Phase Of Mammary Gland Development Research Articles

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

Fgf10/Fgfr2b Signaling in Mammary Gland Development, Homeostasis, and Cancer

Fibroblast growth factor 10 (Fgf10) is a secreted ligand acting via the Fibroblast growth factor receptor 2b (Fgfr2b). Fgf10/Fgfr2b signaling plays important roles both in the epithelium and in the mesenchyme during mammary gland development. Evidence in mice show that Fgf10 is critical for the induction of four out of five of the mammary placodes and for the formation of the white adipose tissue. Fgfr2b ligands also play important function in the maintenance of the terminal end buds, specialized structures at the tip of the ramified ducts during the postnatal phase of mammary gland development. Finally, in humans, FGF10 has been described to be expressed in 10% of the breast adenocarcinoma and activation of FGFR2b signaling correlates with a worse prognostic. Therefore, Fgf10 plays pleiotropic roles in both mammary gland development, homeostasis and cancer and elucidating its mechanism of action and cellular targets will be crucial to either enhance mammary gland development or to find innovative targets to treat aggressive breast cancer.

Impact of perinatal bisphenol A and 17β estradiol exposure: Comparing hormone receptor response.

Hormonal regulation controls mammary gland (MG) development. Therefore some hormone-related factors can disrupt the early phases of MGs development, making the gland more susceptible to long term modifications in its response to circulating hormones. Endocrine disruptors, such as bisphenol A (BPA), are able to cause alterations in hormone receptor expression, leading to changes in the cell proliferation index, which may expose the tissue to neoplastic alterations. Thus, we evaluated the variations in hormone receptor expression in the MG of 6-month old Mongolian gerbils exposed to BPA and 17β estradiol during the perinatal period. Receptors for estrogen alpha (ERα), beta (ERβ), progesterone (PGR), prolactin (PRL-R), and co-localization of connexin 43 (Cx43) and ERα in gerbils were analyzed, and serum concentrations of estradiol and progesterone were assessed. No alterations in body, liver, and ovary-uterus complex weights were observed. However, there was an increase in epithelial ERα expression in the 17β estradiol (E2) group and in PGR in the BPA group. Although immunohistochemistry did not show alterations in ERβ expression, western blotting revealed a decrease in this protein in the BPA group. PRL-R was more present in epithelial cells in the vehicle control (VC), E2, and BPA groups in comparison to the intact control group. Cx43 was more frequent in E2 and BPA groups, suggesting a protective response from the gland against possible malignancy. Serum concentration of estradiol reduced in VC, E2, and BPA groups, confirming that alterations also impacts steroid levels. Consequently, perinatal exposure to BPA and the reference endogenous estrogen, 17β estradiol, are able to increase the tendency of endocrine disruption in MG in a long term manner, since repercussions are observed even 6 months after exposure.

Runx Genes in Breast Cancer and the Mammary Lineage.

A full understanding of RUNX gene function in different epithelial lineages has been thwarted by the lethal phenotypes observed when constitutively knocking out these mammalian genes. However temporal expression of the Runx genes throughout the different phases of mammary gland development is indicative of a functional role in this tissue. A few studies have emerged describing how these genes impact on the fate of mammary epithelial cells by regulating lineage differentiation and stem/progenitor cell potential, with implications for the transformed state. The importance of the RUNX/CBFβ core factor binding complex in breast cancer has very recently been highlighted with both RUNX1 and CBFβ appearing in a comprehensive gene list of predicted breast cancer driver mutations. Nonetheless, the evidence to date shows that the RUNX genes can have dualistic outputs with respect to promoting or constraining breast cancer phenotypes, and that this may be aligned to individual subtypes of the clinical disease. We take this opportunity to review the current literature on RUNX and CBFβ in the normal and neoplastic mammary lineage while appreciating that this is likely to be the tip of the iceberg in our knowledge.

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

Mammary Stroma in Development and Carcinogenesis

Mammary glands of adult human females are secretory organs comprised of interdependent epithelial and mesenchymal cells. These cells constitute an assemblage of collecting ducts that end in terminal duct lobular units with hollow alveolar ductules that can differentiate to produce and expel milk. Systemic and maternal hormones, autocrine and paracrine growth factors, and cytokines regulate virtually all phases of mammary gland development. During organogenesis, epithelial and mesenchymal cells interact to form precursors of the parenchyma and stroma in the mature gland. Organogenesis precedes five stages of postnatal development: puberty, pregnancy, lactation, involution, and menopause. Each stage requires a specific set of morphogenetic changes in glandular structure and function. Cycles of cell proliferation, differentiation, and involution may recur until menopause. In addition, physiological responses such as inflammation and pathological events such as tumorigenesis are remarkable for their similarities to embryonic morphogenesis. Here we take a succinct look at the ever-improving understanding of stroma-epithelial interactions and mesenchyme function in mammary gland biology.

