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Branching morphogenesis is a characteristic feature of many essential organs, such as the lung and kidney, and most glands, and is the net result of two tissue behaviors: branch point initiation and elongation. Each branched organ has a distinct architecture customized to its physiological function, but how patterning occurs in these ramified tubular structures is a fundamental problem of development. Here, we use quantitative 3D morphometrics, time-lapse imaging, manipulation of ex vivo cultured mouse embryonic organs and mice deficient in the planar cell polarity component Vangl2 to address this question in the developing mammary gland. Our results show that the embryonic epithelial trees are highly complex in topology owing to the flexible use of two distinct modes of branch point initiation: lateral branching and tip bifurcation. This non-stereotypy was contrasted by the remarkably constant average branch frequency, indicating a ductal growth invariant, yet stochastic, propensity to branch. The probability of branching was malleable and could be tuned by manipulating the Fgf10 and Tgfβ1 pathways. Finally, our in vivo data and ex vivo time-lapse imaging suggest the involvement of tissue rearrangements in mammary branch elongation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from multipotent stem cells (MaSCs), which are progressively replaced by unipotent progenitors by birth. However, the lack of specific markers for early fate specification has prevented the delineation of the features and spatial localization of MaSC-derived lineage-committed progenitors. Here, using single-cell RNA sequencing from E13.5 to birth, we produced an atlas of matched mouse mammary epithelium and mesenchyme and reconstructed the differentiation trajectories of MaSCs toward basal and luminal fate. We show that murine MaSCs exhibit lineage commitment just prior to the first sprouting events of mammary branching morphogenesis at E15.5. We identify early molecular markers for committed and multipotent MaSCs and define their spatial distribution within the developing tissue. Furthermore, we show that the mammary embryonic mesenchyme is composed of two spatially restricted cell populations, and that dermal mesenchyme-produced FGF10 is essential for embryonic mammary branching morphogenesis. Altogether, our data elucidate the spatiotemporal signals underlying lineage specification of multipotent MaSCs, and uncover the signals from mesenchymal cells that guide mammary branching morphogenesis., (© 2024. The Author(s).)

The Wnt/β-catenin pathway plays a critical role in cell fate specification, morphogenesis, and stem cell activation across diverse tissues, including the skin. In mammals, the embryonic surface epithelium gives rise to the epidermis as well as the associated appendages including hair follicles and mammary glands, both of which depend on epithelial Wnt/β-catenin activity for initiation of their development. Later on, Wnts are thought to enhance mammary gland growth and branching, whereas in hair follicles, they are essential for hair shaft formation. In this study, we report a strong downregulation of epithelial Wnt/β-catenin activity as the mammary bud progresses to branching. We show that forced activation of epithelial β-catenin severely compromises embryonic mammary gland branching. However, the phenotype of conditional Lef1-deficient embryos implies that a low level of Wnt/β-catenin activity is necessary for mammary cell survival. Transcriptomic profiling suggests that sustained high β-catenin activity leads to maintenance of mammary bud gene signature at the expense of outgrowth/branching gene signature. In addition, it leads to upregulation of epidermal differentiation genes. Strikingly, we find a partial switch to hair follicle fate early on upon stabilization of β-catenin, suggesting that the level of epithelial Wnt/β-catenin signaling activity may contribute to the choice between skin appendage identities., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Branching morphogenesis is an evolutionary solution to maximize epithelial function in a compact organ. It involves successive rounds of branch elongation and branch point formation to generate a tubular network. In all organs, branch points can form by tip splitting, but it is unclear how tip cells coordinate elongation and branching. Here, we addressed these questions in the embryonic mammary gland. Live imaging revealed that tips advance by directional cell migration and elongation relies upon differential cell motility that feeds a retrograde flow of lagging cells into the trailing duct, supported by tip proliferation. Tip bifurcation involved localized repression of cell cycle and cell motility at the branch point. Cells in the nascent daughter tips remained proliferative but changed their direction to elongate new branches. We also report the fundamental importance of epithelial cell contractility for mammary branching morphogenesis. The co-localization of cell motility, non-muscle myosin II, and ERK activities at the tip front suggests coordination/cooperation between these functions., (© 2023 Myllymäki et al.)

Multidimensional fluorescence imaging represents a powerful approach for studying the dynamic cellular processes underpinning the development, function, and maintenance of the mammary gland. Here, we describe key multidimensional imaging strategies that enable visualization of mammary branching morphogenesis and epithelial cell fate dynamics during postnatal and embryonic mammary gland development. These include 4-dimensional intravital microscopy and ex vivo imaging of embryonic mammary cultures, in addition to methods that facilitate 3-dimensional imaging of the ductal epithelium at single-cell resolution within its native stroma. Collectively, these approaches provide a window into mammary developmental dynamics, and the perturbations underlying tissue dysfunction and disease., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The mammary gland develops from the surface ectoderm during embryogenesis and proceeds through morphological phases defined as placode, hillock, bud, and bulb stages followed by branching morphogenesis. During this early morphogenesis, the mammary bud undergoes an invagination process where the thickened bud initially protrudes above the surface epithelium and then transforms to a bulb and sinks into the underlying mesenchyme. The signaling pathways regulating the early morphogenetic steps have been identified to some extent, but the underlying cellular mechanisms remain ill defined. Here, we use 3D and 4D confocal microscopy to show that the early growth of the mammary rudiment is accomplished by migration-driven cell influx, with minor contributions of cell hypertrophy and proliferation. We delineate a hitherto undescribed invagination mechanism driven by thin, elongated keratinocytes-ring cells-that form a contractile rim around the mammary bud and likely exert force via the actomyosin network. Furthermore, we show that conditional deletion of nonmuscle myosin IIA (NMIIA) impairs invagination, resulting in abnormal mammary bud shape., (© 2021 Trela et al.)

