Mammary Gland Development Hector Macias and Lindsay Hinck∗

Advanced Review

Mammary gland development Hector Macias and Lindsay Hinck∗

The mammary gland develops through several distinct stages. The first transpires in the embryo as the ectoderm forms a mammary line that resolves into placodes. Regulated by epithelial–mesenchymal interactions, the placodes descend into the underlying mesenchyme and produce the rudimentary ductal structure of the gland present at birth. Subsequent stages of development—pubertal growth, pregnancy, lactation, and involution—occur postnatally under the regulation of hormones. Puberty initiates branching morphogenesis, which requires growth hormone (GH) and estrogen, as well as insulin-like growth factor 1 (IGF1), to create a ductal tree that fills the fat pad. Upon pregnancy, the combined actions of progesterone and prolactin generate alveoli, which secrete milk during lactation. Lack of demand for milk at weaning initiates the process of involution whereby the gland is remodeled back to its prepregnancy state. These processes require numerous signaling pathways that have distinct regulatory functions at different stages of gland development. Signaling pathways also regulate a specialized subpopulation of mammary stem cells that fuel the dramatic changes in the gland occurring with each pregnancy. Our knowledge of mammary gland development and mammary stem cell biology has significantly contributed to our understanding of breast cancer and has advanced the discovery of therapies to treat this disease. © 2012 Wiley Periodicals, Inc.

How to cite this article: WIREs Dev Biol 2012. doi: 10.1002/wdev.35

INTRODUCTION and luminal. The basal epithelium consists of myoepithelial cells, which generate the outer layer of the he mammary gland (breast) distinguishes mam- gland, and a small population of stem cells, which mals from all other animals with its unique T supply the different cell types. The luminal epithelium anatomical structure that secretes milk for the nourish- forms ducts and secretory alveoli and contains pop- ment of the newborn. Mammary glands are epidermal ulations of cells defined by their hormone receptor appendages that evolved over 300 million years ago, status. Together with the myoepithelium, the lumi- most likely from apocrine sweat glands.1 They are nal epithelium generates a bilayered, tubular structure complex secretory organs composed of a number of that allows form to meet function during lactation different cell types: epithelial cells that grow from when the outer myoepithelial cells contract to squeeze the nipple into a fat pad, formed by adipocytes and milk from the inner alveolar luminal cells. infiltrated by vascular endothelial cells, fibroblasts, There are three major stages of breast develop- and immune cells. This article focuses primarily on ment—embryonic, pubertal, and reproductive. Our changes occurring in the epithelial compartment over knowledge of these stages is derived primarily from the lifetime of the animal (Figure 1). During embryo- studies performed in mice, providing insight into the genesis, these changes are directed by signals from the biology of the human breast. Although there are archi- mesenchyme, but during puberty and in adulthood, circulating hormones released from the pituitary and tectural and hormonal differences between mouse ovary provide additional instructive input. Two main and human mammary glands, many researchers use cell types comprise the mammary epithelium: basal the mouse mammary gland as a model system to explore developmental mechanisms because the ∗Correspondence to: [email protected] gland is amenable to sophisticated in vivo and in Department of Molecular, Cell and Developmental Biology, vitro manipulations. These techniques have allowed University of California, Santa Cruz, CA, USA researchers to investigate mechanisms underlying

Fat pad End bud, 1° branch Alveoli Involution Lymph node 2° and 3° branch Stage 1 involution Stage 2 involution Stage 2 Stage 1

LN LN

Newborn 4-week virgin 12 weeks Estrus Lactation

LN LN LN LN LN

GH Progesterone LN Estrogen IGF1 Progesterone Pregnancy prolactin

FIGURE 1| Illustration depicting the stages of postnatal mammary gland development. At birth, the mammary epithelium is rudimentary, consisting of only a few small ducts that grow allometrically until puberty (4 weeks in mice). With the onset of puberty, comes expansive growth in a process called ductal morphogenesis that fills the fat pad with the epithelial mammary tree. This growth is influenced by growth hormone (GH), estrogen, and a growth factor, insulin-like growth factor-1 (IGF1). In the mature virgin, short tertiary branches form under the influence of progesterone, but alveologenesis only occurs upon pregnancy with the induction of prolactin (PRL), which together with progesterone, fuels the growth of alveolar cells. PRL stimulation continues into the stage of lactogenesis, culminating in milk production that continues until a lack of demand at weaning signals involution and the mammary gland is remodeled back to its original adult state. epithelial–mesenchymal interactions, hormonal regu- at the mammary line.3 In humans, the mammary lines latory controls, and cell–cell communication. Here, we form during the first trimester and give rise to a single review how these basic processes shape the structure pair of placodes.4 and function of the gland at each stage of development.

Mammary Line Specification EMBRYONIC MAMMARY GLAND The embryonic mammary mesenchyme underlying the DEVELOPMENT mammary line provides key inductive signals that In the embryo, there are two cellular compartments specify mammary epithelial cell differentiation. This of the mammary gland, the epithelial compartment was demonstrated in tissue recombination experi- and the surrounding stromal compartment. These ments in which E13 mouse mammary mesenchyme tissues are derived embryologically from ectoderm was combined with E13 midventral or dorsal epider- and mesoderm, respectively. The development of the mis and then grafted into host animals. These tissue murine gland begins at embryonic day (E) 10 with recombinants yielded mammary ducts containing both the formation of bilateral stripes (milk lines) of luminal and myoepithelial cells and, if the hosts multilayered ectoderm that run anterior-to-posterior were pregnant upon grafting, lobuloalveolar struc- from the forelimb bud to hindlimb bud on the ventral tures that expressed milk proteins.5 The influence of surface of the embryo. By E11.5, the mammary line mesenchyme on mammary epithelial development was resolves into five pairs of placodes at reproducible also demonstrated in reciprocal tissue recombination locations. These five pairs develop asynchronously experiments in which E12 and E16 mammary epithe- with the third pair of placodes emerging first, followed lium (ME) was combined with salivary mesenchyme by the fourth pair, then the first and fifth pairs (SM) and grown either in culture or under the kidney simultaneously, followed finally by the second pair. capsule.6,7 These tissue recombinants produced an The pairs are not identically determined as evidenced epithelium that was morphologically reminiscent of by both loss and supernumerary formation of different salivary gland. However, during pregnancy and lac- pairs as a consequence of specific genetic mutations. tation of hosts harboring the older (E16 ME/SM) Histologically, placodes appear as a thickened plate recombinants, the grafts displayed lobular–alveo- of ectoderm consisting of several layers of columnar- lar development and milk production.7 These and shaped cells that arise, not from cell proliferation2 other studies show that mesenchyme contains criti- but instead from the migration and subsequent cal inductive signals that produce, through a series aggregation of ectodermal cells into surface clusters of reciprocal epithelial–mesenchymal interactions,

milk line

Ventral LEF1 Dorsal epithelium WNT10B milk line WNT3 WNT6 Rostral epithelium FGFR2b mesenchyme BMP4 WNT10B Ventral Dorsal somites TBX3 NRG3 WNT11 Caudal mesenchyme WNT5A GLI3 FGF10B somite