Effects of restricted feeding of prepubertal ewe lambs on growth performance and mammary gland development

The aim of this study was to determine the effects of restricted feeding before puberty on growth performance and mammary gland development in replacement ewe lambs. At weaning, 72 Dorset ewe lambs were assigned to one of the three diets: an ad libitum control diet with medium-quality forage (MQF; diet A-MQF); a restricted diet with the same forage as A, but less feed concentrate (diet R-MQF); or a high-quality forage (HQF) diet (diet F-HQF). The quantity of concentrate offered to the group R-MQF and F-HQF ewe lambs was adjusted to obtain 70% of the control ewe lambs’ growth rate. The diets were offered for 75 days after weaning to cover the allometric phase of mammary gland development. During this period, average daily gain (ADG) was 223 and 229 g/day for groups R-MQF and F-HQF, respectively, compared to 305 g/day for group A-MQF (P < 0.0001). At the end of this period, 28 ewe lambs were slaughtered and their mammary gland was collected. Parenchymal fresh tissue weight tended to be higher for groups R-MQF and F-HQF compared to group A-MQF (P = 0.09). Stroma weight was greater (P < 0.05) for the group A-MQF ewe lambs than for those in the other treatments. Total DNA and total protein in parenchymal tissue tended to be greater for groups R-MQF and F-HQF (P = 0.09 and P = 0.07, respectively). Dry fat-free tissue was greater for groups R-MQF and F-HQF (P < 0.05). The remaining ewe lambs were fed the same haylage and barley diet until their first lambing. During this period, compensatory growth was observed. ADG was greater (P < 0.01) for groups R-MQF and F-HQF than for group A-MQF, and feed conversion was improved (P < 0.01) for groups R-MQF and F-HQF compared with the control, whereas the dry matter intake was similar for all groups. Live body weight, loin eye depth and backfat depth at breeding and around lambing were similar for all groups. The results of this study suggest that restricted feeding before puberty improves mammary gland development without compromising growth performance in ewe lambs.

Effects of restricted feeding of prepubertal ewe lambs on reproduction and lactation performances over two breeding seasons

The aim of this study was to determine the effects of restricted feeding before puberty on reproduction, lactation and offspring growth performance in replacement ewe lambs over two breeding seasons. At weaning, 41 Dorset ewe lambs were assigned to one of three diets: an ad libitum control diet with medium-quality forage (MQF; 13.3% crude protein (CP), 1.81 Mcal metabolizable energy per kg, 42.8% ADF; diet A-MQF); a restricted diet with the same forage as A but less feed concentrate (diet R-MQF); or a high-quality forage (HQF) diet (14.8% CP, 2.15 Mcal ME/kg, 34.7% ADF; diet F-HQF). The quantity of concentrate offered to the group R-MQF and F-HQF ewe lambs was adjusted to obtain 70% of the control ewe lambs’ growth rate. The diets were offered for 75 days following weaning to cover the allometric phase of mammary gland development. Prepubertal restriction did not affect (P > 0.10) the gestation rate, number of lambs born or the body weight and body condition score of ewes at lambing or at the end of lactation. Ewes from groups R-MQF and F-HQF tended to produce more milk during their first lactation compared to those from group A-MQF (P = 0.07). During the second lactation, groups R-MQF and F-HQF had better standardized milk production than group A-MQF (P < 0.05), and group R-MQF produced more milk than group F-HQF (P < 0.05). Milk fat and protein content were not affected by treatments (P > 0.10) Fat and protein yield were affected by treatments only at the second lactation (P < 0.10 and P < 0.05, respectively). Lamb birth and weaning weights were not affected by prepubertal restriction of feeding in their mother (P > 0.10). However, the average daily gain of second breeding season lambs was higher for the R-MQF group than the F-HQF group (P < 0.05), and a similar trend was observed for total gain (P < 0.10). Restricted feeding before puberty does not impair future reproductive performance; however, it has a positive impact on lactation and on lambs’ growth performance.

