epithelium. Furthermore, treatment of wild-type E13 salivary glands with gefitinib, a small molecular inhibitor of EGF-R, led to apoptosis of the mesenchyme. Interestingly, MMP2 and plasminogen activators were upregulated upon inhibition of EGF-R signaling. To summarize, we show that EGF-R is a physiological regulator of salivary gland development and its main function is to support the proliferation and maturation of the epithelium and the survival of the mesenchyme.     

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.     

Reconstitution of Mammary Epithelial Morphogenesis by Murine Embryonic Stem Cells Undergoing Hematopoietic Stem Cell Differentiation

Background

Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.

Methodology/Principal Findings

To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.

Conclusions/Significance

Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.     

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity^1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood ⁴. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo⁵. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111^6,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands⁸. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.     

Milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction

Epithelial cell differentiation frequently occurs in situ in conjunction with supporting mesenchyme or connective tissue. In embryonic development the importance of the supporting mesenchyme for cytodifferentiation and morphogenesis has been demonstrated in several epithelial tissues, but the importance of epithelial-connective tissue interactions is less well studied in adult epithelial organs. We have investigated the interaction of adult mammary epithelial cells with adipocytes, which compose the normal supporting connective tissue in the mammary gland. Mammary epithelial cells from mice in various physiological states were cultured on cellular substrates of adipocytes formed from cells of the 3T3-L1 preadipocyte cell line. We found that there were two distinct phases to the interaction of epithelial cells with adipocytes. Cytodifferentiation of the epithelial cells and milk protein production were dependent on lactogenic hormones (insulin, hydrocortisone, and prolactin), whereas ductal morphogenesis was lactogenic hormone independent. When cultured on preadipocytes or adipocytes, mammary epithelial cells from never pregnant, pregnant, lactating, and involuting mice responded to lactogenic hormones rapidly by producing and secreting large amounts of alpha-, beta-, and gamma-casein and alpha-lactalbumin. This response was seen in individual as well as in clusters of epithelial cells, but was not seen if the same cells were cultured on tissue culture dishes without adipocytes, on fibroblasts (human newborn foreskin fibroblasts) or in the presence of adipocytes but in the absence of lactogenic hormones. Continued incubation of mammary epithelial cells on adipocytes in the presence or absence of lactogenic hormones resulted in the formation of a branching ductal system. Mammary epithelial cells in ducts that formed in the absence of lactogenic hormones produced no casein, but rapidly synthesized casein when subsequently exposed to these hormones. Ultrastructural studies revealed that the formation of a basement membrane occurs only in co-cultures of mammary epithelium with adipocytes or preadipocytes. Ultrastructural changes associated with secretion occurred only in the presence of lactogenic hormones. We propose that growth and formation of a ductal system in vitro can occur in the absence of lactogenic hormones, but that certain environment-associated events must occur if the epithelium is to become responsive to lactogenic hormones and undergo the cytodifferentiation associated with lactation.     

Morphogenesis of Mouse Mammary Epithelium In Vivo in Response to Biomatrix Prepared from a Stimulatory Fetal Mesenchyme

Insoluble "biomatrix" of mesenchyme is a stimulator of mammary cell differentiation in vitro , but its effect in the morphogenesis is unknown. Fetal salivary mesenchyme induces intense local duct formation when implanted into adult mammary gland. We have therefore tested whether biomatrix prepared from fetal salivary mesenchyme retains this abillity to stimulate duct formation in vivo . Salivary mesenchyme isolated from mouse fetuses at 13.5–14.0 days of gestation, extracted sequentially with water and with 1 M NaCl, then digested with DNAse and RNAse was implanted into mammary glands of female mice and left for periods of 1–35 days. In approximately 40% of recipients, the local epithelium either formed cyst like structures, or else "spikes" of mammary epithelium penetrated the matrix forming a simplified ductwork inside it. Similar responses were elicited by salivary mesenchyme killed by freezing and also by biomatrix prepared from fetal mammary fat pad precursor tissue, mesenchyme of fetal lung, and fetal heart, liver, and brain. However when mesenchyme was either fixed with glutaraldehyde or sonicated and embedded in polymer blocks before implantation, no epithelial response was noted. These observations suggest that the biomatrix provides a passive scaffolding that contributes to morphogenesis of mammary ducts, is insufficient to support normal morphogenesis.     

Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects

The cytokine-transforming growth factor beta1 (TGFB1) is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We compared mammary gland morphogenesis in virgin Tgfb1(+/+), Tgfb1(+/-), and Tgfb1(-/-) mice and transplanted Tgfb1(+/+) and Tgfb1(-/-) epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated relative to the timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly, irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1(-/-) mice. The terminal end buds appeared to be normal morphologically, and a comparable amount of epithelial proliferation was evident. When transplanted into wild-type recipients, however, Tgfb1(-/-) epithelium showed accelerated invasion compared with Tgfb1(+/+) epithelium. This suggests that the normal rate of ductal extension in Tgfb1(-/-) null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1(-/-) mice were morphologically similar to those in Tgfb1(+/+) and Tgfb1(+/-) animals, with a normal branching pattern, and the tissue differentiated into early alveolar structures in the diestrous phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis: autocrine inhibition of epithelial ductal growth, and endocrine or paracrine stimulation of epithelial ductal growth.     

Mammary development in the embryo and adult: a journey of morphogenesis and commitment

Mammary gland development occurs through distinctive stages throughout embryonic and pubertal development and reproductive life. At each stage, different signals are required to induce changes in both the epithelium and the surrounding mesenchyme/stroma. Recent studies have provided new insights into the origin, specification and fate of mammary stem and progenitor cells and into how the differentiated lineages that comprise the functional mammary gland are determined. The development of new tools and culture techniques has also enabled the factors that influence branching morphogenesis in the embryonic and pubertal gland to be identified. A surprising recent discovery has been that mammary epithelial cells commit to differentiated lineages using the same signalling pathways that regulate lineage determination in T helper cells.     

Mammary epithelial‐specific knockout of the ephrin‐B2 gene leads to precocious epithelial cell death at lactation

The family of Eph receptor tyrosine kinases and their membrane bound ligands, the ephrins, are involved in a wide variety of morphogenic processes during embryonic development and adult tissue homeostasis. Receptor‐ligand interaction requires direct cell–cell contact and results in forward and reverse signaling originating from the receptor and ligand, respectively. We have previously shown that EphB4 and ephrinB2 are differentially expressed during the development of the adult mammary parenchyma. Overexpression of EphB4 in the mammary epithelium of transgenic mice leads to perturbations in mammary epithelial morphology, motility and growth. To investigate the role of ephrinB2 signaling in mammary gland biology, we have established transgenic mice exhibiting conditional ephrinB2 knockout in the mammary epithelium. In homozygote double transgenic CreLox mice, specific knockout of ephrinB2 occurred in the mammary epithelium during the first pregnancy‐lactating period. Abolishing ephrinB2 function led to severe interference with the architecture and functioning of the mammary gland at lactation. The morphology of the transgenic lactating glands resembled that of involuting controls, with decreased epithelial cell number and collapsed lobulo‐alveolar structures. Accordingly, massive epithelial cell death and expression of involution‐specific genes were observed. Interestingly, in parallel to cell death, significant cell proliferation was apparent, suggestive of tissue regeneration.     

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43^−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43^−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43^−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.     