FIGURE 2| Illustration depicting the generation of the mammary milk line. The position and specification of the multilayered epithelial ridge, the milk line, in an embryo at E10.5. The milk line is apparent between the forelimbs and hindlimbs that demarcate the rostral and caudal extension of the line. Specification of the line requires early Wnt signaling in the epithelium and in the mesenchyme that flanks the milk line. Tbx3 expression is required at early time points vertically under the line, and it controls subsequent Wnt signaling within the line that is required for the development of mammary placodes. Tbx3 expression is regulated by fibroblast growth factor 10 (FGF10) that emanates from somites underlying the line, as well as Wnt signaling in the flank and bone morphogenetic protein 4 (BMP4) signaling localized at the ventral border. For details, see text. (Reprinted with permission from Ref 30. Copyright 2010 Cold Spring Harbor Laboratory Press) epithelium that is progressively restricted along the (Figure 2). This pattern of expression suggests that mammary lineage.8 In turn, the epithelium influ- noncanonical Wnt signaling may restrict canonical ences mesenchymal maturation such that by E14 the signaling, limiting the response to Wnts to the ectoder- mammary mesenchyme has condensed to form a few mal ridge that becomes the mammary line. Lef1−/− layers of fibroblast-rich cells, closely surrounding the mice display a milder phenotype compared to mice epithelial rudiment and distinct from the fat pad pre- overexpressing Dkk1. Instead of the absence of pla- cursor mesenchyme, which develops from more deeply code development, as is the case with Dkk1 over- located subcutaneous mesenchymal cells.9 expression, three pairs of small placodes form in The mammary line has been marked molec- Lef1−/− mice.12 This suggests that DKK1 quenches ularly with Wnt10b andalsotheWnt signaling signaling from multiple TCF/LEF1 transcription fac- reporter TOP (T-cell factor (TCF) optimal promoter) tors that mediate the response to Wnts during mam- GAL (β-galactosidase), which reports the transcrip- mary line specification and placode development, tional activity of the Tcf3/Lef1 target genes (Figure 2). underscoring the complexity of the signaling required Interestingly, TOPGAL activity appears 1 day earlier to pattern these specialized regions of ectoderm. than Wnt10b expression in both the ectoderm and mesenchyme,10 indicating that an earlier Wnt signal emanates from the mesenchyme and initiates specifica- Placode Development tion of the mammary lines. The essential nature of this Once the mammary line has been delineated, signals Wnt signal in mammogenesis was demonstrated by the from the mesenchyme regulate the dorsal/ventral pat- overexpression of a secreted Wnt inhibitor, Dickkopf terning of the placodes. The normal developmental (DKK1), in the ectoderm. This completely blocked outcome of numerous signaling cascades is five pairs the formation of mammary placodes and antagonized (one in humans) of symmetrically localized placodes, the local expression of Wnt10b, TOPGAL, and other which have a button-like appearance on the surface early markers such as the T-box transcription factor, of the ectoderm. TBX3 is a factor expressed very Tbx3.10 Despite the importance of this initial signal early, at E10.25, in a restricted region of mesenchyme in mammary gland specification, the identity of the underlying the presumptive mammary line.13 and later Wnts that generate this initial signal is unknown, accumulating in the placodes. Mice homozygous for although there are several candidates. Wnt3, Wnt6, a null allele of Tbx3 exhibit an almost complete and Wnt10b are all expressed in the broad band of failure to develop mammary placodes, whereas the ectoderm flanking the mammary line10,11 and Wnt5a heterozygous deficiency results in poorly developed and Wnt11, which signal in a noncanonical fashion, or missing glands.13 In humans, a similar defect are expressed in the underlying flank mesenchyme10 is caused by haploinsufficiency for TBX3, resulting

© 2012 Wiley Periodicals, Inc. Advanced Review wires.wiley.com/devbio in ulnar–mammary syndrome characterized by upper that generally functions as a transcriptional repressor limb deficiency and severe mammary hypoplasia.14 in the hedgehog signaling pathway, although a These phenotypes show that Tbx3 plays a funda- hedgehog-independent role as a transcriptional activa- mental role during mammary gland development, tor has also been reported.21 The similarities between and other studies have demonstrated that its effects the Gli3Xt/Xt and Fgf10−/− phenotypes suggest that are exerted through the regulation of Wnt signal- GLI3 positively regulates Fgf10 expression, similar to ing. Wnt10b and Lef1 expression are expanded its regulation of other FGF family members in differ- in mice engineered to ectopically express Tbx3.15 ent developmental contexts22 (Figure 2). This idea is whereas their expression is absent in Tbx3−/− supported by the rescue of both Wnt10b expression, mice.16 These data show that TBX3 has the abil- albeit at low frequency, and ectodermal thickening ity to position Lef1 expression and thereby define that occurs when FGF10 beads are implanted into the the mammary line. This raises the question as to flanks of Gli3xt/xt mutants.17 the identity of the signal responsible for localizing These data have allowed researchers to sketch Tbx3. Bone morphogenetic protein 4, BMP4, which a rudimentary signaling cascade in which GLI3 regu- is expressed along the ventral border of the mammary lates FGF10 expression in the somite (Figure 2). The line, is one candidate for regulating the ventral extent FGF10 signal is transduced through the mesenchyme of Tbx3 expression15 (Figure 2). BMP4 antagonizes to regulate Wnt10b expression in the presumptive Tbx3 expression and vice versa, leading to a model of mammary epithelium. There are likely intervening reciprocal antagonism, whereby the mammary line is factors that relay the signals from Gli3 to Fgf10, delineated, at least in part, by the actions of these two and, an as yet unidentified mechanism, which facili- factors restricting the expression of Wnt10b. tates the interaction between FGF10, expressed by the BMP4 and TBX3, however, do not work alone underlying somite, and FGFR2b, which is expressed in because fibroblast growth factors (FGFs) also pattern the mammary line. One model proposes that FGF10- the dorsal/ventral placement of placodes by regulat- expressing cells are brought in direct contact with ing the expression of Wnt10b and Lef1 (Figure 2). the mammary line by changes in cell adhesion that Fgf10 is expressed at E10.5 in the central and ventral allow somitic cells, expressing FGF10, to delami- regions of thoracic somites underlying the mammary nate, migrate, and directly contact placode epithelium line.17 Identification of the potential role for this cue that is expressing FGFR2b.17 Neuregulin-3 (NRG3), a and its cognate receptor, FGFR2b, in mammary line member of the epidermal growth factor (EGF) family, formation came from the analysis of the knockout is a candidate regulator of these cell–cell contacts. mice, which develop only the fourth placode.18 Since It is expressed in the mesenchyme underlying the then, additional mutations have been discovered that mammary line and regulates the expression of cell similarly affect mammary placode development by adhesion receptors and extracellular matrix (ECM) inhibiting Fgf10 expression, either directly by affect- components such as tenascin, which is involved in tis- ing its expression or indirectly by disrupting thoracic sue remodeling.23 In hypomorphic Nrg3Ska mutants, somite development. For example, Fgf10 expression is the third placode is small or absent and there is ectopic abolished in the Pax3 mutant, which fails to undergo placodal development near the fourth placode.24 ventral extension of thoracic somites, eliminating the There is no loss of Fgf10 or mesenchymal Tbx3 somatic source of FGF10 and generating a corre- expression in Nrg3Ska animals, indicating that NRG3 sponding dorsal shift in lateral line formation.17 acts downstream of these factors. However, Wnt10b A similar birth defect, Poland syndrome, occurs in and Lef1 are altered, displaying decreased or absent humans and is characterized by the underdevelop- expression at the thirdplacode and ectopic expres- ment of the somite-derived pectoral muscle on one sion at the supernumerary buds, data that suggest an side of the body and a corresponding hypoplasia of upstream role for NRG3 in regulating these factors. the overlying breast on that side.19 These defects arise Concordantly, NRG3 coated beads or overexpression from disruption in FGF10 signaling, rather than loss of of Nrg3 in basal epithelia under the control of a ker- the somite, because somitic development is normal in atin 14 (Krt14) promoter induces Wnt signaling and Fgf10 heterozygote animals, but the mammary glands expression in the mammary line and generates super- in these animals are characterized by the absence numerary placodes that develop nipples and glandular of Wnt10b and Lef1 expression, reduced thicken- epithelia.23,25 Together, these studies suggest a role for ing of the ectoderm along the mammary line, and NRG3 in signaling to its epithelial ERBB4 receptor subsequent loss of buds 3 and 5.17,20 A similar pheno- and modulating cell adhesion that promotes transduc- type occurs in mutant mice with no functional GLI3 tion of somatic FGF10 signals and development of (extratoes, Gli3Xt/Xt). GLI3 is a transcription factor placodal epithelium.