Patterns of cell signaling pathway activation that characterize mammary development

Previous work has detailed the histological and biochemical changes associated with mammary development and remodeling. We have now made use of gene expression profiling, and in particular of the previously described signatures of cell signaling pathway activation, to explore the events associated with mammary gland development. We find that there is elevated E2F-specific pathway activity prior to lactation and relatively low levels of other important signaling pathways, such as RAS, MYC and SRC. Upon lactation and continuing into the involution phase, these patterns reverse with a dramatic increase in RAS, SRC and MYC pathway activity and a decline in E2F activity. At the end of involution, these patterns return to that of the adult non-lactating mammary gland. The importance of the changes in E2F pathway activity, particularly during the proliferative phase of mammary development, was confirmed through the analysis of mice deficient for various E2F proteins. Taken together, these results reveal a complex pattern of pathway activity in relation to the various phases of mammary gland development.

Transcriptional Changes Underlying the Secretory Activation Phase of Mammary Gland Development

The secretory activation stage of mammary gland development occurs after parturition and converts inactive lobuloalveoli to active milk secretion. This process is triggered by progestin withdrawal and depends upon augmented prolactin (Prl) signaling. Little is known about the Prl-induced transcriptional changes that occur in the mammary gland to drive this process. To examine changes in the mammary transcriptome responsible for secretory activation, we have used transcript profiling of three mouse models that exhibit failure of secretory activation: knockout of galanin (a regulator of pituitary Prl production and a mammary cell autonomous modulator of Prl action); treatment with S179D Prl (a phosphoprolactin mimic); and knockout of a single Prl receptor allele. A significant reduction in expression was observed in genes belonging to 46 gene ontologies including those representing milk proteins, metabolism, lipid, cholesterol and fatty acid biosynthetic enzymes, immune response, and key transcription factors. A set of 35 genes, commonly regulated in all three models, was identified and their role in lactogenesis was validated by examining their expression in response to Prl stimulation or signal transducer and activator of transcription 5 knockdown in the HC11 mouse mammary cell culture model. The transcript profiles provided by these experiments identify 35 key genes (many for the first time) involved in the secretory activation phase of mammary gland development, show that S179D acts as an antagonist of Prl action, and provide insight into the partial penetrance of failed lactation in Prl receptor heterozygous females.

Next stop, the twilight zone: hedgehog network regulation of mammary gland development.

The hedgehog signal transduction network is a critical mediator of cell-cell communication during embryonic development. Evidence also suggests that properly regulated hedgehog network function is required in some adult organs for stem cell maintenance or renewal. Mutation, or misexpression, of network genes is implicated in the development of several different types of cancer, particularly that of skin, brain, lung, and pancreas. Recent studies in the mouse mammary gland have demonstrated roles for hedgehog network genes at virtually every phase of mammary gland development where it regulates such diverse processes as embryonic mammary gland induction, establishment of ductal histoarchitecture, and functional differentiation in lactation. Further, studies suggest a role for misregulated network function in the progression of breast cancer.

Developmental role of the SNF1-related kinase Hunk in pregnancy-induced changes in the mammary gland.

The steroid hormones 17 beta-estradiol and progesterone play a central role in the pathogenesis of breast cancer and regulate key phases of mammary gland development. This suggests that developmental regulatory molecules whose activity is influenced by ovarian hormones may also contribute to mammary carcinogenesis. In a screen designed to identify protein kinases expressed in the mammary gland, we previously identified a novel SNF1-related serine/threonine kinase, Hunk (hormonally upregulated Neu-associated kinase). During postnatal mammary development, Hunk mRNA expression is restricted to a subset of mammary epithelial cells and is temporally regulated with highest levels of expression occurring during early pregnancy. In addition, treatment of mice with 17 beta-estradiol and progesterone results in the rapid and synergistic upregulation of Hunk expression in a subset of mammary epithelial cells, suggesting that the expression of this kinase may be regulated by ovarian hormones. Consistent with the tightly regulated pattern of Hunk expression during pregnancy, mammary glands from transgenic mice engineered to misexpress Hunk in the mammary epithelium manifest temporally distinct defects in epithelial proliferation and differentiation during pregnancy, and fail to undergo normal lobuloalveolar development. Together, these observations suggest that Hunk may contribute to changes in the mammary gland that occur during pregnancy in response to ovarian hormones.

Gene expression during involution of mammary gland (review).

