Mammary Gland Morphogenesis Research Articles

Inducible Knockout of Twist1 in Young and Adult Mice Prolongs Hair Growth Cycle and Has Mild Effects on General Health, Supporting Twist1 as a Preferential Cancer Target

Twist1 promotes epithelial-mesenchymal transition, invasion, metastasis, stemness, and chemotherapy resistance in cancer cells and thus is a potential target for cancer therapy. However, Twist1-null mice are embryonic lethal, and people with one Twist1 germline mutant allele develop Saethre-Chotzen syndrome; it is questionable whether Twist1 can be targeted in patients without severe adverse effects. We found that Twist1 is expressed in several tissues, including fibroblasts of the mammary glands and dermal papilla cells of the hair follicles. We developed a tamoxifen-inducible Twist1 knockout mouse model; Twist1 knockout in 6-week-old female mice did not affect mammary gland morphogenesis and function during pregnancy and lactation. In both males and females, the knockout did not influence body weight gain, heart rate, or total lean and fat components. The knockout also did not alter blood pressure in males, although it slightly reduced blood pressure in females. Although Twist1 is not cyclically expressed in dermal papilla cells, knockout of Twist1 at postnatal day 13 (when hair follicles have developed) drastically extended the anagen phase and accelerated hair growth. These results indicate that Twist1 is not essential for maintaining an overall healthy condition in young and adult mice and that loss of function facilitates hair growth in adulthood, supporting Twist1 as a preferential target for cancer therapy.

Prenatal morphogenesis of mammary glands in mouse and rabbit.

Our understanding of prenatal morphogenesis of mammary glands has recently greatly advanced. This review focuses on morphogenesis proper, as well as cellular processes and tissue interactions involved in the progression of the embryonic mammary gland through sequential morphogenic stages in both the mouse and rabbit embryo. We provide a synthesis of both historical and more recent studies of embryonic mammary gland development, as well as arguments to revise old concepts about mechanisms of mammary line and rudiment formation. Finally, we highlight outstanding issues that remain to be addressed.

P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation

Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-β receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-β paracrine signaling.

Embryonic Mammary Gland Development; a Domain of Fundamental Research with High Relevance for Breast Cancer Research

Mammary gland development is often presented as a series of events happening during puberty, pregnancy, lactation and involution; even though it starts in utero, and extensive morphogenetic events occur prior to birth. During the last century, research on the embryonic phase of mammary gland development received little attention as lactation and breast cancer investigations experienced greater emphasis. Nonetheless, important contributions were made during the 1950’s–1980’s by Boris Balinsky, Albert Raynaud, Alain Propper, Klaus Kratochwil and Teruyo Sakakura and respective coworkers in understanding mammary development in rabbit and mouse embryos. In part due to interests in developmental biology and birth defects per se, and in part due to the recognition of mechanistic parallels between organogenesis and carcinogenesis, the prenatal phase of mammary gland development has become more intensively studied again over the past decade. We set out to assemble this issue in such a way that it could serve as a textbook of prenatal mammary gland development, bringing together the older literature with the more recent insights. Though we succeeded in doing that for mammary development in mouse and rabbit embryos, admittedly and regrettably, this issue underrepresents insights in prenatal mammary development in monotremes, marsupials, and placentals other than mice and rabbit, including human. This is in part due to these species being less available for study, although interesting work has been and is currently being done on them. For a comprehensive review on progress in research on human breast development, we recommend a recent publication elsewhere by Gusterson and Stein [1]. We have been very fortunate in receiving the enthusiastic cooperation of many authors who have all greatly contributed to our current understanding of prenatal mammary gland development. We are particularly indebted to several esteemed Emeritus Professors, whom despite their “officially retired” status, generously dedicated their time and treasures of knowledge to this special issue. These scientists performed classical experimental embryology several decades ago and produced insights that are seminal to our current understanding of embryonic mammary development. Among them are Alain Propper who, using rabbit embryos as a model, was the first to demonstrate that the inductive signals for mammary gland development reside in the dermis (1967), and to propose that ‘wandering cells’ along the mammary line contribute to growth of the mammary rudiments (1978). He joined us in co-authoring a review of morphogenesis of the embryonic mammary gland in rabbit and mouse embryos, which also serves as an introduction of developmental stages and nomenclature used in other reviews in this issue. Evolutionary biologist Olav Oftedal and developmental biologist Danielle Dhouailly teamed up especially for this occasion. Olav received the inspiration to study the evolution of the mammary gland and lactation biology from the longest and perhaps least known research efforts (1890–1922) of his lineal ancestor Alexander Graham Bell to selectively (and successfully) breed sheep for increased teat number. Danielle was a naturalist at a very young age and dedicated her career to understanding skin appendage and pattern B. A. Howard (*) Division of Breast Cancer Research, Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK e-mail: beatrice.howard@icr.ac.uk