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Embryonic mammary gland development involves the formation of mammary placodes, invagination of flask-shaped mammary buds and development of miniature bi-layered ductal trees. Currently there is a good understanding of the factors that contribute to ectodermal cell movements to create these appendages and of pathways that lead to mammary specification and commitment. Gene expression profiles of early bipotent mammary stem cells populations as well as cell surface proteins and transcription factors that promote the emergence of unipotent progenitors have been identified. Analyses of these populations has illuminated not only embryonic mammary development, but highlighted parallel processes in breast cancer. Here we provide an overview of the highly conserved pathways that shape the embryonic mammary gland. Understanding the dynamic signaling events that occur during normal mammary development holds considerable promise to advance attempts to eliminate cancer by restoring differentiative signals., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Little is known about the role of Sox11 in the regulation of mammary progenitor cells. Sox11 is expressed by mammary bud epithelial cells during embryonic mammary gland development and is not detected in mammary epithelial cells after birth. As Sox11 is an oncofetal gene, we investigated the effects of reducing Sox11 levels in embryonic mammary progenitor cells and found that Sox11 regulates proliferative state, stem cell activity and lineage marker expression. We also investigated the effect of reducing Sox11 levels in two transplantable Brca1-deficient oestrogen receptor-negative mouse mammary tumour cell lines, to assess whether Sox11 regulates similar functions in tumour progenitor cells. When Sox11 levels were reduced in one Brca1-deficient mammary tumour cell line that expressed both epithelial and mesenchymal markers, similar effects on proliferation, stem cell activity and expression of lineage markers to those seen in the embryonic mammary progenitor cells were observed. Orthotopic grafting of mammary tumour cells with reduced Sox11 levels led to alterations in tumour-initiating capacity, latency, expression of lineage markers and metastatic burden. Our results support a model in which tumours expressing higher levels of Sox11 have more stem and tumour-initiating cells, and are less proliferative, whereas tumours expressing lower levels of Sox11 become more proliferative and capable of morphogenetic/metastatic growth, similar to what occurs during embryonic mammary developmental progression., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The mammary gland is a unique tissue and the defining feature of the class Mammalia. It is a late-evolving epidermal appendage that has the primary function of providing nutrition for the young, although recent studies have highlighted additional benefits of milk including the provision of passive immunity and a microbiome and, in humans, the psychosocial benefits of breastfeeding. In this Review, we outline the various stages of mammary gland development in the mouse, with a particular focus on lineage specification and the new insights that have been gained by the application of recent technological advances in imaging in both real-time and three-dimensions, and in single cell RNA sequencing. These studies have revealed the complexity of subpopulations of cells that contribute to the mammary stem and progenitor cell hierarchy and we suggest a new terminology to distinguish these cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Loss of expression of the transcription regulator DC-SCRIPT (Zfp366) is a prominent prognostic event in estrogen receptor-positive breast cancer patients. Studying the inherent link between breast morphogenesis and tumorigenesis, we recently reported that DC-SCRIPT affects normal mammary branching morphogenesis and mammary epithelium homeostasis. Here we investigated the molecular mechanism involved in DC-SCRIPT mediated regulation of FGF2 induced mammary branching morphogenesis in a 3D organoid culture system. Our data show that the delayed mammary organoid branching observed in DC-SCRIPT -/- organoids cannot be compensated for by increasing FGF2 levels. Interestingly, FGFR1, the dominant FGF2 receptor, was expressed at a significantly lower level in basal epithelial cells of DC-SCRIPT deficient organoids relative to wildtype organoids. A potential link between DC-SCRIPT and FGFR1 was further supported by the predicted locations of the DC-SCRIPT DNA binding motif at the Fgfr1 gene. Moreover, ERK1/2 phosphorylation downstream of the FGFR1 pathway was decreased in basal epithelial cells of DC-SCRIPT deficient organoids. Altogether, this study shows a relationship between DC-SCRIPT and FGFR1 related pERK signaling in modulating the branching morphogenesis of mammary organoids in vitro., Competing Interests: Declaration of competing interest The authors declare no competing of financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Over the past 5 years, several studies have begun to uncover the links between the classical signal transduction pathways and the physical mechanisms that are used to sculpt branched tissues. These advances have been made, in part, thanks to innovations in live imaging and reporter animals. With modern research tools, our conceptual models of branching morphogenesis are rapidly evolving, and the differences in branching mechanisms between each organ are becoming increasingly apparent. Here, we highlight four branched epithelia that develop at different spatial scales, within different surrounding tissues and via divergent physical mechanisms. Each of these organs has evolved to employ unique branching strategies to achieve a specialized final architecture., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