Mammary placode Mammary bud Ductal tree

Ectoderm Nipple sheath

Hair follicle Mammary Mammary formation Rudimentary mesenchyme epithelium mammary tree Fat pad MSX2 Dermal PTHLH E11.5 mesenchyme Mammary PTH1R BMPR1A mesenchyme BMP4 E15.5

Newborn

FIGURE 3| A schematic representation portraying embryonic mammary gland development. Mammary placodes expand into a ball of cells that descends into the underlying mesenchyme. Parathyroid hormone-related protein (PTHLH) signals from the epithelium to the mesenchyme to increase the expression of bone morphogenetic protein receptor-1A (BMPR1A). Bone morphogenetic protein 4 (BMP4) expressed in the mesenchyme signals through BMPR1A to MSX2 and inhibits hair follicle formation at the developing nipple sheath. The mammary epithelium grows into a small, simple tree-like structure containing an open lumen and remains in this form until birth. For details, see text. (Reprinted with permission from Ref 30. Copyright 2010 Cold Spring Harbor Laboratory Press)

Additional FGF signaling, mediated through descends into the underlying mesenchyme by E1430 FGFR1b and/or FGFR2c in the ectoderm, is required (Figure 3). This sphere of cells continues to descend to induce and maintain Tbx3 expression, first in the as a stalk is formed connecting the mammary bud mesenchyme underlying the mammary line and later to the epidermis. Surrounding the epithelium is the in the placode.26 Positioning of the TBX3 signal in the condensed mesenchyme comprising a thin layer of region underlying the nascent placode is accomplished fibroblastic cells. At E16, a solid cord of epithelial cells 17 by the positive input of FGF10, and the negative reg- extends from the mammary bud and grows through 15 ulation of BMP4 in the ventral mesenchyme. Once this condensed mesenchyme into the fat pad precursor Tbx3 expression becomes localized, it generates a sec- mesenchyme, which at this stage is a small collection ond wave of Wnt signaling, mediated by WNT10b of preadipocytes. Once the epithelial sprout reaches that ultimately specifies the fate of the mammary the fat pad, it begins to branch by equal division of epithelium by promoting the pseudostratification of the terminal bud. This dichotomous branching yields ectodermal cells to generate placodes. The signaling a rudimentary ductal system of 10–15 branches that is pathways that lie downstream of Lef activation in the generated without hormonal input and remains largely mammary placode are poorly understood, but likely quiescent until puberty. In humans, instead of a single include the target Edar, a receptor for ectodysplasin-A epithelial sprout extending from the mammary bud, (EDA), which is a member of the tumor necrosis factor 27 several sprouts form creating multiple mammary trees (TNF) superfamily. Overexpression of Eda results that unite at the nipple. in the formation of supernumerary placodes located 28 Before embryonic development concludes, three between the normal third and fourth placodes. additional morphological processes occur: formation Mutations in the corresponding gene in humans cause of the ductal lumen, generation of the nipple hypohidrotic ectodermal dysplasia (HED), character- structure, and, in mice, specification of male versus ized by the lack of breast and other tissues that female mammary glands. Lumen formation is initially develop from the ectoderm (e.g., skin, hair, nails, observed at E16 when intercellular spaces develop teeth, and sweat glands).29 Together, these studies within the cords. These spaces increase in size and illustrate the important insights into placode develop- number until a distinct lumen is apparent at E18.31 ment that have been derived from the study of mouse Studies performed in cultured mammary cells grown mutants. However, it is also clear from these studies in a three-dimensional matrix have implicated three that our understanding is incomplete and that each processes in mammary lumen formation: apoptosis, pair of placodes in mice appears to have both shared autophagy, and cellular remodeling.32–34; however, and distinct genetic requirements for their formation. the precise mechanisms giving rise to open luminal space in the embryonic epithelial rudiments have Mammary Bud and Rudimentary Duct not yet been delineated. Nipple generation occurs Formation by modifications of the skin overlying the primary During the next stage of development, mammary mammary mesenchyme and involves thickening of the placodes expand to form a round ball of cells that epidermis, suppression of hair follicles, and generation