Epithelial induction of stromal tenascin in the mouse mammary gland: from embryogenesis to carcinogenesis

The distribution of the extracellular matrix glycoprotein tenascin was studied by immunofluorescence in the developmental history of the mouse mammary gland from embryogenesis to carcinogenesis. Tenascin appeared only in the mesenchyme immediately surrounding the epithelia just starting morphogenesis, that is, in embryonic mammary glands from 13th to 16th day of gestation, in mammary endbuds which are a characteristic structure starting development during maturation of the mammary gland, and in the stroma of malignant mammary tumors. However, tenascin was absent in the elongating ducts of embryonic, adult, proliferating, and involuting mammary glands and preneoplastic hyperplastic alveolar nodules. The transplantation of embryonic submandibular mesenchyme into adult mammary glands induces the development of duct-alveolus nodules, which morphologically resemble developing endbuds. Tenascin reappeared around those nodules during the initial stages of their development. Tenascin expression could be induced experimentally in several ways. First, tenascin was detected at the site where the first mammary tumor cells GMT-L metastasized. Second, tenascin was detected in the connective tissue in the tumors derived from the injected C3H mammary tumor cell line CMT315 into Balb/c nude mouse. Cross-strain marker anti-CSA antiserum clearly showed that the tenascin-positive fibroblasts were of Balb/c origin. Third, when embryonic mammary epithelium was explanted on to embryonic mammary fat pad cultures, the mesenchymal cells condensed immediately surrounding the epithelium. Tenascin was detected in these condensed cells. From these three observations we conclude that both embryonic and neoplastic epithelium induced tenascin synthesis in their surrounding mesenchyme.     

Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 microns. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.     

Inappropriate P-cadherin expression in the mouse mammary epithelium is compatible with normal mammary gland function

Cadherins comprise a family of cell-cell adhesion proteins critical to the architecture and function of tissues. Expression of family members E-, N-, and P-cadherin is regulated in a spatial and temporal fashion in the developing and adult organism. Using in vivo and in vitro experimental systems, perturbation of cadherin expression by genetic deletion, overexpression, mutant dominant-negative constructs, and, to a lesser degree, expression of an inappropriate cadherin have all been shown to alter embryogenesis, tissue architecture, and cell behavior. Here we studied how expression of an inappropriate cadherin affects the adult mouse mammary gland. Human P-cadherin was expressed in mammary epithelial cells under control of the mouse mammary tumor virus (MMTV) promoter, and the effect on mammary gland behavior was studied. Typically, E-cadherin is expressed by mammary epithelial cells, whereas P-cadherin is found in myoepithelial cells and cap cells of the ductal terminal end bud. However, breast cancers frequently express P-cadherin, even though they are thought to arise from epithelial cells, and it is a marker of poor prognosis. We developed two independent transgenic mouse lines that exhibited high levels of P-cadherin protein expression in the mammary epithelium. P-cadherin was detected in most, but not all, luminal epithelial cells, and was appropriately localized to cell-cell borders. It was detected in the mammary glands of virgin, pregnant, lactating, post-lactation, and aged parous female mice. Despite the robust and widespread expression of an inappropriate cadherin, no effect was observed on mammary gland morphogenesis, architecture, lactation, or involution in transgenic mice compared to wild-type mice. No mammary tumors formed spontaneously in either wild-type or transgenic mice. Moreover, mammary tumors induced by the neu oncogene, which was introduced by a breeding strategy, showed no differences between mice with or without hP-cadherin. Surprisingly, however, none of the tumors expressed hP-cadherin protein. Together, our studies show no apparent effect on adult mammary gland or tumor behavior by inappropriate expression of P-cadherin in normal mammary epithelial cells.     

Effects of mesenchyme on epithelial tissue architecture revealed by tissue recombination experiments between the submandibular gland and lung of embryonic mice

Lung epithelium during morphogenesis maintains a sheet structure of polarized cells lining a lumen, in which E-cadherin, β-catenin and tight junctional proteins are localized at the cell–cell contact sites. On the other hand, the submandibular gland epithelium at early stages of development forms a non-cavitated mass of cells where E-cadherin/β-catenin are present on the entire cell surfaces and tight junctional proteins are almost absent or weakly scattered. In the present study, tissue recombination experiments were performed between the two organs to explore roles of mesenchyme in the architectural development of the epithelium. Homotypic recombinants of both submandibular gland and lung showed the tissue architecture as observed in the intact organs. In contrast, 11-day lung epithelium cultured with 13-day submandibular mesenchyme formed multilayers of cells with the lumen being less visible. It was accompanied by redistribution of E-cadherin/β-catenin along the entire cell surfaces and by an irregular distribution of tight junctional proteins. A similar redistribution of these molecules was observed in 15-day lung epithelium cultured with the submandibular mesenchyme, although the epithelial sheet structure lining the lumen was formed. On the other hand, the tissue architecture of submandibular gland epithelium was little affected by lung mesenchyme, although the epithelium was flattened and showed branching morphogenesis.     