17β-Estradiol induces the postnatal development of mammary gland and influences breast carcinogenesis by binding to the estrogen receptor ERα. ERα acts as a transcription factor but also elicits rapid signaling through a fraction of ERα expressed at the membrane. Here, we have used the C451A-ERα mouse model mutated for the palmitoylation site to understand how ERα membrane signaling affects mammary gland development. Although the overall structure of physiological mammary gland development is slightly affected, both epithelial fragments and basal cells isolated from C451A-ERα mammary glands failed to grow when engrafted into cleared wild-type fat pads, even in pregnant hosts. Similarly, basal cells purified from hormone-stimulated ovariectomized C451A-ERα mice did not produce normal outgrowths. Ex vivo , C451A-ERα basal cells displayed reduced matrix degradation capacities, suggesting altered migration properties. More importantly, C451A-ERα basal cells recovered in vivo repopulating ability when co-transplanted with wild-type luminal cells and specifically with ERα-positive luminal cells. Transcriptional profiling identified crucial paracrine luminal-to-basal signals. Altogether, our findings uncover an important role for membrane ERα expression in promoting intercellular communications that are essential for mammary gland development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fibroblast growth factor (FGF) signaling is crucial for mammary gland development. Although multiple roles for FGF signaling in the epithelium have been described, the function of FGF signaling in mammary stroma has not been elucidated. In this study, we investigated FGF signaling in mammary fibroblasts. We found that murine mammary fibroblasts express FGF receptors FGFR1 and FGFR2 and respond to FGF ligands. In particular, FGF2 and FGF9 induce sustained ERK1/2 signaling and promote fibroblast proliferation and migration in 2D cultures. Intriguingly, only FGF2 induces fibroblast migration in 3D extracellular matrix (ECM) through regulation of actomyosin cytoskeleton and promotes force-mediated collagen remodeling by mammary fibroblasts. Moreover, FGF2 regulates production of ECM proteins by mammary fibroblasts, including collagens, fibronectin, osteopontin and matrix metalloproteinases. Finally, using organotypic 3D co-cultures we show that FGF2 and FGF9 signaling in mammary fibroblasts enhances fibroblast-induced branching of mammary epithelium by modulating paracrine signaling, and that knockdown of Fgfr1 and Fgfr2 in mammary fibroblasts reduces branching of mammary epithelium. Our results demonstrate a pleiotropic role for FGF signaling in mammary fibroblasts, with implications for regulation of mammary stromal functions and epithelial branching morphogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Branching morphogenesis is a fundamental developmental program that generates large epithelial surfaces in a limited three-dimensional space. It is regulated by inductive tissue interactions whose effects are mediated by soluble signaling molecules, and cell-cell and cell-extracellular matrix interactions. Here, we will review recent studies on inductive signaling interactions governing branching morphogenesis in light of phenotypes of mouse mutants and ex vivo organ culture studies with emphasis on developing mammary and salivary glands. We will highlight advances in understanding how cell fate decisions are intimately linked with branching morphogenesis. We will also discuss novel insights into the molecular control of cellular mechanisms driving the formation of these arborized ductal structures and reflect upon how distinct spatial patterns are generated., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The mammary gland develops from the placode at ectodermal invagination. The rudimentary parenchyma (mammary bud) develops mammary trees and alveolar structures, suggesting that the mammary bud consists of stem/progenitor cells. Here, we established a clonal stem cell line from a mammary bud of a p53 null female embryo at day 14.5. FP5-3-1 line was a homogeneous cell population with polygonal epithelial morphology and spontaneously became heterogeneous during passages. Recloning gave rise to four sublines; three sublines have basal epithelial property and one subline has luminal epithelial property. The former sublines generate functional mammary glands when injected into cleared fat pads and the latter subline does not. The cell lines also express many stemness-related genes. The clonal cell lines established in the present study are shown to be mammary stem cells and not tumorigenic. They provide useful models for normal and tumor biology of the mammary gland in vivo and in vitro.

Humans are exposed to estrogenic chemicals in food and food packaging, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS; 4,4'-sulfonyldiphenol), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. Here, we quantified the effects of perinatal exposures to BPS on the male mouse mammary gland. In our first study, pregnant CD-1 mice were orally exposed to BPS (2 or 200 μg/kg/day) starting on pregnancy day 9 through lactation day 20, and male mammary glands were evaluated on embryonic day 16, prior to puberty, and in early adulthood. We observed modest changes in tissue organization in the fetal gland, and significant increases in growth of the gland induced by developmental BPS exposure in adulthood. In our second study, pregnant CD-1 mice were orally exposed to BPS (2, 200 or 2000 μg/kg/day) starting on pregnancy day 9 through lactational day 2. After weaning, the male pups were administered either oil (vehicle) or an estrogen challenge (1 μg ethinyl estradiol/kg/day) for ten days starting prior to puberty. After the 10-day estrogen challenge, we examined hormone-sensitive outcomes including anogenital index (AGI), weight of the seminal vesicles, and morphological parameters of the mammary gland. Although AGI and seminal vesicle weight were not affected by BPS, we observed dose-specific effects on the response of male mammary glands to the peripubertal estrogen challenge. Because male mammary glands are structurally less developed compared to females, they may provide a simple model tissue to evaluate the effects of putative xenoestrogens., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Milk production by the sow is a major factor limiting the growth and survival of her litter. Understanding the process of morphogenesis of the sow's mammary gland and the factors that regulate mammary development are important for designing successful management tools that may enhance milk production. Primordia of the mammary glands are first observable in the porcine embryo at approximately 23 days of gestation. The glands then progress through a series of morphologically distinct developmental stages such that, at birth, each mammary gland is composed of the teat, an organized fat pad and two separate lactiferous ducts each with a few ducts branching into the fat pad. The glands continue to grow slowly until about 90 days of age when the rate of growth increases significantly. The increased rate of mammary gland growth coincides with the appearance of large ovarian follicles and an increase in circulating estrogen. After puberty, the continued growth of the gland and elongation and branching of the duct system into the fat pad takes place in response to the elevated levels of estrogen occurring as part of the estrous cycles. After conception, parenchymal mass of each gland increases slowly during early pregnancy and then grows increasingly rapidly during the final trimester. This growth is in response to estrogen, progesterone, prolactin and relaxin. Lobuloalveolar development occurs primarily during late pregnancy. By parturition, the fat pad of the mammary gland has been replaced by colostrum-secreting epithelial cells that line the lumen of the alveoli, lobules and small ducts. All mammary glands develop during pregnancy, however, the extent of development is dependent on the location of the mammary gland on the sow's underline. The mammary glands undergo significant functional differentiation immediately before and after farrowing with the formation of colostrum and the transition through the stages of lactogenesis. Further growth of the glands during lactation is stimulated by milk removal. Individual glands may grow or transiently regress in response to the intensity of suckling during the initial days postpartum. Attempts to enhance milk production by manipulation of mammary development at stages before lactation generally have met with limited success. A more in depth understanding of the processes regulating porcine mammary gland morphogenesis at all stages of development is needed to make further progress.