Parathyroid Hormone-Related Protein Specifies the Mammary Mesenchyme and Regulates Embryonic Mammary Development

Parathyroid Hormone related Protein (PTHrP) is a critical regulator of mammary gland morphogenesis in the mouse embryo. Loss of PTHrP, or its receptor, PTHR1, results in arrested mammary buds at day 15 of embryonic development (E15). In contrast, overexpression of PTHrP converts the ventral epidermis into hairless nipple skin. PTHrP signaling appears to be critical for mammary mesenchyme specification, which in turn maintains mammary epithelial identity, directs bud outgrowth, disrupts the male mammary rudiment and specifies the formation of the nipple. In the embryonic mammary bud, PTHrP exerts its effects on morphogenesis, in part, through epithelial-stromal crosstalk mediated by Wnt and BMP signaling. Recently, PTHLH has been identified as a strong candidate for a novel breast cancer susceptibility locus, although PTHrP's role in breast cancer has not been clearly defined. The effects of PTHrP on the growth of the embryonic mammary rudiment and its invasion into the dermis may, in turn, have connections to the role of PTHrP in breast cancer.

Ectodysplasin/NF-κB Signaling in Embryonic Mammary Gland Development

The ectodysplasin (Eda) signaling pathway consists of a TNF-like ligand Eda, its receptor Edar, and an adaptor protein Edaradd and its activation leads to NF-κB mediated transcription. In humans, mutations in the EDA pathway genes cause hypohidrotic ectodermal dysplasia, a disorder characterized by defective formation of hair follicles, teeth, and several exocrine glands including the breast. Embryonic mammary gland development proceeds via placode, bud, bulb and sprout stages before the onset of branching morphogenesis. Studies on mouse models have linked Eda with two aspects of embryonic mammary gland morphogenesis: placode induction and ductal growth and branching. Here we summarize the current knowledge on the role of Eda/NF-κB in mammary gland development.

Abstract 862: Purified recombinant Maspin is processed through the endosomal pathway in breast cancer cells.

Abstract Maspin (Mammary Serine Protease Inhibitor), a gene involved in mammary gland development and morphogenesis, has been shown to act as a tumor suppressor in multiple cancers by inhibiting the aggressive cancer cell phenotype. Recombinant Maspin (rMas) added to cell culture media is internalized by cancer cells and capable of recapitulating many of the effects of endogenously re-expressed Maspin in Maspin-null cancer cells, including the inhibition of cell invasion, migration and induction of angiogenesis. Importantly, these effects are mediated by purified rMas without the need for post-translational modifications prior to application, increasing the future therapeutic potential for utilizing rMas as an adjuvant therapy. However, the therapeutic use of rMas is limited by its rapid clearance from cancer cells, and many of the molecular mechanisms regarding rMas internalization and processing remain to be determined. Building on preliminary data, we hypothesized that rMas is processed by the endosomal pathway. We demonstrate localization of rMas in Rab9 late-endosomes and Lamp-1 positive lysosomes following treatment of 10 μg/ml rMas in BT549, Hs578T and MDA-MB-231 human breast cancer cell lines. rMas internalization was reduced when incubated at 4oC, and increased at temperatures of 20oC and 37oC, with the highest rates of internalization at 37oC, indicative of endocytosis. Furthermore, depletion of K+ decreased rMas internalization in Hs578T and MDA-MB-231, suggestive of clarthrin-mediated endocytosis. Our data show that rMas is capable of acting at the membrane to mediate effects on invasion and migration, but may not be functional once internalized due to rapid degradation by the lysosome. Interestingly, this may provide a plausible explanation for previously reported data in which rMas was not able to reproduce the effects of endogenous Maspin re-expression on cancer cell apoptosis sensitivity, which require cytosolic and mitochondrial mechanisms. Our data provide evidence that strategies which induce endosomal escape of rMas will intensify its anti-tumor effects on cancer cells, and further enhance its potential therapeutic applications. Citation Format: Thomas M. Bodenstine, Richard E. B. Seftor, Elisabeth A. Seftor, Zhila Khalkhali-Ellis, Nicole A. Samii, J. Cesar Monarrez, Philip A. Pemberton, Mary J. C. Hendrix. Purified recombinant Maspin is processed through the endosomal pathway in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 862. doi:10.1158/1538-7445.AM2013-862

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus)

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERβ. Yet throughout pregnancy, ERα and ERβ levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.