© 2012 Wiley Periodicals, Inc. Advanced Review wires.wiley.com/devbio of a nipple sheath from keratinocytes at the site where downregulated in both the epithelium and the epi- the primary duct connects to the skin surface.35 Sexual dermis, whereas it is upregulated in the mammary dimorphism of the mammary gland is also achieved mesenchyme. In Pthlh−/− and Pth1r−/− glands, this during this stage of embryonic development in mice, developmental down regulation of Lef1 does not but later in humans. In male mice, the mesenchyme occur. Instead, expression of Lef1 is retained in the surrounding the stalk continues to condense until it epithelium and epidermal cells surrounding the pre- severs the bud resulting in a greatly diminished ductal sumptive nipple; yet it is absent in the mesenchyme.42 system.36 These latter two processes, nipple generation These studies suggest that PTHLH, expressed in the and sexual dimorphism, require signaling that is epithelium, signals through mesenchymal PTH1R to mediated by the parathyroid hormone-related protein modulate Wnt signaling and, consequently, cell fate. (PTHLH) (described later). In humans, sexual This idea is supported by experiments in which dimorphism is accomplished via a different mechanism Pthlh expression is expanded into basal keratinocytes and the bud is not severed during embryogenesis. by driving its expression under the regulation of a Instead, male and female glands undergo comparable Krt14 promoter. In these animals, Lef1 expression is development until puberty when their regulation by retained in the ventral epidermis, which becomes in different hormones specifies their size.4 Thus, human essence hairless nipple skin.42 These effects of PTHLH male breasts retain a normal growth potential as appear to require BMP4 signaling, which is augmented evidenced by the incidence of gynecomastia, the through the upregulation of the BMP receptor-1A, benign enlargement of breast tissue in males as a BMPR1A, by PTHLH in the mesenchyme35 (Figure 3). consequence of altered estrogen–androgen balance. BMP4 rescues the outgrowth of Pthlh−/− mammary The parathyroid hormone-related protein buds in culture35 It also increases the expression of (PTHLH) plays a central role in regulating the transcription factor Msx2 in mesenchyme, a regula- transition from budding to branching. PTHLH is tory event that is required for PTHLH suppression produced by the mammary epithelium. It signals of hair follicle formation upon the nipple, as demon- through the G-protein coupled receptor, Type1 strated by the recovery of hair follicle development in 37 PTH/PTHLH receptor (PTH1R) , expressed in the Krt14-Pthlh transgenic mice that have been crossed 38 mammary mesenchyme. The absence of either with Msx2−/− mice.35 PTHLH or PTH1R in knockout mice results in chon- Altogether, these studies on PTHLH signaling drodystrophy, which is characterized by numerous underscore the importance of epithelial–mesenchymal embryonic defects, including a lack of nipples and interactions in regulating processes such as mammary mammary ducts, that ultimately result in neona- bud formation. PTHLH, produced by the epithelia, tal death. A similar phenotype is also observed in acts on its receptor expressed in the surrounding mes- humans suffering from loss-of-function mutations in enchyme to modulate Wnt and BMP signaling and the PTH1R gene, resulting in a rare, lethal form of induces the production of a specialized condensed 39 dwarfism known as Blomstrand chondrodysplasia, mesenchyme that maintains mammary epithelial cell characterized by severe defects in endochondral fate. This triggers nipple sheath formation in the over- bone formation and the absence of nipple or duct lying epidermis and initiates ductal outgrowth. By the 40 − − development. Careful examination of Pthlh / and end of embryonic development, these and other regu- − − Pth1r / mutant glands reveals normal development latory processes generate a rudimentary ductal system throughout the mammary bud stage, but subsequent that provides the framework for mammary outgrowth failure of the bud to undergo the initial phase of during puberty. branching at E16.41 This results in degeneration of mammary epithelial cells, such that by birth only the scattered remnants of ductal structures remain, PUBERTAL MAMMARY GLAND although the fat pad and vasculature remain intact. DEVELOPMENT Moreover, mutant glands lack nipple structures, which fail to develop on the ventral epidermal surface. At birth, the gland is just a rudimentary ductal system, Further investigation into PTHLH signaling has yet it is competent to produce milk. Fetal exposure to revealed that the pathway maintains epithelial cell maternal hormones can cause milk expression, which fate and triggers nipple differentiation, in part by is sometimes referred to as witch’s milk. As these orchestrating events mediated by other signaling sys- endocrine influences subside, the gland undergoes a tems, notably Wnt and Bmp. As discussed in the period of allometric growth, keeping up with overall preceeding section, Wnt signaling plays an essential body development, until puberty when expansive pro- role in mammary gland specification during placode liferation occurs, filling the fat pad under the influence development, but by E14, Lef1 expression is of hormones and growth factors (Figure 1). Terminal

Pituitary Gland Liver Ovary

GHs Estrogen

E GHR Stromal Cell ESR1 IGF1 Myoepithelial EGFR EGFR Cell

IGF1 AREG AREG FGFs ProAREG IGF1R Luminal ADAM17

E FGFR2 Epithelial Cell ESR1

Cell Survival Cell Proliferation and Proliferation and Branching ESR1+

Luminal epithelial cell

Myoepithelial cell SLIT2 NTN1 FGFs Stromal Cell RELN Adipocyte TGFB1 Extracellular Fat Pad matrix

TEB LN Fat Pad

FIGURE 4| Schematized view of the events occurring during pubertal development. Terminal end buds (TEBs) grow through the mammary fat pad, fueled by cell proliferation (diagram at the bottom). Growth hormone (GH) regulates cell proliferation by inducing the expression of insulin-like growth factor-1 (IGF1) in both the liver and mammary stroma. IGF1 acts, together with estrogen secreted from the ovary, to induce epithelial cell proliferation (diagram at the top). Estrogen signaling through its receptor (ESR1) acts via a paracrine fashion to stimulate the release of epidermal growth factor (EGF) family member, amphiregulin (AREG), which proceeds to bind its receptor on stromal cells and induce expression of FGFs. FGFs, in turn, to stimulate luminal cell proliferation. Other factors, such as TGFB1, Reelin (RELN), Slit2, and Netrin1 (NTN1) contribute to mammary architecture by either positively or negatively regulating cell proliferation or maintaining cell–cell interactions. For details, see text. end buds (TEBs) are club-shaped structures at the tips tubular ductal bilayer that encircles inner luminal of growing ducts that penetrate the fat pad, driven cells.43 Secondary branches sprout laterally from the by the proliferation of a single layer of cap cells at primary ducts until a tree-like pattern of ducts occu- the tip of the TEB and by the underlying prelumenal pies up to 60% of the available fatty stroma, leaving epithelium (Figure 4). The primary ductal structure is abundant space for inﬁlling that will occur under the generated by TEB bifurcation and is regulated by the inﬂuence of pregnancy hormones. Under cyclical ovar- surrounding stroma. Cap cells of the TEB differentiate ian stimulation, short tertiary branches will form, but into myoepithelial cells, forming the outer layer of the full blossoming of the alveolar buds into units capable

© 2012 Wiley Periodicals, Inc. Advanced Review wires.wiley.com/devbio of milk secretion occurs only under the influence of containing Ghr−/− epithelium/WT stroma appear pregnancy hormones (Figure 1). In humans, the puber- identical to WT/WT recombinants, demonstrating tal breast contains a similarly extensive mammary that GH signaling is required in the mammary stroma tree, but the lateral branches lead to terminal ducts but not in the mammary epithelium.45 The current that give rise to terminal ductal–lobular units com- model is that GH signals through GHR in stromal prising numerous blind-ended ductules, called acini.4 fibroblasts, inducing IGF1 that then signals to the These acini are embedded in fibroblastic, intralobular mammary epithelium (Figure 5). This model is sup- stroma that is far more pronounced in the human ported by studies on Igf1−/− mice which exhibit breast than in the adipocyte-rich stroma surrounding greatly diminished ductal development, similar to the the branches of the rodent mammary tree. phenotype observed in Ghr−/− mice.46 Furthermore, Igf1−/− mice produce normal levels of GH and their Hormones and Growth Factors Control defects in mammary development can be rescued by Ductal Morphogenesis Studies, performed as early as the 1930s, demonstrated that extracts of the pituitary gland regulate mammary gland function, with researchers observ- ing enhanced mammogenesis and lactogenesis upon 44 Ovary Pituitary its administration. Two hormones were identified gland that are responsible for these effects: growth hormone and prolactin (PRL). Later studies showed that mammary pubertal development was disrupted in Progesterone PRL mice lacking Gh, insulin-like growth factor 1 (Igf1) RANKL

or estrogen receptor (α) (Esr1), genes that mediate PRLR SIRPA Integrin } pathways regulating ductal outgrowth and morpho- JAK2 RANK

genesis. In contrast, development of the adoles- STAT5 NFKB Socs1} cent gland occurs normally in mice lacking Prl or P Socs2 PGR Socs3 Csnb iB Rankl progesterone receptor (Pgr), genes that mediate sig- Csnb Wap } naling pathways regulating alveologenesis (described Rankl in the following section).