Terminal end bud maintenance in mammary gland is dependent upon FGFR2b signaling

We previously demonstrated that Fibroblast Growth Factor 10 (FGF10) and its receptor FGFR2b play a key role in controlling the very early stages of mammary gland development during embryogenesis [Mailleux, A.A., Spencer-Dene, B., Dillon, C., Ndiaye, D., Savona-Baron, C., Itoh, N., Kato, S., Dickson, C., Thiery, J.P., and Bellusci, S. (2002). Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. Development 129, 53-60. Veltmaat, J. M., Relaix, F., Le, L.T., Kratochwil, K., Sala, F.G., van Veelen, W., Rice, R., Spencer-Dene, B., Mailleux, A.A., Rice, D.P., Thiery, J.P., and Bellusci, S. (2006). Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes. Development 133, 2325-35.]. However, the role of FGFR2b signaling in postnatal mammary gland development is still elusive. We show that FGF10 is expressed at high level throughout the adipose tissue in the mammary gland of young virgin female mice whereas its main receptor FGFR2 is found mostly in the epithelium. Using a rtTA transactivator/tetracycline promoter approach allowing inducible and reversible attenuation of the FGFR2b signaling throughout the adult mouse, we are now reporting that FGFR2b signaling is also critical during postnatal mammary gland development. Ubiquitous attenuation of FGFR2b signaling in the postnatal mouse for 6 weeks starting immediately after birth is not lethal and leads to minor defects in the animal. Upon dissection of the mammary glands, a 40% reduction in size compared to the WT control is observed. Further examination shows a rudimentary mammary epithelial tree with completely absent terminal end buds (TEBs), compared to a well-branched structure observed in wild type. Transplantation of mammary gland explants into cleared fat pad of wild type mouse recipients indicates that the observed abnormal branching results from defective FGFR2b signaling in the epithelium. We also demonstrate that this rudimentary tree reforms TEBs and resumes branching upon removal of doxycycline suggesting that the regenerative capacities of the mammary epithelial progenitor cells were still functional despite long-term inactivation of the FGFR2b pathway. At the cellular level, upon FGFR2b attenuation, we show an increase in apoptosis associated with a decrease in the proliferation of the mammary luminal epithelium. We conclude that during puberty, there is a differential requirement for FGFR2b signaling in ductal vs. TEBs epithelium. FGFR2b signaling is crucial for the survival and proliferation of the mammary luminal epithelial cells, but does not affect the regenerative potential of the mammary epithelial progenitor cells.     

Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland

Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates cell proliferation, apoptosis and immune system responses. In the breast, the mammary epithelium is the primary source of TGFB1 and increased expression is associated with increased breast cancer risk. This study was conducted to investigate the roles of epithelial cell-derived TGFB1 in regulation of epithelial cell activity and macrophage phenotype in the mammary gland. Tgfb1 null mutant and wildtype mammary epithelium was transplanted into contra-lateral sides of the cleared mammary gland of TGFB1 replete scid mice. Transplanted tissue was analysed for markers of proliferation and apoptosis to determine the effect of Tgfb1 null mutation on epithelial cell turnover, and was analysed by immunohistochemistry to investigate the location, abundance and phenotype of macrophages. The number of proliferating and dying ductal epithelial cells, determined by BrdU and TUNEL, was increased by 35% and 3.3-fold respectively in mammary gland transplanted with Tgfb1 null epithelium compared to wildtype epithelium (p < 0.05). Abundance of F4/80+ macrophages in between Tgfb1 null epithelial cells compared to wildtype epithelial cells was increased by 50%. The number of iNOS+ and CCR7+ cells in the stroma surrounding Tgfb1 null alveolar epithelium was increased by 78% and 2-fold respectively, and dendriform MHC class II+ cells within ductal epithelium were decreased by 30%. We conclude that epithelial cell-derived TGFB1 in the mammary gland has two functions: (1) regulation of cellular turnover of epithelial cells, and (2) regulation of local macrophage phenotype. These findings shed new light on the diversity of roles of TGFB1 in the mammary gland which are likely to impact on breast cancer risk.     

Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development

Prior reports have demonstrated that both parathyroid hormone-related protein (PTHrP) and the type I PTH/PTHrP receptor are necessary for the proper development of the embryonic mammary gland in mice. Using a combination of loss-of-function and gain-of-function models, we now report that PTHrP regulates a series of cell fate decisions that are central to the survival and morphogenesis of the mammary epithelium and the formation of the nipple. PTHrP is made in the epithelial cells of the mammary bud and, during embryonic mammary development, it interacts with the surrounding mesenchymal cells to induce the formation of the dense mammary mesenchyme. In response, these mammary-specific mesenchymal cells support the maintenance of mammary epithelial cell fate, trigger epithelial morphogenesis and induce the overlying epidermis to form the nipple. In the absence of PTHrP signaling, the mammary epithelial cells revert to an epidermal fate, no mammary ducts are formed and the nipple does not form. In the presence of diffuse epidermal PTHrP signaling, the ventral dermis is transformed into mammary mesenchyme and the entire ventral epidermis becomes nipple skin. These alterations in cell fate require that PTHrP be expressed during development and they require the presence of the PTH/PTHrP receptor. Finally, PTHrP signaling regulates the epidermal and mesenchymal expression of LEF1 and (&bgr;)-catenin, suggesting that these changes in cell fate involve an interaction between the PTHrP and Wnt signaling pathways.     

Development of Foreign Mammary Epithelial Morphology in the Stroma of Immunodeficient Mice

Systemic growth and branching stimuli, and appropriate interactions with the host stroma are essential for the development of foreign epithelia in the mammary gland of immunodeficient mice. These factors were manipulated to promote and investigate the generation of representative bovine epithelial morphology in the transplanted mouse mammary stroma. The bovine mammary epithelium is unique in its commitment to rapid proliferation and high rate of differentiation. Its morphological organization within a fibrotic stroma resembles that of the human breast, and differs significantly from the rudimentary ductal network that penetrates a fatty stroma in mice. Transplantation of bovine mammary epithelial cells into the cleared mammary fat pad of NOD-SCID mice led to continuous growth of epithelial structures. Multilayered hollow spheres developed within fibrotic areas, but in contrast to mice, no epithelial organization was formed between adipocytes. The multilayered spheres shared characteristics with the heifer gland’s epithelium, including lumen size, cell proliferation, cytokeratin orientation, estrogen/progesterone receptor expression and localization, and milk protein synthesis. However, they did not extend into the mouse fat pad via ductal morphology. Pre-transplantation of fibroblasts increased the number of spheres, but did not promote extension of bovine morphology. The bovine cells preserved their fate and rarely participated in chimeric mouse–bovine outgrowths. Nevertheless, a single case of terminal ductal lobuloalveolar unit (TDLU) development was recorded in mice treated with estrogen and progesterone, implying the feasibility of this representative bovine morphology’s development. In vitro extension of these studies revealed paracrine inhibition of bovine epithelial mammosphere development by adipocytes, which was also generalized to breast epithelial mammosphere formation. The rescue of mammosphere development by fibroblast growth factor administration evidences an active equilibrium between inhibitory and supportive effects exerted by the adipose and fibrotic regions of the stroma, respectively, which determines the development of foreign epithelium.