The pig teat traits are important indices of genetic improvement in pig breeding, which belong to reproductive traits and can directly affect the sows lactation rate and piglet survival rate. Understanding the genetic mechanism underlying the variation of teat traits is of immense value for the improvement of pig reproductive performance. However, the genetic mechanism underlying teat traits (including teat number, type, location distribution, and fluctuating asymmetry) remains elusive. In this review, we summarize the studies on physiology and genetics of teat traits in pigs, including the development process of the mammary gland, the QTL mapping, and candidate gene researches. This review aims to provide a new perspective for the identification of causal mutations and major genes affecting the teat traits and revealing the complex genetic mechanism of the differences in teat number, type and location distribution during embryonic development in pigs.

Commercial artificial sweeteners present in the market are usually made of combination of nutritive and artificial sweeteners such as sorbitol and aspartame. The aim of this research was to study the effect of in utero exposure to commercial artificial sweeteners on the mouse development and on mammary gland in different stages (18-day embryos and 4-week-old mice). Pregnant mice of treated groups were given 50 mg/kg body weight of commercial artificial sweetener. The dose was given on day 1 of pregnancy until 3-week nursing, while the controls were given distilled water. Congenital malformations were seen in treated 18-day fetus and 4-week-old mice, such as a significant decrease in the diameter of the placenta and the weight of the fetuses, while in 4-week-old mice, a significant decrease in the length of the body, limbs, and tail was seen compared to the controls. The result of this study showed that in 18-day fetuses, clusters of mammary gland in the treated mice seemed to be more differentiated than the controls. In 4-week-old mice, the number of mammary gland ducts in the treated group was significantly more than the control group, and the lumen of the ducts in the treated sections seemed to be narrower than the controls, also many regressing terminal end buds (TEBs) were seen in the treated group. A significant increase in the mammary gland area of treated group was seen compared to the controls.

The innervation of the mammary gland is controlled by brain-derived neurotrophic factor (BDNF), and sexually dimorphic sequestering of BDNF by the truncated form of TrkB (TrkB.T1) directs male-specific axonal pruning in mice. It is unknown whether other cues modulate these processes. We detected specific, non-dimorphic, expression of Semaphorin family members in the mouse mammary gland, which signal through PlexinA4. PlexinA4 deletion in both female and male embryos caused developmental hyperinnervation of the gland, which could be reduced by genetic co-reduction of BDNF. Moreover, in males, PlexinA4 ablation delayed axonal pruning, independently of the initial levels of innervation. In support of this, in vitro reduction of BDNF induced axonal hypersensitivity to PlexinA4 signaling. Overall, our study shows that precise sensory innervation of the mammary gland is regulated by the balance between trophic and repulsive signaling. Upon inhibition of trophic signaling, these repulsive factors may promote axonal pruning., Competing Interests: HS, RG, YG, AY No competing interests declared, (© 2019, Sar Shalom et al.)

Stem cell biology offers promise for understanding the origins of the mammary gland. However, the distribution of mammary stem cell (MaSC) activities at earlier embryonic stages has not been fully identified. The markers for sorting adult MaSC, CD24, CD29, and CD49f have been applied to analyze fetal MaSCs. Here we explored mammary anlagen MaSCs by investigating the expression of CD24 and CD49f. According to the comparative analysis between adult mammary gland and fetal mammary anlagen, we found that fetal mouse mammary anlagen may possess a high percentage of potential MaSCs. Flow cytometry analysis revealed 2 distinct mammary anlagen populations: Lin - CD24 med and Lin - CD24 high . Sphere-forming and mammary repopulating assays confirmed that the stem cell activity of E14.5 mouse mammary anlagen was restricted to the Lin - CD24 med cell population. Furthermore, CD24 med mammary anlagen cells were separated into Lin - CD24 med CD49f + and Lin - CD24 med CD49f - populations and identified, respectively. The results proved that the mammary anlagen Lin - CD24 med CD49f + cell population possesses more stem cell activities than the Lin - CD24 med CD49f - cell population. However, a limited numbers of stem cells and large numbers of stromal cells were identified in mammary anlagen in the Lin - CD24 med cell population.

Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type-specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states., Competing Interests: The authors have declared that no competing interests exist.

Mammary glands develop through primary ductal elongation and side branching to maximize the spatial area. Although primary ducts are generated by bifurcation of terminal end buds, the mechanism through which side branching occurs is still largely unclear. Here, we show that inhibitor of DNA-binding 2 (ID2) drives side branch formation through the differentiation of K6 + bipotent progenitor cells (BPs) into CD61 + luminal progenitor cells (LPs). Id2 -null mice had side-branching defects, along with developmental blockage of the differentiation of K6 + BPs into CD61 + LPs. Notably, CD61 + LPs were found in budding and side branches, but not in terminal end buds. Hormone reconstitution studies using ovariectomized MMTV-hemagglutinin-nuclear localized sequence-tagged Id2 transgenic mice revealed that ID2 is a key mediator of progesterone, which drives luminal lineage differentiation and side branching. Our results suggest that CD61 is a marker of side branches and that ID2 regulates side branch formation by inducing luminal lineage commitment from K6 + BPs to CD61 + LPs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The mammary gland is composed of basal cells and luminal cells. It is generally believed that the mammary gland arises from embryonic multipotent progenitors, but it remains unclear when lineage restriction occurs and what mechanisms are responsible for the switch from multipotency to unipotency during its morphogenesis. Here, we perform multicolour lineage tracing and assess the fate of single progenitors, and demonstrate the existence of a developmental switch from multipotency to unipotency during embryonic mammary gland development. Molecular profiling and single cell RNA-seq revealed that embryonic multipotent progenitors express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in embryonic multipotent progenitors promotes unipotent basal cell fate and was sufficient to reprogram adult luminal cells into basal cells by promoting an intermediate hybrid multipotent-like state. Altogether, this study identifies the timing and the mechanisms mediating early lineage segregation of multipotent progenitors during mammary gland development.