Growth Hormone and IGF1 are Global Directors of Postnatal Development Growth hormone, secreted from the pituitary gland, LN is an important global regulator of mammary gland development, but current evidence suggests that its effect on the mammary gland is mediated largely, or Alveoli even entirely, through IGF1 whose production it stimulates in the stroma45,46 (Figure 4). Growth hormone receptor (Ghr) knockout mice are small for their age, Adipocyte creating a murine model of human Laron syndrome,a Alveolar cell Myoepithelial cell Milk globule hereditary dwarﬁsm disorder resulting from defects in FIGURE 5| Schematized view of the events that generate lactation 47 GHR. Ghr−/− mice also display a dramatic (90%) competence during pregnancy. Alveoli develop into milk-secreting reduction in serum IGF1 levels,47 and delayed mam- lobules regulated by prolactin (PRL) that works together with mary gland development with eventual outgrowth of progesterone. Both hormones regulate transcriptional programs that only a sparse tree.45 To evaluate the compartment include the control of Rankl TNFSF11), which signals in a paracrine of the mammary gland (epithelial or stromal) that fashion to stimulate proliferation by upregulating expression of target is affected by the loss of Ghr, tissue recombination genes such as Cyclin delta 1 (Ccnd1) through the RANK receptor on experiments were performed in which Ghr−/− KO neighboring cells. Progesterone stimulates secondary and tertiary and wild type (WT) epithelia were grafted into WT branching, while PRL integrates many signals, including those from the extracellular matrix (ECM) by interacting with integrin through stromal fat pads. In these experiments, the endogenous transmembrane, signal regulatory protein α (SIRPA). PRL transduces WT epithelium is precleared from the host fat pad to this information through pathways, such as those mediated by prevent confusion arising from a fat pad containing JAK2/STAT5, whose downstream targets include milk genes casein β both host WT and graft KO epithelium. Recombinants (Csnb) and whey acidic protein (Wap). For details, see text.

IGF1 treatment, demonstrating the essential nature of The first studies to show the direct influence of IGF1 on mammary gland development downstream estrogen on mammary gland development involved of GH. the delivery of estrogen or antiestrogen directly In the last decade, researchers have tried to to the gland by combining these substances with determine the relative importance in mammary gland Elvax a biologically compatible polymer that can be development of IGF1 produced locally versus that pro- implanted into tissue.53 These studies demonstrated duced systemically by the liver. Initial results suggested that estrogen stimulates mammary ductal growth in that local IGF1, functioning in a paracrine, rather both ovariectomized and intact animals, and that than endocrine, manner plays a central role. This was this stimulation is blocked by the estrogen receptor inferred from studies on mice that carry an insertional antagonist, tamoxifen.54,55 This was the first mutation in the Igf1 gene, Igf1tm2Ts also known as identification of the local actions of estrogen on estro- the Igf1 midi allele, resulting in a 70%, rather than gen receptors, which are broadly expressed in both 100%, reduction in Igf1 expression.48 These mice dis- epithelial and stromal compartments of the gland. play reduced mammary ductal branching, confirming Estrogen acts together with IGF1 to regulate the importance of Igf1. In contrast, mice harbor- ductal morphogenesis, as evidenced by the synergistic ing a liver specific deletion, which reduces the levels enhancement of ductal outgrowth in Igf1−/− mice of circulating IGF1 by 75%, display normal duc- treated with estrogen together with IGF1.46 There are tal morphogenesis, suggesting that circulating IGF1 is two forms of intracellular estrogen receptors, α and nonessential.48 However, neither of these mouse mod- β, encoded by different genes, Esr1 and Esr2, respec- els directly address the effects of circulating IGF1 in tively. ESR1 is the major receptor operating during the presence or absence of local IGF1. To accomplish ductal morphogenesis as determined by the analysis of this, mice were generated that produce only circulating knockout phenotypes.56,57 Mice lacking Esr1, similar IGF1 by overexpressing an Igf1 transgene specifically to mice lacking Igf1, only form a rudimentary ductal in the liver of Igf1−/− mice.49 In these mice, not only system that fails to undergo the estrogen-mediated body size but also mammary gland development is growth spurt that accompanies puberty.56 Yet only rescued, showing that systemic IGF1 can compensate a subpopulation of luminal epithelial cells expresses for the loss of local IGF1 production. Nevertheless, ESR1, with fewer than 40% of these cells incorporat- 58 there appears to be interplay between systemic and ing the cell proliferation marker, BrdU. One expla- local sources because control mice, expressing both nation for this observation is that ESR1-positive cells normal levels of mammary IGF1 and transgenic levels drive proliferation by signaling in a paracrine fashion of serum IGF1, display increased cellular proliferation and, in this case, only a few cells would be required and enhanced mammary development.49 These results to initiate signaling. Alternatively, ESR1 expression suggest that an overabundance of IGF1 can inappro- may be rapidly downregulated in response to estrogen priately drive mammary epithelial cell growth, for and, as a result, ESR1-positive cells would have been example, in a tumor setting. Indeed, high levels of missed in an experiment looking for coexpressed ESR1 58 IGF1 have been associated with an increased risk of and BrdU. To distinguish between these possibilities, malignancy,50,51 but the relative contribution of local researchers again turned to the technique of transplan- versus circulating IGF1 to tumorigenesis has not yet tation, but this time they generated mosaic epithelial − − been determined. outgrowths by mixing Esr1 / cells with WT (Esr1- positive) cells and grafting them into precleared fat pads.59 These studies demonstrated that a subpopula- Estrogen Manages Local Cell Growth tion of WT cells rescues the knockout phenotype when During Puberty mixed together with Esr1−/− cells, supporting the The ovarian hormone, estrogen, is another criti- notion that ESR1-positive cells signal in a paracrine cal regulator of pubertal mammary development fashion to stimulate the proliferation of neighboring and is responsible for the tremendous surge in ESR1-negative cells. Amphiregulin (AREG), a member growth occurring during this period that generates of the EGF family, is currently the leading candidate a functional mammary gland (Figure 4). Estrogen, a for the factor that is released by ESR1-positive cells membrane-soluble ligand, is released from the ovary and signals cell proliferation (see section on Growth and activates gene expression through intracellular Factors). In addition to this role in stimulating cell receptors. For a long time, it was unclear whether growth during ductal morphogenesis, estrogen also hormones such as estrogen had direct effects on functions during pregnancy in the growth and mainte- mammary gland development or whether, instead, nance of alveolar cells as demonstrated over 50 years they functioned indirectly to stimulate the release ago in hormone replacement studies on ovariec- of hormones such as PRL from the pituitary.52 tomized mice.60,61 These observations were confirmed