Abstract B42: Dichotomus role of profilin in early vs. late steps of breast cancer metastasis

Abstract Profilin1 (Pfn1), an essential control element for actin polymerization, plays indispensable role in migration and proliferation of normal cells. Seemingly contrary to these essential cellular functions of Pfn1, its expression is downregulated in breast cancer, the significance of which is unclear. In this study, expression profiling of Pfn1 in human breast cancer specimens of various histopathological stages correlate lower Pfn1 expression levels with propensity to metastasize. Xenograft studies uncover a unique dichotomous role of Pfn1 in early vs late steps of breast cancer metastasis. Specifically, loss of Pfn1 expression promotes dissemination of breast cancer cells from the primary mammary tumor but metastatic colonization of breast cancer cells is critically dependent on Pfn1's function. In the context of dissemination, breast cancer cells exhibit increased sustenance of PI3K signaling and acquire a hyper-invasive phenotype (marked by MMP9 upregulation, faster invasion through collagen matrix) involving a PI3K-PI(3,4)P2 signaling axis when Pfn1 expression is suppressed. In addition, Pfn1-deficient breast cancer cells display increased proficiency to cross through an endothelial cell barrier (an obligatory step for vascular dissemination) in a VEGF-dependent manner, respectively. Gene expression profiling combined with biochemical analyses of secreted proteins show that loss of Pfn1 impacts expression of several other pro-invasive and pro-metastatic genes. Finally, correlated with hyper-invasive phenotype of breast cancer cells caused by Pfn1 depletion, we found that mammary-specific deletion of Pfn1 gene expedites ductal outgrowth. In summary, contrasting the well-established pro-migratory role of Pfn1 in the physiological contexts, our findings set the first in vivo example of a context-specific effect of loss of Pfn1 in cell motility-dependent processes such as mammary gland morphogenesis and metastatic dissemination. Citation Format: Partha Roy. Dichotomus role of profilin in early vs. late steps of breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B42.

Abstract PR14: ErbB2-induced epithelium to mesenchyme transition (EMT) is blocked by p38α/β signaling to restrict early dissemination in breast cancer

Abstract Our aim is to better understand the mechanisms dictating the early dissemination of cells from pre-malignant lesions, as well as those controlling cell fate decisions at secondary sites to develop more effective therapies for eradicating these populations. The majority of breast cancer patients die from metastatic disease, and not from their primary tumor. The current paradigm argues that dissemination of tumor cells can only occur from invasive lesions observed during late stages of the disease. However, clinical evidence suggests that “pre-malignant” mammary epithelial cells can disseminate and accumulate in target organs like the bone. This can occur from non-invasive lesions such as those found in Ductal Carcinoma in Situ (DCIS) patients. Thus, “early” disseminated tumor cells (DTCs) might be critical contributors to metastasis, but the mechanisms behind these events remain largely unknown. We had shown that p38-alpha/beta signaling maintains mammary gland ductal architecture by promoting anoikis and preventing disorganization of the luminal and basal epithelial compartments. We propose that, in addition to regulating mammary gland morphogenesis, p38-alpha/beta regulates a switch controlling early dissemination. In clinical samples we have found that ErbB2+ DCIS or invasive lesions show a down-regulation of the p38-signaling pathway, suggesting a role for the inhibition of p38-alpha/beta during cancer progression. In MMTV-Neu mammary glands ErbB2 is inversely correlated to membranous beta-catenin (inactive beta-catenin). Further, loss of P-ATF2 (p38 target) also coincides with reduced E-cadherin in this ErbB2+ epithelium. We also reveal that inhibition of p38-alpha/beta in MCF10A-ErbB2 or “pre-malignant” MMTV-ErbB2 acini results in increased invasion and disruption of normal acinar structure. In the MCF10A-ErbB2 model this correlated with E-cadherin down-regulation and induction of an 11-gene Wnt signaling EMT signature (e.g. up-regulation of Wnt5a/b, Wnt11, SNAIL, TWIST, N-cadherin, ZEB2, and SLUG). This EMT signature was also induced simply by p38-alpha/beta inhibition or ErbB2 overexpression in parental MCF10A cells. Further, treatment of MCF10A cells with Wnt3a inhibited p38-alpha and ATF2 phosphorylation. Inhibition of p38-alpha/beta signaling in MMTV-ErbB2 mice strongly increased the accumulation of pre-malignant MECs (detected by surface ErbB2) in the lungs and bone marrow. However, at least in lungs, >80% of these DTCs remained dormant as measure by P-Rb staining. We propose that in pre-malignant lesions p38-alpha/beta acts as an inhibitor to ErbB2-induced EMT, anoikis resistance and early dissemination. We further propose that Wnt signaling executes the EMT and also the silencing of p38-alpha/beta signaling to allow for early dissemination. This abstract is also presented as Poster B74. Citation Format: Kathryn Harper, Maria Soledad Sosa, Alvaro Avivar-Valderas, Huei-Chi Wen, Julio Aguirre-Ghiso. ErbB2-induced epithelium to mesenchyme transition (EMT) is blocked by p38α/β signaling to restrict early dissemination in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr PR14.