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

Fatty Acid Elongase 7 (ELOVL7) is the newly discovered protein on human that catalyzes the rate-limiting step towards the synthesis of very long-chain fatty acids and exhibits the highest activity toward C18: 3 (n-3) acyl-CoAs, which is the precursor of eicosapentaenoic acid (EPA, 20: 5n-3). However, in ruminants, an overall understanding of ELOVLs gene family and the transcriptional regulation of ELOVL7 remain unknown. The purpose of this study is to investigate the transcriptional regulation and the influence of bovine ELOVL7 in bovine mammary epithelial cells (bMECs). Quantitative real-time PCR analysis demonstrated that ELOVLs gene family had differential expression patterns in bMECs, and bovine ELOVL7 was expressed in a tissue-specific manner, which was high in kidney, followed by in abdominal fat and in bMECs. Promoter analysis of bovine ELOVL7, including bioinformatics analyzes, dual-luciferase reporter assays, protein pull-down assay, Western blot assay, over-expression and RNA interference assay, have independently and synthetically demonstrated that transcription factor Sp1 (SP1) specifically interacted with the GC-box at -143 to -128 base pair on ELOVL7 promoter. Furthermore, the exogenous α-linolenic acid (ALA, 18: 3n-3), strengthened the binding of SP1 to the ELOVL7 proximal promoter, resulting in the accumulation of lipid droplets in bMECs. In conclusion, these data suggest that the transcription of bovine ELOVL7 is affected by the binding of SP1 and the treatment of ALA, moreover, enlightening us the profound role of SP1 in modulating lipid synthesis of the mammary gland in cattle., (© 2017 Wiley Periodicals, Inc.)

Epithelial cells contribute to the development of various vital organs by generating tubular and/or glandular architectures. The fully developed forms of ductal organs depend on processes of branching morphogenesis, whereby frequency, total number, and complexity of the branching tissue define the final architecture in the organ. Some ductal tissues, like the mammary gland during pregnancy and lactation, disintegrate and regenerate through periodic cycles. Differentiation of branched epithelia is driven by antagonistic actions of parallel growth factor systems that mediate epithelial-mesenchymal communication. Transforming growth factor-β (TGF-β) family members and their extracellular antagonists are prominently involved in both normal and disease-associated (e.g., malignant or fibrotic) ductal tissue patterning. Here, we discuss collective knowledge that permeates the roles of TGF-β family members in the control of the ductal tissues in the vertebrate body., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

Prep1 (pKnox1) is a homeodomain transcription factor essential for in utero and post-natal development and an oncosuppressor gene in human and adult mice. We have analyzed its role in the development of the mouse mammary gland. We used Prep1 i/i hypomorphic and Prep1 F/F -Ker5CRE crosses to analyze the role of Prep1 in vivo in adult mouse mammary gland development. We also cultured mammary gland stem/progenitor cells in mammospheres to perform biochemical studies. Prep1 was expressed in mammary gland progenitors and fully differentiated mammary gland cells. Using different Prep1-deficient mouse models we show that in vivo Prep1 contributes to mammary gland branching since the branching efficiency of the mammary gland in Prep1-deleted or Prep1 hypomorphic mice was largely reduced. In-vitro, Prep1 sustained functions of the mammary stem/progenitor compartment. Prep1-deficient mammary stem/progenitor cells showed reduced ability to form mammospheres; they were not able to branch in a 3D assay, and exhibited reduced expression of Snail1, Snail2 and vimentin. The branching phenotype associated with increased Tp53-dependent apoptosis and inability to properly activate signals involved in branching morphogenesis. Finally, Prep1 formed complexes with Snail2, a transcription factor essential in branching morphogenesis, and its absence destabilizes and promotes Snail2 proteasome-mediated degradation. We conclude that Prep1 is required for normal adult mammary gland development, in particular at its branching morphogenesis step. By binding Snail2, Prep1 protects it from the proteasomal degradation.