© 2012 Wiley Periodicals, Inc. Advanced Review wires.wiley.com/devbio by conditional ablation of Esr1 in alveoli following does not disrupt ductal development, while deletion ductal elongation, resulting in defective lobuloalveolar of stromal EGFR results in dramatically reduced duc- development and inadequate milk production.62 tal outgrowth,68 further supporting the notion that Altogether, these studies on GH, IGF1, and paracrine circuitry of the estrogen response occurs via estrogen have led to a model in which pituitary GH crosstalk with the stroma. FGFs are attractive candi- induces the production of IGF1 by the liver (Figure 4). dates to mediate the mitogenic signal because they are These factors, together with locally produced IGF1 upregulated in the stroma during puberty,69 and their in mammary stroma and epithelium, act via their cognate receptor, FGFR2, is required on epithelial receptors in the mammary gland to stimulate TEB cells for ductal elongation.70 formation and ductal branching. Estrogen acts in con- While positive factors that elicit growth are cert with IGF1 to generate the burst of proliferation clearly important for creating the ductal architec- required for ductal morphogenesis. ESR1 operates in ture, negative regulators are equally important and a subset of epithelial cells and induces the release of particularly crucial in the mammary gland, which has AREG that signals through the stroma, generating an open ductal architecture, characterized by ample additional growth factors (e.g., FGFs) that contribute space between branches for pregnancy-induced alveo- to the rapid period of mammary gland growth occur- lar infilling. Transforming growth factor β1 (TGFB1) ring at puberty (see below). has been identified as a major negative regulator of mammary branching and ductal elongation, and Growth Factors Sculpt Ductal Tree it restricts end bud bifurcation and branch formation by limiting epithelial proliferation.71–73 Although Architecture some of the antiproliferative effects of TGFB1 are Members of the EGF and FGF families of growth fac- mediated through stromal receptors,74,75 numerous tors signal through their respective tyrosine kinase studies have demonstrated significant inhibitory sig- receptors to positively influence mammary gland naling occurring within the epithelium. For example, ductal morphogenesis. In terms of the EGF fam- transplantation of either Tgfb1−/− epithelium73 into ily, AREG is the only obligatory ligand, although WT mammary fat pads results in increased epithelial several EGF family members can affect ductal out- proliferation and ductal extension. A similar result growth. For example, transforming growth factor α can be obtained using Tgfb1+/−epithelium, which (TGFA), NRG1, and EGF are all capable of rescuing has dramatically reduced TGFB1 levels due to the ductal development in ovariectomized and Esr1−/− absence of positive feedback {Ewan, 2002 #826}, mice,63,64 but only mice lacking Areg have a mam- while in contrast overexpression of dominant neg- mary gland phenotype that closely matches that of ative TGF β receptor II (TGFBR2) in the epithelium the Esr1 KO.65,66 The expression of Areg is strongly leads to alveolar hyperplasia.76 Studies using three- induced in mammary epithelial cells by estrogen at dimensional micropatterned collagen gel assays that puberty, but this transmembrane protein binds to control the shape of mammary epithelial tubules in the epidermal growth factor receptor (EGFR) that is culture demonstrate that TGFB1 can be released in located at a distance on stromal cells across the gradients capable of inhibiting the sites of branching.77 basement membrane. Studies have shown that the WNT5A is one downstream mediator of these growth transmembrane metalloproteinase Adam 17 (formerly inhibitory effects, and it acts by regulating prolifer- known as TNFalpha converting enzyme (TACE), ation through the LEF/TCF pathway.78 These and TNFα converting enzyme) releases AREG from mam- other studies demonstrate that mammary branching mary epithelial cells, allowing for the paracrine morphogenesis requires a balance of positive and neg- activation of stromal EGFR.67 Evidence supporting ative factors that generate, by still largely unknown this sequence of events includes the observation that mechanisms, the stochastic pattern of widely spaced defects in ductal outgrowth observed in Adam17−/− branch outgrowth observed in mammary trees. glands resemble those in Areg−/− and Esr1−/− In addition to members of the major growth fac- glands and the fact that local administration of sol- tor families, a host of additional secreted proteins have uble AREG rescues the development of Adam17−/− been identified that regulate mammary morphogene- glands.67 Nevertheless, despite the delineation of these sis. Examples include members of neurogenic families. events, the growth factor that actually drives cell These factors were originally discovered in the nervous proliferation at puberty has not been definitively iden- system, but regulate developmental events in other tified. AREG signals to its receptor on stromal cells organs. Netrin1 (NTN1), Slit2 (SLIT2), and Reelin and can, therefore, only indirectly affect mammary (RELN) are all proteins that regulate axon guidance epithelial cells. Indeed, tissue recombination experi- and cell migration during neural development, and ments have confirmed that epithelial deletion of EGFR also regulate mammary ductal morphogenesis. Ntn1