After cessation of lactation, the mammary gland undergoes involution, regressing to a state resembling that of a virgin animal. This phase of mammary gland development is characterized by epithelial cell death and tissue remodeling. To understand molecular mechanisms of mammary gland involution, we identified involution-induced clones by differential screening of a mouse mammary gland cDNA library. Several known genes were induced during mammary gland involution: sulfated glycoprotein-2 (SGP-2), WDNM1, lactoferrin, ferritin heavy chain (FHC), lysozyme and osteopontin genes. Involution of the mammary gland is presumed to be mediated by a decrease in serum prolactin level induced by weaning, but may also involve changes in paracrine or autocrine growth factors. Effects of lactogenic hormones and EGF on the expression of the involution-induced genes were examined in mammary epithelial cells. Insulin, dexamethasone, and prolactin did not influence the expression of the FHC, WDNM1 and SGP-2 genes. However, EGF strongly inhibited the expression of WDNM1 and SGP-2 genes. Our recent results are reviewed and discussed.

Programmed cell death and mammary neoplasia.

Normal development and maintenance of tissue size is dependent on a balance between cell proliferation and cell death. Apoptosis, or programmed cell death, plays important roles in mammary gland development, from the embryonic development of sexual dimorphism to senescent involution of the mammary gland. The involution of secretory epithelium following pregnancy and lactation is the most dramatic example of the role of apoptosis in mammary gland development. This phase of mammary gland development provides an important physiological and pathological context in which to study the programmed death of epithelium, because a failure in cell death appears to contribute to neoplastic development [1–3]. Although considerable effort has been directed toward understanding the role of cell proliferation in neoplastic development, much less is known about the process of apoptotic cell death in either the control of normal tissue homeostasis or its potential influence on neoplastic development.

Localization and Quantification of Wnt-2 Gene Expression in Mouse Mammary Development

The Wnt gene family encodes a group of proteins probably involved in cell-cell communication during several stages of vertebrate development. More than 10 members of this family have been identified and shown to be expressed mainly in developing neural tissue. Using a reverse transcription-polymerase chain reaction (RT-PCR)-based approach with degenerate oligonucleotides directed against conserved sequences in the Wnt genes, Wnt-2 transcripts were detected in RNA isolated from mammary glands of 4-to 6-week-old virgin C3H mice, a period characterized by extensive end bud and ductal proliferation. The spatial and temporal expression of Wnt-2 in the developing mouse mammary gland was studied by in situ hybridization, quantitative RT-PCR, and Northern analysis. Wnt-2 is expressed during the ductal phase of mammary gland development, primarily in the basal layer of mammary ducts and in the body cells of end buds. Wnt-2 RNA transcripts were readily detected in poly(A) RNA isolated from 5-week-old C3H and Balb/c mice. RNA transcript levels measured as molecules per nanogram of total RNA by RT-PCR decreased 10- to 40-fold within 2 days after the onset of pregnancy and remained low during pregnancy and lactation. This is in contrast to the patterns of expression of other Wnt family members, Wnt-5a and -5b, whose expression was either barely or not detectable in the 4- to 6-week-old mammary gland, but increased markedly during pregnancy. These results confirm the differential expression of Wnt gene family members during mammary gland development. Furthermore, they suggest that Wnt-2, as well as several other family members, may play a role in pattern formation during early mammary gland development.

Adipocyte development in subcutaneous tissues of the young rat.

The relationship between mammary gland development and adipocyte formation in rat subcutaneous tissue was studied. Tissue samples from newborn rats and young rats were prepared for histological and histochemical analysis. The development of subcutaneous adipocytes in the young rat is associated temporally and spatially with mammary gland development. The growing mammary gland ducts and the blood vessels and nerves that serve the ducts may physically interact with connective tissue cells, resulting in a population of undifferentiated cells. Adipocytes and a wide variety of cell types may be derived from this pool of undifferentiated cells. Histochemical characteristics of cells of immature ends of mammary gland ducts indicated hypoxic conditions. These conditions may have directed a vascularization process that resulted in network of adipocytes and capillaries around mammary gland ducts. Thus, under normal conditions, adipocyte formation is an integral phase of mammary gland development in the fetal and young rat.

Further study on the relation between area and desoxyribonucleic acid content of the mammary gland in mice.

The relation between area and DNA content of the mammary gland was investigated using C3H/He and C57BL/6 strains of virgin mice 50 to 230 days after birth in order to study whether mammary gland area (MGA) measured with the whole mount preparation can be used as an index of mammary gland development. Correlation coefficients were calculated in the pooled data of certain ages according to the phase of mammary gland development. In C3H/He mice, significant correlations were obtained in the 50 to 70 and the 170 to 230 day-old age groups, but not in the 90 to 150 day-old group. In C57BL/6 mice, there were significant correlations not only in the 50 to 90 day-old group but also in the 110 to 210 day-old group, the coefficient being larger in the former. The present results revealed that MGA would be a fairly good index of quantitative development of the mammary gland during the sharp growing phases of the gland, especially of the duct system.