Poultry embryos are easily affected by environmental changings during incubation, thereinto, the temperature modification is the most important one, but the mechanism of temperature effects on bird eggs is not clear. By using RNA-seq, we have previously found that endoplasmic reticulum stress (ERS) may involve in regulating embryonic muscle development of duck under the influence of temperature alteration. To further clarify the role of ERS in the effect, in the present study, we detected the impact of increasing the incubation temperature by 1℃ during embryonic days 10-27 (E10-27) on the development of duck embryos, and investigated the changes in mRNA and protein expression of ERS marker genes and muscle-related genes under the thermal manipulation (TM). The results of relative weight comparison showed that only the relative weight of breast muscle was steadily decreased by TM from E10 to the first day after hatching (W0). Meanwhile, the real-time PCR and western-blot analysis revealed that raising the incubation temperature stimulated the expression of ERS marker genes in breast muscle at E20. The mRNA expressions of muscle hypertrophy and atrophy-related genes were also detected, and were not changed regularly, however, the protein expressions of hypertrophy-related genes were all decreased at both E20 and W0, and the protein expression of atrophy-related genes were up-regulated at E20. The protein expression of muscle proliferation-related genes were also decreased at E20. Additionally, these results were the same as that in the ERS positive control groups. Taken together, these results indicated that long-term TM during late embryonic period could block the development of duck breast muscle by inhibiting muscle hypertrophy and proliferation, and promoting muscle atrophy at a post-transcriptional level via the activation of ERS., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The hierarchical relationships between various stem and progenitor cell subpopulations driving mammary gland morphogenesis and homoeostasis are poorly understood. Conditional inactivation experiments previously demonstrated that expression of the zinc finger transcriptional repressor Blimp1/PRDM1 is essential for the establishment of epithelial cell polarity and functional maturation of alveolar cells. Here we exploit a Prdm1.CreERT2-LacZ reporter allele for lineage tracing experiments. Blimp1 expression marks a rare subpopulation of unipotent luminal stem cells that initially appear in the embryonic mammary gland at around E17.5 coincident with the segregation of the luminal and basal compartments. Fate mapping at multiple time points in combination with whole-mount confocal imaging revealed these long-lived unipotent luminal stem cells survive consecutive involutions and retain their identity throughout adult life. Blimp1 + luminal stem cells give rise to Blimp1 - progeny that are invariably Elf5 + ERα - PR - . Thus, Blimp1 expression defines a mammary stem cell subpopulation with unique functional characteristics.

In utero exposure to the endocrine disrupting compound bisphenol A (BPA) is known to disrupt mammary gland development and increase tumor susceptibility in rodents. It is unclear whether different periods of in utero development might be more susceptible to BPA exposure. We exposed pregnant CD-1 mice to BPA at different times during gestation that correspond to specific milestones of in utero mammary gland development. The mammary glands of early-life and adult female mice, exposed in utero to BPA, were morphologically and molecularly (estrogen receptor-α and Ki67) evaluated for developmental abnormalities. We found that BPA treatment occurring before mammary bud invasion into the mesenchyme [embryonic day (E)12.5] incompletely resulted in the measured phenotypes of mammary gland defects. Exposing mice up to the point at which the epithelium extends into the precursor fat pad (E16.5) resulted in a nearly complete BPA phenotype and exposure during epithelial extension (E15.5 to E18.5) resulted in a partial phenotype. Furthermore, the relative differences in phenotypes between exposure windows highlight the substantial correlations between early-life molecular changes (estrogen receptor-α and Ki67) in the stroma and the epithelial elongation defects in mammary development. These data further implicate BPA action in the stroma as a critical mediator of epithelial phenotypes., (Copyright © 2017 Endocrine Society.)

Obesity and alterations in metabolic programming from early diet exposures can affect the propensity to disease in later life. Through dietary manipulation, developing mouse pups were exposed to a hyperinsulinemic, hyperglycemic milieu during three developmental phases: gestation, lactation, and postweaning. Analyses showed that a postweaning high fat/high sugar (HF/HS) diet had the main negative effect on adult body weight, glucose tolerance, and insulin resistance. However, dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis revealed that animals born to a mother fed a HF/HS gestation diet, nursed by a mother on a mildly diet-restricted, low fat/low sugar diet (DR) and weaned onto a HF/HS diet (HF/DR/HF) had the highest mammary tumor incidence, while HF/HF/DR had the lowest tumor incidence. Cox proportional hazards analysis showed that a HF/HS postweaning diet doubled mammary cancer risk, and a HF/HS diet during gestation and postweaning increased risk 5.5 times. Exposure to a HF/HS diet during gestation, when combined with a postweaning DR diet, had a protective effect, reducing mammary tumor risk by 86% (HR = 0.142). Serum adipocytokine analysis revealed significant diet-dependent differences in leptin/adiponectin ratio and IGF-1. Flow cytometry analysis of cells isolated from mammary glands from a high tumor incidence group, DR/HF/HF, showed a significant increase in the size of the mammary stem cell compartment compared with a low tumor group, HF/HF/DR. These results indicate that dietary reprogramming induces an expansion of the mammary stem cell compartment during mammary development, increasing likely carcinogen targets and mammary cancer risk. Cancer Prev Res; 10(10); 553-62. ©2017 AACR See related editorial by Freedland, p. 551-2 ., (©2017 American Association for Cancer Research.)

Macrophages are required for proper mammary gland development and maintaining tissue homeostasis. However, the mechanisms by which macrophages regulate this process remain unclear. Here, we identify STAT5 as an important regulator of macrophage function in the developing mammary gland. Analysis of mammary glands from mice with STAT5-deficient macrophages demonstrates delayed ductal elongation, enhanced ductal branching and increased epithelial proliferation. Further analysis reveals that STAT5 deletion in macrophages leads to enhanced expression of proliferative factors such as Cyp19a1/aromatase and IL-6. Mechanistic studies demonstrate that STAT5 binds directly to the Cyp19a1 promoter in macrophages to suppress gene expression and that loss of STAT5 results in enhanced stromal expression of aromatase. Finally, we demonstrate that STAT5 deletion in macrophages cooperates with oncogenic initiation in mammary epithelium to accelerate the formation of estrogen receptor (ER)-positive hyperplasias. These studies establish the importance of STAT5 in macrophages during ductal morphogenesis in the mammary gland and demonstrate that altering STAT5 function in macrophages can affect the development of tissue-specific disease., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.