© 2012 Wiley Periodicals, Inc. WIREs Developmental Biology Mammary gland development and Slit2 are expressed in both luminal and myoep- ADULT MAMMARY GLAND ithelial cells, but their receptors are restricted to the DEVELOPMENT outer myoepithelial layer. A loss-of-function mutation in these genes, individually, results in TEB abnormal- Pregnancy and Lactation are Accompanied ities and, together, in ductal defects characterized by by Major Changes as the Mammary Tree delamination of the epithelial cell layers.79,80 In con- Prepares to Bear Fruit trast, RELN is expressed by the periductal stromal and The mammary gland must undergo numerous changes its receptor is expressed in both cell types of the epithe- to prepare for lactation. These changes require both lium. Reln knockouts contain only rudimentary ductal gland maturation and alveologenesis and are primar- structures that exhibit decreased terminal branching ily under the control of progesterone and PRL. The 81 and a disorganized luminal layer. Other proteins, ﬁrst transformation of pregnancy is a tremendous such as milk fat globule-EGF factor 8 (MFGE8) and increase in secondary and tertiary ductal branching, matrix metalloproteinases (MMPs), function not only providing ductal arbors for the second transforma- during branching morphogenesis, but also have roles tion, alveolar development. Proliferating epithelial in other stages of mammary gland development cells generate alveolar buds that progressively cleave such as involution. MFGE8 is expressed in the and differentiate into distinct alveoli, which become epithelium, regulating cell proliferation during ductal milk-secreting lobules during lactation. Interstitial adi- outgrowth82 and the elimination of apoptotic cells 83 pose tissue disappears as the proliferating epithelial during involution. In contrast, stromal MMPs func- cells occupy the interductal spaces. Increased vascu- tion similarly at both developmental time points and larization occurs and by midpregnancy, each alveolus are involved in remodeling the basement membrane is surrounded by a basket-like network of capillaries.86 to assist with architectural changes required for mor- By late pregnancy, the alveoli encompass the major- phogenesis. ity of the fat pad and show some secretory activity Altogether, these lines of investigation have led as pregnancy approaches term. Some of these changes to a model in which estrogen binding to ESR1 in also occur during estrus cycles when the gland exhibits sensor cells induces the expression of transmembrane mild proliferation and differentiation that includes AREG, which is released from the cell surface by the limited expression of milk proteins, followed by Adam17 (Figure 4). Cleaved AREG makes its way, involution. by unknown mechanisms, across the basement membrane to stromal ﬁbroblasts expressing its receptor, EGFR. Activation of EGFR induces the expression Progesterone Makes Crucial Contributions of FGFs that, in turn, stimulate cell proliferation through FGFR2 expressed on the mammary epithe- to Alveologenesis lium. TGFB1 functions as a negative regulator of duc- Progesterone, like estrogen, is a membrane-soluble, tal morphogenesis by inhibiting cell proliferation and ovarian hormone that signals through intracellular restricting branch formation. Other factors also play receptors to generate epithelial growth in the mam- important roles, and additional information needs to mary gland (Figure 5). Progesterone is responsible be gathered on the many, diverse extracellular signals for the extensive sidebranching and alveologenesis that contribute to the morphogenesis of the adolescent required to create a lactation-competent gland. In gland. An important question raised by these studies combination with PRL, it also promotes the dif- is how do quiescent ESR1-positive cells become prolif- ferentiation of specialized structures, alveoli, which erative in hormone-dependent, ESR1-positive human synthesize and secrete milk during lactation. The breast tumors. The answer may be through the robust importance of progesterone in both these processes crosstalk that occurs between epithelial ERα and stro- was revealed by the analysis of progesterone recep- mal EGFR signaling. Studies on hyperplastic enlarged tor knockout mice. Pgr−/− glands produce only a lobular units (HELUs), considered the earliest lesion simple epithelial tree, even following the surge of with premalignant potential, have shown that both growth at puberty, and upon pregnancy they do not Esr1 and Areg are upregulated, and together they may undergo the associated ductal proliferation or lobu- generate a self-propagating growth-regulatory loop loalveolar differentiation.87,88 Transplantation studies that fuels tumorigenesis.84 Despite the clinical success were performed to address whether PGR is required of estrogen receptor antagonist, tamoxifen, there is in the epithelial, stromal, or both compartments and great interest in adding to the arsenal of drugs that will revealed that Pgr expression in the epithelium only slow the growth of ER-positive tumors. Recent studies is required for sidebranching and alveologenesis88 In suggest that AREG may be an attractive therapeutic the epithelium, the pattern of Pgr expression changes target for breast cancer intervention.85 from a uniform pattern of expression in the juvenile

© 2012 Wiley Periodicals, Inc. Advanced Review wires.wiley.com/devbio to a scattered expression pattern in a subset of epithe- alveologenesis during pregnancy,95 a phenotype that 89 − − lial cells in the adult. This suggests that, similar to is very similar to that described in Pgrib / glands. estrogen signaling, progesterone signaling occurs in a Moreover, the phenotype of such glands can be paracrine fashion. This was evaluated using mosaic rescued by the ectopic expression of Rankl, an exper- analysis in which Pgr−/− cells were mixed with iment that was performed by both viral infections WT (Pgr-positive) cells and grafted into precleared of bulk mammary epithelial cells,96 andinamore fat pads, similar to the experiments performed with refined manner by using an inducible bigenic system Esr1−/− cells.88 The Pgr−/−/WT mammary gland that drives Rankl expression specifically in the cells outgrowths appear normal, demonstrating that WT that normally express it.97 Numerous studies have cells rescue the KO phenotype and supporting the idea shown that RANKL is induced by progesterone,98 that progesterone signals via a paracrine mechanism. specifically in PGR-positive luminal epithelial cells,91 Studies on progesterone receptors have been and recent data suggest that it is the paracrine complicated by the fact that there are two receptor signal that tells neighboring PGR-negative cells to 96 isoforms (PGRiA and PGRiB), produced by alternate proliferate (Figure 5). This central role of RANKL promoter usage from a single gene. They are identical in generating the pro-growth response to progesterone except that PGRiB contains 164 additional N-terminal is illustrated by recent studies demonstrating its residues. Both isoforms of PGR are expressed in responsibility in driving cell proliferation in progestin- the mammary gland of the virgin mouse and dur- dependent breast cancers. This has led to the idea 90 ing pregnancy, with PGRiA levels exceeding those that RANKL inhibitors, a therapy currently being of PGRiB. Each of these isoforms has been knocked pursued for arthritis, periodontal disease, and osteo- out individually, and they have distinct physiological porosis, should also be considered for breast cancer functions in some reproductive tissues. However, in treatment.99,100 the mammary gland, loss of Pgria does not produce a phenotype, whereas loss of Pgrib results in sig- PRL Collaborates with Progesterone nificantly reduced sidebranching and alveologenesis to Generate a Lactation-Competent Gland 91,92 during pregnancy These data show that PGRiB is PRL is the major generator of lactational competence uniquely required in the mammary gland, but they do during pregnancy, and it functions both indirectly, not rule out a distinct function for PGRiA, which may through its regulation of ovarian progesterone produc- be compensated by the presence of PGRiB. Indeed, tion, and directly via its effect on mammary epithelial expression profiling studies on human breast cancer cells. PRL is a small polypeptide hormone produced cell lines engineered to express one or the other iso- by the pituitary, as well as other sites, such as mam- form show that PGRiA and PGRiB regulate distinct mary epithelium. It binds to a member of the class and overlapping sets of genes.93 This pattern of reg- I cytokine receptor superfamily, resulting in the acti- ulation may have clinical significance because human vation of a number of signaling pathways, including breast cancers expressing altered ratios of the two Jak/Stat, Map Kinase,andphosphoinositide (PI) 3 PGR isoforms have different, ratio-specific clinical kinase (Figure 5). The role of PRL during mammary outcomes.94 gland morphogenesis has been investigated using Prl There is still much to be learned about signal- and Prolactin Receptor (Prlr) knockout mice101,102 ing downstream of progesterone receptors, but recent Embryonic and postnatal developments appear to be data identify tumor necrosis factor ligand superfamily, normal in these mice, and there is typical outgrowth member 11 (TNFSF11), also known as RANKL of the mammary tree until puberty, whereupon a (receptor activator of NFKB1 ligand), as a lack of sidebranching and alveolar budding results key paracrine-mediator of progesterone-induced in a sparse tree, similar to that observed in Pgr−/− proliferation (Figure 5). RANKL signals through mice. To determine whether this defect is intrinsic to tumor necrosis factor receptor superfamily, member the mammary epithelium or due to systemic effects, 11a (TNFRSF11A), also known as RANK, to regulate transplantation experiments were performed in which a broad range of physiological processes, including Prl−/− epithelium was grafted into precleared WT fat osteoclastogenesis and bone-remodeling, by stimulat- pads.103,104 These experiments reveal normal developing NFKB1 in neighboring cells. Given this role in ment in the transplanted tissue, suggesting that defects regulating skeletal calcium release, perhaps it is not in knockout glands are due to the lack of PRL/PRLR surprising that RANKL should play such an essen- signaling in other tissues. This requirement for sys- tial role in regulating alveologenesis, the first step temic PRL was confirmed by experiments in which in transmitting maternal calcium to infant mam- the sidebranching and alveolar defects of Prl−/− mals. Mice lacking Rankl or Rank fail to undergo mice were rescued by engrafting Prl+/− pituitaries