Endocrine Disruptors and Breast Cancer Risk - Time to Consider the Environment

The term endocrine disruptors is used to describe a variety of natural and manmade substances that have the capacity to potentially interfere with and modify the normal physiology of endocrine system either by mimicking, blocking or modulating the actions of natural endogenous hormones. The rising incidence of breast cancer over the last 50 years and the documented higher incidence in urban as compared to rural areas suggest a relationship to the introduction and increased use of xenoestrogens in our environment. The literature has developed over the last decades where initial experiments on endocrine disruptors did not support an involvement in breast cancer, and then evidence mounted implicating various environmental factors including hormones, endocrine disrupting chemicals and non-endocrine disrupting environmental carcinogens in the pathogenesis of breast cancer. Available data support the hypothesis that exposure to endocrine disruptors in utero leaves a signature on mammary gland morphogenesis so that the resulting dysgenic gland becomes more predisposed to develop tumors upon exposures to additional insults later on during life. Exceptionally, exposure to phytoestrogens could be beneficial to human health. Most of the available data are from well developed countries while the developing countries are still understudied regarding these issues. Here, we raise a note of caution about potential role of environmental toxins including endocrine disruptors in breast cancer development and call for serious measures to be taken by all involved parties in the developing world.

Slug Controls Stem/Progenitor Cell Growth Dynamics during Mammary Gland Morphogenesis

BackgroundMorphogenesis results from the coordination of distinct cell signaling pathways controlling migration, differentiation, apoptosis, and proliferation, along stem/progenitor cell dynamics. To decipher this puzzle, we focused on epithelial-mesenchymal transition (EMT) “master genes”. EMT has emerged as a unifying concept, involving cell-cell adhesion, migration and apoptotic pathways. EMT also appears to mingle with stemness. However, very little is known on the physiological role and relevance of EMT master-genes. We addressed this question during mammary morphogenesis. Recently, a link between Slug/Snai2 and stemness has been described in mammary epithelial cells, but EMT master genes actual localization, role and targets during mammary gland morphogenesis are not known and we focused on this basic question.Methodology/Principal FindingsUsing a Slug–lacZ transgenic model and immunolocalization, we located Slug in a distinct subpopulation covering about 10–20% basal cap and duct cells, mostly cycling cells, coexpressed with basal markers P-cadherin, CK5 and CD49f. During puberty, Slug-deficient mammary epithelium exhibited a delayed development after transplantation, contained less cycling cells, and overexpressed CK8/18, ER, GATA3 and BMI1 genes, linked to luminal lineage. Other EMT master genes were overexpressed, suggesting compensation mechanisms. Gain/loss-of-function in vitro experiments confirmed Slug control of mammary epithelial cell luminal differentiation and proliferation. In addition, they showed that Slug enhances specifically clonal mammosphere emergence and growth, cell motility, and represses apoptosis. Strikingly, Slug-deprived mammary epithelial cells lost their potential to generate secondary clonal mammospheres.Conclusions/SignificanceWe conclude that Slug pathway controls the growth dynamics of a subpopulation of cycling progenitor basal cells during mammary morphogenesis. Overall, our data better define a key mechanism coordinating cell lineage dynamics and morphogenesis, and provide physiological relevance to broadening EMT pathways.