Progression of mammalian cells through the G1 and S phases of the cell cycle is driven by the D-type and E-type cyclins. According to the current models, at least one of these cyclin families must be present to allow cell proliferation. Here, we show that several cell types can proliferate in the absence of all G1 cyclins. However, following ablation of G1 cyclins, embryonic stem (ES) cells attenuated their pluripotent characteristics, with the majority of cells acquiring the trophectodermal cell fate. We established that G1 cyclins, together with their associated cyclin-dependent kinases (CDKs), phosphorylate and stabilize the core pluripotency factors Nanog, Sox2 and Oct4. Treatment of murine ES cells, patient-derived glioblastoma tumour-initiating cells, or triple-negative breast cancer cells with a CDK inhibitor strongly decreased Sox2 and Oct4 levels. Our findings suggest that CDK inhibition might represent an attractive therapeutic strategy by targeting glioblastoma tumour-initiating cells, which depend on Sox2 to maintain their tumorigenic potential.

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα + luminal cells and not the mammary stem cells (MaSCs) that are ERα neg . Since ERα + luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα + subset of EpCAM + /CD24 + /CD49f hi MaSCs. We show that the MaSC population has a distinct SCA-1 + population that is abundant in pre-pubertal mammary glands. The SCA-1 + MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1 neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1 + MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα + , estrogen-sensitive subpopulation within the CD24 + /CD49f hi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Macrophages are important regulators of branching morphogenesis during development and postnatally in the mammary gland. Regulation of macrophage dynamics during these processes can therefore have a profound impact on development. We demonstrate here that the developing mammary gland expresses high levels of inflammatory CC-chemokines, which are essential in vivo regulators of macrophage migration. We further demonstrate that the atypical chemokine receptor ACKR2, which scavenges inflammatory CC-chemokines, is differentially expressed during mammary gland development. We have previously shown that ACKR2 regulates macrophage dynamics during lymphatic vessel development. Here, we extend these observations to reveal a novel role for ACKR2 in regulating the postnatal development of the mammary gland. Specifically, we show that Ackr2 -/- mice display precocious mammary gland development. This is associated with increased macrophage recruitment to the developing gland and increased density of the ductal epithelial network. These data demonstrate that ACKR2 is an important regulator of branching morphogenesis in diverse biological contexts and provide the first evidence of a role for chemokines and their receptors in postnatal development processes., Competing Interests: The authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Left-right (L-R) differences in mammographic parenchymal patterns are an early predictor of breast cancer risk; however, the basis for this asymmetry is unknown. Here, we use retinoid X receptor alpha heterozygous null (RXRα +/- ) mice to propose a developmental origin: perturbation of coordinated anterior-posterior (A-P) and L-R axial body patterning. We hypothesized that by analogy to somitogenesis-in which retinoic acid (RA) attenuation causes anterior somite pairs to develop L-R asynchronously-that RA pathway perturbation would likewise result in asymmetric mammary development. To test this, mammary glands of RXRα +/- mice were quantitatively assessed to compare left- versus right-side ductal epithelial networks. Unlike wild-type controls, half of the RXRα +/- thoracic mammary gland (TMG) pairs exhibited significant L-R asymmetry, with left-side reduction in network size. In RXRα +/- TMGs in which symmetry was maintained, networks had bilaterally increased size, with left networks showing greater variability in area and pattern. Reminiscent of posterior somites, whose bilateral symmetry is refractory to RA attenuation, inguinal mammary glands (IMGs) also had bilaterally increased network size, but no loss of symmetry. Together, these results demonstrate that mammary glands exhibit differential A-P sensitivity to RXRα heterozygosity, with ductal network symmetry markedly compromised in anterior but not posterior glands. As TMGs more closely model human breast development than IMGs, these findings raise the possibility that for some women, breast cancer risk may initiate with subtle axial patterning defects that result in L-R asymmetric growth and pattern of the mammary ductal epithelium.This article is part of the themed issue 'Provocative questions in left-right asymmetry'., (© 2016 The Author(s).)

Vertebrate Hox genes encode transcription factors operating during the development of multiple organs and structures. However, the evolutionary mechanism underlying this remarkable pleiotropy remains to be fully understood. Here, we show that Hoxd8 and Hoxd9, two genes of the HoxD complex, are transcribed during mammary bud (MB) development. However, unlike in other developmental contexts, their coexpression does not rely on the same regulatory mechanism. Hoxd8 is regulated by the combined activity of closely located sequences and the most distant telomeric gene desert. On the other hand, Hoxd9 is controlled by an enhancer-rich region that is also located within the telomeric gene desert but has no impact on Hoxd8 transcription, thus constituting an exception to the global regulatory logic systematically observed at this locus. The latter DNA region is also involved in Hoxd gene regulation in other contexts and strongly interacts with Hoxd9 in all tissues analyzed thus far, indicating that its regulatory activity was already operational before the appearance of mammary glands. Within this DNA region and neighboring a strong limb enhancer, we identified a short sequence conserved in therian mammals and capable of enhancer activity in the MBs. We propose that Hoxd gene regulation in embryonic MBs evolved by hijacking a preexisting regulatory landscape that was already at work before the emergence of mammals in structures such as the limbs or the intestinal tract., Competing Interests: The authors declare no conflict of interest.

