A Unifying Theory of Branching Morphogenesis.

The morphogenesis of branched organs remains a subject of abiding interest. Although much is known about the underlying signaling pathways, it remains unclear how macroscopic features of branched organs, including their size, network topology, and spatial patterning, are encoded. Here, we show that, in mouse mammary gland, kidney, and human prostate, these features can be explained quantitatively within a single unifying framework of branching and annihilating random walks. Based on quantitative analyses of large-scale organ reconstructions and proliferation kinetics measurements, we propose that morphogenesis follows from the proliferative activity of equipotent tips that stochastically branch and randomly explore their environment but compete neutrally for space, becoming proliferatively inactive when in proximity with neighboring ducts. These results show that complex branched epithelial structures develop as a self-organized process, reliant upon a strikingly simple but generic rule, without recourse to a rigid and deterministic sequence of genetically programmed events.

Embryonic mammary gland development.

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.

Mammary development in the embryo and adult: new insights into the journey of morphogenesis and commitment.

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.

Branching morphogenesis.

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.

Maternal pineal melatonin in gestation and lactation physiology, and in fetal development and programming.

Pregnancy and lactation are reproductive processes that rely on physiological adaptations that should be timely and adequately triggered to guarantee both maternal and fetal health. Pineal melatonin is a hormone that presents daily and seasonal variations that synchronizes the organism's physiology to the different demands across time through its specific mechanisms and ways of action. The reproductive system is a notable target for melatonin as it actively participates on reproductive physiology and regulates the hypothalamus-pituitary-gonads axis, influencing gonadotropins and sexual hormones synthesis and release. For its antioxidant properties, melatonin is also vital for the oocytes and spermatozoa quality and viability, and for blastocyst development. Maternal pineal melatonin blood levels increase during pregnancy and triggers the maternal physiological alterations in energy metabolism both during pregnancy and lactation to cope with the energy demands of both periods and to promote adequate mammary gland development. Moreover, maternal melatonin freely crosses the placenta and is the only source of this hormone to the fetus. It importantly times the conceptus physiology and influences its development and programing of several functions that depend on neural and brain development, ultimately priming adult behavior and energy and glucose metabolism. The present review aims to explain the above listed melatonin functions, including the potential alterations observed in the progeny gestated under maternal chronodisruption and/or hypomelatoninemia.

Form and function: how estrogen and progesterone regulate the mammary epithelial hierarchy.

The mammary gland undergoes dramatic post-natal growth beginning at puberty, followed by full development occurring during pregnancy and lactation. Following lactation, the alveoli undergo apoptosis, and the mammary gland reverses back to resemble the nonparous gland. This process of growth and regression occurs for multiple pregnancies, suggesting the presence of a hierarchy of stem and progenitor cells that are able to regenerate specialized populations of mammary epithelial cells. Expansion of epithelial cell populations in the mammary gland is regulated by ovarian steroids, in particular estrogen acting through its receptor estrogen receptor alpha (ERα) and progesterone signaling through progesterone receptor (PR). A diverse number of stem and progenitor cells have been identified based on expression of cell surface markers and functional assays. Here we review the current understanding of how estrogen and progesterone act together and separately to regulate stem and progenitor cells within the human and mouse mammary tissues. Better understanding of the hierarchal organization of epithelial cell populations in the mammary gland and how the hormonal milieu affects its regulation may provide important insights into the origins of different subtypes of breast cancer.

In the beginning: the establishment of the mammary lineage during embryogenesis.

The mammary primordium is comprised of an aggregate of immature, undifferentiated mammary epithelial cells and its associated mammary mesenchyme, a specialised tissue which harbours mammary-inductive capacity. The mammary primordium forms during embryogenesis as a result of inductive interactions between its two component tissues, the mammary mesenchyme and epithelium. These two tissues constitute a signalling centre that directs the formation of the mammary gland through a series of reciprocal mesenchymal-epithelial interactions. A rudimentary mammary ductal tree and stroma is formed prior to birth as a result of these interactions. The subsequent mammary outgrowths that arise upon hormonal stimulation during puberty originate from this rudimentary tissue. The initial appearance of the embryonic mammary primordium during embryogenesis represents the earliest morphological evidence of commitment to the mammary lineage. Classic tissue recombination studies of mouse mammary primordial cells have demonstrated that the epithelial cells are already functionally determined as mammary at the embryonic mammary bud stage. Recent studies have determined the molecular identity of the embryonic mammary cells by transcriptomic profiling and these have provided new insights into signalling components that mediate early embryonic mammary inductive signalling and lineage commitment. This review highlights what is currently known about the morphogenesis, function, and behaviour of embryonic mammary cells and examine current knowledge of the genetics underlying mammary cell fate and establishment of the mammary lineage during embryogenesis.

Polythelia: simple atavistic remnant or a suspicious clinical sign for investigation?