An integrin–ILK–microtubule network orients cell polarity and lumen formation in glandular epithelium

The extracellular matrix has a crucial role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues, however the underlying mechanisms remain elusive. By using Cre-Lox deletion we show that β1-integrins are required for normal mammary gland morphogenesis and lumen formation, both in vivo and in a 3D primary culture model where epithelial cells directly contact basement membrane. Downstream of basement membrane-β1-integrins, Rac1 is not involved, however ILK is needed to polarize microtubule plus ends at the basolateral membrane and disrupting each of these components prevents lumen formation. The integrin-microtubule axis is necessary for the endocytic removal of apical proteins from the basement membrane-cell interface and for internal Golgi positioning. We propose that this integrin-signalling network controls the delivery of apical components to the correct surface and thereby governs the orientation of polarity and development of lumens.

P130Cas Over-Expression Impairs Mammary Branching Morphogenesis in Response to Estrogen and EGF

p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the in vivo consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.

Development of MammoQuant: An Automated Quantitative Tool for Standardized Image Analysis of Murine Mammary Gland Morphogenesis

Development of MammoQuant: An Automated Quantitative Tool for Standardized Image Analysis of Murine Mammary Gland Morphogenesis

In utero and lactational exposure to vinclozolin and genistein induces genomic changes in the rat mammary gland

Exposure to low doses of environmental estrogens such as bisphenol A and genistein (G) alters mammary gland development. The effects of environmental anti-androgens, such as the fungicide vinclozolin (V), on mammary gland morphogenesis are unknown. We previously reported that perinatal exposure to G, V, and the GV combination causes histological changes in the mammary gland during the peripubertal period, suggesting alterations to the peripubertal hormone response. We now investigate whether perinatal exposure to these compounds alters the gene expression profiles of the developing glands to identify the dysregulated signaling pathways and the underlying mechanisms. G, V, or GV (1 mg/kg body weight per day) was added to diet of Wistar rats, from conception to weaning; female offspring mammary glands were collected at postnatal days (PNDs) 35 and 50. Genes displaying differential expression and belonging to different functional categories were validated by quantitative PCR and immunocytochemistry. At PND35, G had little effect; the slight changes noted were in genes related to morphogenesis. The changes following exposure to V concerned the functional categories associated with development (Cldn1, Krt17, and Sprr1a), carbohydrate metabolism, and steroidogenesis. The GV mixture upregulated genes (Krt17, Pvalb, and Tnni2) involved in muscle development, indicating effects on myoepithelial cells during mammary gland morphogenesis. Importantly, at PND50, cycling females exposed to GV showed an increase in the expression of genes (Csn2, Wap, and Elf5) related to differentiation, consistent with the previously reported abnormal lobuloalveolar development previously described. Thus, perinatal exposure to GV alters the mammary gland hormone response differently at PND35 (puberty) and in animals with established cycles.

Remodeling of the Lymphatic Vasculature during Mouse Mammary Gland Morphogenesis Is Mediated via Epithelial-Derived Lymphangiogenic Stimuli

Despite the key roles of lymphatic vessels in homeostasis and disease, the cellular sources of signals that direct lymphatic vascular growth and patterning remain unknown. Using high-resolution imaging in two and three dimensions, we demonstrated that postnatal mouse mammary gland lymphatic vessels share an intimate spatial association with epithelial ducts and large blood vessels. We further demonstrated that the lymphatic vasculature is remodeled together with the mammary epithelial tree and blood vasculature during postnatal mouse mammary gland morphogenesis. Neither estrogen receptor α nor progesterone receptor were detected in lymphatic endothelial cells in the mouse mammary gland, suggesting that mammary gland lymphangiogenesis is not likely regulated directly by these steroid hormones. Epithelial cells, especially myoepithelial cells, were determined to be a rich source of prolymphangiogenic stimuli including VEGF-C and VEGF-D with temporally regulated expression levels during mammary gland morphogenesis. Blockade of VEGFR-3 signaling using a small-molecule inhibitor inhibited the proliferation of primary lymphatic endothelial cells promoted by mammary gland conditioned medium, suggesting that lymphangiogenesis in the mammary gland is likely driven by myoepithelial-derived VEGF-C and/or VEGF-D. These findings provide new insight into the architecture of the lymphatic vasculature in the mouse mammary gland and, by uncovering the proximity of lymphatic vessels to the epithelial tree, suggest a potential mechanism by which metastatic tumor cells access the lymphatic vasculature.