Among all tissues and organs, the mammary gland is unique because most of its development occurs in adulthood. Notch signaling has a major role in mammary gland development and has been implicated in breast cancer. The vacuolar-ATPase (V-ATPase) is a proton pump responsible for the regulation and control of pH in intracellular vesicles and the extracellular milieu. We have previously reported that a2V-ATPase (a2V), an isoform of 'a' subunit of V-ATPase, regulates processing of Notch receptor and alters Notch signaling in breast cancer. To study the role of a2V in mammary gland development, we generated an a2V-KO model (conditional mammary knockout a2V mouse strain). During normal mammary gland development, the basal level expression of a2V increased from puberty, virginity, and pregnancy through the lactation stage and then decreased during involution. Litters of a2V-KO mice weighed significantly less when compared with litters from wild-type mice and showed reduced expression of the lactation marker β-casein. Whole-mount analysis of mammary glands demonstrated impaired ductal elongation and bifurcation in a2V-KO mice. Consequently, we found disintegrated mammary epithelium as seen by basal and luminal epithelial staining, although the rate of proliferation remained unchanged. Delayed mammary morphogenesis in a2V-KO mice was associated with aberrant activation of Notch and TGF-β (transforming growth factor-β) pathways. Notably, Hey1 (hairy/enhancer-of-split related with YRPW motif) and Smad2, the key downstream mediators of Notch and TGF-β pathways, respectively, were upregulated in a2V-KO mice and also in human mammary epithelial cells treated with a2V siRNA. Taken together, our results show that a2V deficiency disrupts the endolysosomal route in Notch and TGF signaling, thereby impairing mammary gland development. Our findings have broader implications in developmental and oncogenic cellular environments where V-ATPase, Notch and TGF-β are crucial for cell survival.

Macrophages are required for normal embryogenesis, tissue homeostasis and immunity against microorganisms and tumours. Adult tissue-resident macrophages largely originate from long-lived, self-renewing embryonic precursors and not from haematopoietic stem-cell activity in the bone marrow. Although fate-mapping studies have uncovered a great amount of detail on the origin and kinetics of fetal macrophage development in the yolk sac and liver, the molecules that govern the tissue-specific migration of these cells remain completely unknown. Here we show that an endothelium-specific molecule, plasmalemma vesicle-associated protein (PLVAP), regulates the seeding of fetal monocyte-derived macrophages to tissues in mice. We found that PLVAP-deficient mice have completely normal levels of both yolk-sac- and bone-marrow-derived macrophages, but that fetal liver monocyte-derived macrophage populations were practically missing from tissues. Adult PLVAP-deficient mice show major alterations in macrophage-dependent iron recycling and mammary branching morphogenesis. PLVAP forms diaphragms in the fenestrae of liver sinusoidal endothelium during embryogenesis, interacts with chemoattractants and adhesion molecules and regulates the egress of fetal liver monocytes to the systemic vasculature. Thus, PLVAP selectively controls the exit of macrophage precursors from the fetal liver and, to our knowledge, is the first molecule identified in any organ as regulating the migratory events during embryonic macrophage ontogeny.

There are currently no reports describing mammary gland development in the Harlan Sprague-Dawley (HSD) rat, the current strain of choice for National Toxicology Program (NTP) testing. Our goals were to empower the NTP, contract labs, and other researchers in understanding and interpreting chemical effects in this rat strain. To delineate similarities/differences between the female and male mammary gland, data were compiled starting on embryonic day 15.5 through postnatal day 70. Mammary gland whole mounts, histology sections, and immunohistochemically stained tissues for estrogen, progesterone, and androgen receptors were evaluated in both sexes; qualitative and quantitative differences are highlighted using a comprehensive visual timeline. Research on endocrine disrupting chemicals in animal models has highlighted chemically induced mammary gland anomalies that may potentially impact human health. In order to investigate these effects within the HSD strain, 2,3,7,8-tetrachlorodibenzo-p-dioxin, diethylstilbestrol, or vehicle control was gavage dosed on gestation day 15 and 18 to demonstrate delayed, accelerated, and control mammary gland growth in offspring, respectively. We provide illustrations of normal and chemically altered mammary gland development in HSD male and female rats to help inform researchers unfamiliar with the tissue and may facilitate enhanced evaluation of both male and female mammary glands in juvenile toxicity studies., (© The Author(s) 2016.)

Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally regulated. In vitro analyses indicate that SerpinB5 phosphorylation is regulated by EGFR ligands, but EGF appears to be the only able to induce SerpinB5 nuclear localization.

Mammary gland morphogenesis depends on a tight balance between cell proliferation, differentiation and apoptosis, to create a defined functional hierarchy within the epithelia. The limited availability of stem cell/progenitor markers has made it challenging to decipher lineage relationships. Here, we identify a rare subset of luminal progenitors that express the zinc finger transcriptional repressor Blimp1, and demonstrate that this subset of highly clonogenic luminal progenitors is required for mammary gland development. Conditional inactivation experiments using K14-Cre and WAPi-Cre deleter strains revealed essential functions at multiple developmental stages. Thus, Blimp1 regulates proliferation, apoptosis and alveolar cell maturation during puberty and pregnancy. Loss of Blimp1 disrupts epithelial architecture and lumen formation both in vivo and in three-dimensional (3D) primary cell cultures. Collectively, these results demonstrate that Blimp1 is required to maintain a highly proliferative luminal subset necessary for mammary gland development and homeostasis., (© 2016. Published by The Company of Biologists Ltd.)

The mammary gland is a dynamic organ that undergoes extensive morphogenesis during the different stages of embryonic development, puberty, estrus, pregnancy, lactation and involution. Systemic and local cues underlie this constant tissue remodeling and act by eliciting an intricate pattern of responses in the mammary epithelial and stromal cells. Decades of studies utilizing methods such as transplantation and lineage-tracing have identified a complex hierarchy of mammary stem cells, progenitors and differentiated epithelial cells that fuel mammary epithelial development. Importantly, these studies have extended our understanding of the molecular crosstalk between cell types and the signaling pathways maintaining normal homeostasis that often are deregulated during tumorigenesis. While several questions remain, this research has many implications for breast cancer. Fundamental among these are the identification of the cells of origin for the multiple subtypes of breast cancer and the understanding of tumor heterogeneity. A deeper understanding of these critical questions will unveil novel breast cancer drug targets and treatment paradigms. In this review, we provide a current overview of normal mammary development and tumorigenesis from a stem cell perspective., (© 2015 Society for Endocrinology.)