Supernumerary nipples (or polythelia) usually appear along the embryonic milk lines or in other sites including the back, thigh, vulva, neck etc. The frequency of polythelia ranges from 0.2% to 5.6%. Despite the plethora of published cases concerning its association with other congenital malformations or syndromes with different patterns of inheritance, polythelia still remains a controversial and theoretical issue. Although most reports describe a link between supernumerary nipples and kidney/urinary tract anomalies, a potential relationship with other congenital anomalies or malignancies has also been speculated. Additionally, polythelia has been associated with genodermatoses, thus being related with an increased malignant potential, as well as with an increased risk for solid tumors such as renal adenocarcinoma, testicular cancer, prostate cancer, and urinary bladder carcinoma. The fact that the Scaramanga (ska) mutant mice presented with ectopic breast tissue imply that misregulation of the neuregulin-3 signaling pathway may be critical in the occurrence of polythelia. This is an attempt to review existing literature in order to (a) draw reliable conclusions whether polythelia is a manifestation of simple atavism or may be associated with concomitant severe conditions needing further investigation and/or management, (b) elucidate its aetiology and (c) establish appropriate clinical and laboratory approach.

Evo-devo of the mammary gland.

We propose a new scenario for mammary evolution based on comparative review of early mammary development among mammals. Mammary development proceeds through homologous phases across taxa, but evolutionary modifications in early development produce different final morphologies. In monotremes, the mammary placode spreads out to form a plate-like mammary bulb from which more than 100 primary sprouts descend into mesenchyme. At their distal ends, secondary sprouts develop, including pilosebaceous anlagen, resulting in a mature structure in which mammary lobules and sebaceous glands empty into the infundibula of hair follicles; these structural triads (mammolobular-pilo-sebaceous units or MPSUs) represent an ancestral condition. In marsupials a flask-like mammary bulb elongates as a sprout, but then hollows out; its secondary sprouts include hair and sebaceous anlagen (MPSUs), but the hairs are shed during nipple formation. In some eutherians (cat, horse, human) MPSUs form at the distal ends of primary sprouts; pilosebaceous components either regress or develop into mature structures. We propose that a preexisting structural triad (the apocrine-pilo-sebaceous unit) was incorporated into the evolving mammary structure, and coupled to additional developmental processes that form the mammary line, placode, bulb and primary sprout. In this scenario only mammary ductal trees and secretory tissue derive from ancestral apocrine-like glands. The mammary gland appears to have coopted signaling pathways and genes for secretory products from even earlier integumentary structures, such as odontode (tooth-like) or odontode-derived structures. We speculate that modifications in signal use (such as PTHrP and BMP4) may contribute to taxonomic differences in MPSU development.

Protocol: ex vivo culture of mouse embryonic mammary buds.

The explant culture techniques of embryonic tissues allow continuous monitoring of organ growth and morphogenesis ex vivo. The effect of growth factors and other soluble molecules can be examined by applying them to the culture medium. Relatively few studies have reported application of tissue culture techniques to analysis of embryonic mammary glands. Here we describe a protocol for murine mammary rudiments that permits ex vivo development up to branching stage.

A method for electroporation to study gene function in mammary gland development.

To reveal the specific functions of various genes during embryonic development, the manipulation of genes using techniques such as electroporation is of fundamental importance for providing direct evidence concerning function or downstream activation of signaling networks. In vitro embryo culture and electroporation are useful techniques to introduce foreign genes, for developmental biology studies. Among the various mammalian culture techniques, Trowell culture is suitable for studies of embryonic mammary gland development because of its stability and ease of use in conjunction with electroporation technique application. The manipulation of gene expression using electroporation is a useful technique for the functional analysis of a particular gene. In this protocol, full steps for electroporation and in vitro embryo culture have been described for use in embryonic mammary gland development research.

Wnt-signalling in the embryonic mammary gland.

The first member of the Wnt-family ligands was identified 30 years ago as a factor in mouse mammary tumours whose expression was deregulated due to the promoter activity emanating from the proximal integration of the Mouse Mammary Tumour Virus genome (Nusse and Varmus, Embo J 31:2670-84, 2012). The Wnt-ligands invoke a number of molecular-genetic signalling cascades fundamental to the patterning of developing tissues and organs during embryogenesis as well as during postnatal development. The Wnt-signalling cascade that controls the activities of ß-catenin and the T-cell Factor (Tcf)/Lympoid enhancer factor (Lef1) plays a fundamental role in control of all stages of embryonic mammary gland development. We provide here a brief overview of the known aspects of Wnt-signalling activities in the embryonic mammary gland and its interactions with other signalling cascades in this developing tissue.

Parathyroid hormone-related protein specifies the mammary mesenchyme and regulates embryonic mammary development.

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

A whole-mount immunofluorescence protocol for three-dimensional imaging of the embryonic mammary primordium.