Mammary gland development is delayed in mice deficient for aminopeptidase N

Development of the mammary gland requires the coordinated action of proteolytic enzymes during two phases of remodelling. Firstly, new ducts and side-branches thereof need to be established during pregnancy to generate an extensive ductal tree allowing the secretion and transport of milk. A second wave of remodelling occurs during mammary involution after weaning. We have analysed the role of the cell surface protease aminopeptidase N (Anpep, APN, CD13) during these processes using Anpep deficient and Anpep over-expressing mice. We find that APN deficiency significantly delays mammary gland morphogenesis during gestation. The defect is characterised by a reduction in alveolar buds and duct branching at mid-pregnancy. Conversely over-expression of Anpep leads to accelerated ductal development. This indicates that Anpep plays a critical role in the proteolytic remodelling of mammary tissue during adult mammary development.

Mammary epithelial-restricted expression of activated c-src rescues the block to mammary gland morphogenesis due to the deletion of the C-terminus of Patched-1

Mesenchymal dysplasia (mes) mice expressing a C-terminally truncated version of the Hedgehog (Hh)-ligand receptor, Patched-1 (Ptch1), exhibit a limited spectrum of developmental defects including blocked ductal morphogenesis of the mammary gland during puberty. Given that the Hh-ligands can stimulate signalling cascades distinct from the canonical pathway involving Smo and the Gli-family proteins and that Ptch1 binds to factors harbouring SH3-domains, we determined whether the mes mammary gland defect could be rescued by activating non-canonical signalling pathways downstream of Ptch1. We demonstrate here that expression of constitutively active c-src (c-srcAct) in mammary epithelial cells overcomes the block to mammary epithelial morphogenesis in mes mice. Specifically, MMTV-directed expression of c-srcAct rescued blocked ductal morphogenesis in mes mice, albeit only after animals were more than 15 weeks of age. The overall morphology resembled wild type mice expressing c-srcAct although 40% of mes/MMTV-c-srcAct mice exhibited terminal end buds at 24 weeks of age. C-srcAct restored the proliferative capacity of mes epithelial cells, self-renewal capacity of mammary progenitor cells and increased the expression of Esr1, Ptch1 and Gli1. These data reveal the cooperative interactions between signalling cascades involving c-src and Ptch1 and suggest that Hh-signalling may be permissive for c-src/Esr1-dependent mammary gland morphogenesis.

Overexpression of Follistatin in the Mouse Epididymis Disrupts Fluid Resorption and Sperm Transit in Testicular Excurrent Ducts1

Activin is a well-established modulator of male and female reproduction that stimulates the synthesis and secretion of follicle-stimulating hormone. Nonpituitary effects of activin have also been reported, although the paracrine actions of this growth factor in several reproductive tissues are not well understood. To identify the paracrine functions of activin during mammary gland morphogenesis and tumor progression, we produced transgenic mice that overexpress follistatin (FST), an intrinsic inhibitor of activin, under control of the mouse mammary tumor virus (MMTV) promoter. Although the MMTV-Fst mice were constructed to assess the role of activin in females, expression of the transgene was also observed in the testes and epididymides of males. While all 17 transgenic founder males exhibited copulatory behavior and produced vaginal plugs in females, only one produced live offspring. In contrast, transgenic females were fertile, permitting expansion of transgenic mouse lines. Light and transmission electron microscopic examination of the transgenic testes and epididymides revealed impairment of fluid resorption and sperm transit in the efferent ducts and initial segment of the epididymis, as indicated by accumulation of fluid and sperm stasis. Consequently, a variety of degenerative lesions were observed in the seminiferous epithelium, such as vacuolation and early stages of mineralization and fibrosis. Sperm collected from the caudae epididymidis of MMTV-Fst males had detached heads and were immotile. Together, these data reveal that activin signaling is essential for normal testicular excurrent duct function and that its blockade impairs fertility. These results also suggest that selective inhibitors of activin signaling may provide a useful approach for the development of male contraceptives without compromising androgen synthesis and actions.