Whole-mount immunofluorescent staining facilitates the profiling of protein expression patterns within diverse and complex tissues. Thanks to the application of antibodies on whole mounted instead of sectioned specimens, this technique has many advantages with respect to the preservation of biological and pathological features of specimens when compared to conventional immunohistological methods. Here, we describe a protocol and optimal conditions of whole-mount immunofluorescence for studying the formation of mammary primordia. We also show an example three-dimensional reconstruction of a mammary primordium based on z-stacked images of a whole-mount stained specimen using confocal microscopy and image analysis software.

Pleiotropic functions of fibroblast growth factor signaling in embryonic mammary gland development.

The mammary gland is an ectodermal appendage and a defining feature of mammals. Consistent with it being a recent evolutionary novelty, many of the molecules essential for the ontogeny and morphogenesis of various vertebrate organs, including those in the fibroblast growth factor (FGF) signaling pathway, are co-opted for induction, maintenance and morphogenesis of the mammary glands. Understanding the mechanism whereby FGF signaling regulates the fundamental cell behavior during normal mammary gland develop may facilitate determination of the consequences of its deregulation during breast cancer progression.

Neuregulin 3 and erbb signalling networks in embryonic mammary gland development.

We review the role of Neuregulin 3 (Nrg3) and Erbb receptor signalling in embryonic mammary gland development. Neuregulins are growth factors that bind and activate its cognate Erbb receptor tyrosine kinases, which form a signalling network with established roles in breast development and breast cancer. Studies have shown that Nrg3 expression profoundly impacts early stages of embryonic mammary development. Network analysis shows how Nrg/Erbb signals could integrate with other major regulators of embryonic mammary development to elicit the morphogenetic processes and cell fate decisions that occur as the mammary lineage is established.

Hedgehog and Gli signaling in embryonic mammary gland development.

The first mouse mutation associated with a heritable defect in embryonic mammary gland development was Extratoes. It represents a functional null-mutation of the gene encoding Gli3, which is best known as a transcription factor mediating canonical Hedgehog (Hh) signaling. Here we review the roles of Hh and Gli proteins in murine embryonic mammary development. We propose that an off-state for Hh signaling, mediated by Gli3-repressor, is determinant for induction of a mammary instead of hair follicle fate in the trunk surface ectoderm.

Ectodysplasin/NF-κB signaling in embryonic mammary gland development.

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

Progesterone--promoter or inhibitor of breast cancer.

Based on the results of a French cohort of postmenopausal women, it has been claimed that micronized progesterone does not enhance breast cancer risk. The impact of reproductive factors on breast cancer risk and a high prevalence of occult breast carcinomas at the time of menopause suggest an involvement of endogenous progesterone in the development of breast cancer. High mammographic density in the luteal phase and during treatment with estrogen/progestogen combinations reflect a change in the composition of mammary stroma and an increased water accumulation in the extracellular matrix which is caused by hygroscopic hyaluronan-proteoglycan aggregates. Proteoglycans are also involved in the regulation of proliferation, migration, and differentiation of epithelial cells and angiogenesis, and may influence malignant transformation of breast cells and progression of tumors. Reports on a lack of effect of estrogen/progesterone therapy on breast cancer risk may be rooted in a selective prescription to overweight women and/or to the very low progesterone serum levels after oral administration owing to a strong inactivation rate. The contradictory results concerning the proliferative effect of progesterone may be associated with a different local metabolism in normal compared to malignant breast tissue. Similar to other progestogens, hormone replacement therapy with progesterone seems to promote the development of breast cancer, provided that the progesterone serum levels have reached the threshold for endometrial protection.

Anatomy of the human mammary gland: Current status of knowledge.

Mammary glands are unique to mammals, with the specific function of synthesizing, secreting, and delivering milk to the newborn. Given this function, it is only during a pregnancy/lactation cycle that the gland reaches a mature developmental state via hormonal influences at the cellular level that effect drastic modifications in the micro- and macro-anatomy of the gland, resulting in remodeling of the gland into a milk-secretory organ. Pubertal and post-pubertal development of the breast in females aids in preparing it to assume a functional state during pregnancy and lactation. Remarkably, this organ has the capacity to regress to a resting state upon cessation of lactation, and then undergo the same cycle of expansion and regression again in subsequent pregnancies during reproductive life. This plasticity suggests tight hormonal regulation, which is paramount for the normal function of the gland. This review presents the current status of knowledge of the normal macro- and micro-anatomy of the human mammary gland and the distinct changes it undergoes during the key developmental stages that characterize it, from embryonic life through to post-menopausal age. In addition, it discusses recent advances in our understanding of the normal function of the breast during lactation, with special reference to breastmilk, its composition, and how it can be utilized as a tool to advance knowledge on normal and aberrant breast development and function. Finally, anatomical and molecular traits associated with aberrant expansion of the breast are discussed to set the basis for future comparisons that may illuminate the origin of breast cancer.