© 2012 Wiley Periodicals, Inc. WIREs Developmental Biology Mammary gland development under the kidney capsules.105 One of the systemic similar phenotype: decreased sidebranching and dis- consequences of losing PRL signaling is low serum rupted alveologenesis. Moreover, transgenic expres- levels of hormones due to impaired secretion from sion of Rankl, alone, in the virgin gland is sufficient to the ovary. Researchers found that simply restor- elicit the characteristics of pregnancy, producing both ing progesterone levels in ovariectomized Prl−/− ductal sidebranching and alveolar budding, functions mice restored ductal sidebranching,105 providing fur- ascribed to PRL and progesterone.112 Negative regula- ther affirmation that PRL and progesterone work tion of PRLR signaling is achieved by members of the together to generate lobuloalveolar growth. Proges- suppressors of cytokine signaling (SOCS) family via terone treatment also rescued the infertility observed a classical negative feedback loop. PRL induces the in Prl−/− mice, allowing researchers to examine the expression of SOCS1, which in turn binds to JAK2 effect of Prl loss, alone, on mammary lobuloalveolar and inhibits its association with STAT5. Socs1−/− development. Surprisingly, alveologenesis is normal in mice display enhanced alveolar development and milk these animals, but this can be explained by the pres- production during pregnancy. Thus, SIRPA, RANKL, ence of prolactin family 3, subfamily d, member 1 and SOCS are three examples of many that illustrate (PRL3D1), also known as placental lactogen, which the complexity of the signaling pathways that regulate can also bind and activate PRLR. Consequently, the response of cells to PRL.113 This web of path- definitive demonstration of a requirement for PRL ways results in a finely tuned transcriptional program signaling in alveologenesis was achieved by transplan- that, in turn, regulates many signaling networks and tation experiments that delete PRLR, by engrafting ultimately controls the differentiation of alveolar cells Prlr−/− epithelium into WT mice. Ductal morphogen- and their production of milk. esis appeared normal in these mice, as progesterone In sum, by helping mothers cope with unreliable levels were normal, but alveologenesis failed, reveal- food stores, lactation has made a critical contribu- ing an essential, epithelial intrinsic role for PRL/PRLR tion to mammalian evolution. Progesterone and PRL signaling in lobular–alveolar development and milk orchestrate this essential reproductive event by engag- production. ing in a complicated and intertwined relationship The JAK2/STAT5 signaling pathway is acti- (Figure 5). Each regulates the other’s expression, and vated downstream of PRL/PRLR, and it plays an each controls overlapping and unique transcriptional essential role in establishing alveologenesis (Figure 5). regulatory networks that govern both the prolifera- This was demonstrated by the analysis of Jak2−/− tion and differentiation of mammary epithelial cells. and Stat5−/− mice whose mammary gland defects RANKL serves as one point of crosstalk between these phenocopy those observed in Prlr−/− glands. More- two signaling pathways, but there are likely additional over, conditional loss of Stat5 following PRL- pathways that function in cooperative and antago- induced differentiation of alveolar cells also showed nist ways to transform progesterone and PRL signals that this pathway is required for maintenance into the structural and functional changes required to of the pregnancy program.106–108 Signaling via generate and deliver milk. the PRLR/JAK2/STAT5 pathway culminates in the expression of milk protein genes, including casein β Involution Remodels the Tree Back (Csn2)andwhey acidic protein (Wap), which con- to Its Original Adult State tain STAT5 responsive elements in their promoters. The lack of demand at weaning causes milk to stag- The pathway is subject to precise control by a num- nate in the mammary epithelium, initiating the process ber of positive and negative regulators, including of involution that removes milk-producing epithelial integrins, which are the major class of ECM recep- cells and remodels the epithelial tree back to a sim- tors and are required for full STAT5 activation.109 ple ductal architecture (Figures 1 and 6). Research on Crosstalk between integrin and PRLR is mediated by involution has been facilitated by forced weaning pro- signal regulatory protein α (SIRPA), a transmembrane tocols that involve the removal of suckling mouse pups glycoprotein. SIRPA integrates the gland’s response to at the height of lactation. This induces synchronous the ECM during lactation, generating a complex that involution, allowing the elucidation of morphologi- includes JAK2 and regulates the response of PRLR as cal and molecular events underlying the process. Two a function of integrin activation in response to various phases of involution have been defined (Figure 6). The ECM components.110 Downstream of PRLR signaling first is reversible, such that resumption of suckling re- is RANKL and, as in the case of PGR signaling, it is establishes the milk supply. This step is accompanied a paracrine-acting target of PRL.111 The regulation of by apoptosis, alveolar cell detachment, and, within Rankl by both progesterone and PRL explains how 12 h, the accumulation of shed cells into the lumen. the homozygous deletion of each gene results in a Remarkably, no major morphological changes occur

WIREs Developmental Biology Mammary gland development with alveolar apoptosis resulting in lactation failure. the expression of two inhibitory subunits of the class Yet, constant suckling in this model, generated by IA PI3 kinase, p50α and p55α, which are gen- serial litter replacement, protects the alveoli, leaving erated by alternative splicing of the Pik3r3 gene. open lumens and intact lobules, demonstrating that This alters the subunit composition of PI3 kinase, systemic hormones can preserve lobuloalveolar struc- restricting its activity and, consequently, the activity ture, even in the midst of significant programmed of AKT.128 Another mechanism by which STAT3 cell death. Other studies have identified both intrinsic antagonizes pro-survival signaling is upregulation (mitochondrial) and extrinsic (death receptor) apop- of the insulin-like growth factor binding protein-5 totic pathways as those responsible for generating the (IGFBP5). IGFBP5 is thought to sequester IGF1 in massive cell death occurring during this initial phase of casein micelles and, as a result, interfere with pro- involution.118–120 Recent studies, however, show that proliferative IGF1 signaling.129 Overexpression of the earliest steps are independent of these apoptotic IGFBP5 in the mammary gland results in accel- processes and rely instead on lysosomal membrane erated involution that can be reversed by exoge- permeabilization.121 Despite these many studies, the nous IGF1 treatment, whereas, deletion of IGFBP5 130 actual trigger of apoptosis is still unknown. Alveolar leads to delayed mammary gland involution. Sim- stretch, the buildup of apoptotic factors in static milk ilarly, STAT3 promotes apoptotic pathways through and/or disruption of cell–ECM contacts are all likely the transcriptional control of numerous targets. For to play a role. Altogether, these studies demonstrate example, it upregulates the expression of lysosomal that local cues regulate the first phase of involu- proteases cathepsin B and cathepsin L, while down- 121 tion, whereas the second phase depends on the fall regulating their endogenous inhibitor Serpin2A. of systemic hormones and can be blocked by the Upregulation of these proteases results in lysosomal administration of glucocorticoids.116 membrane permeabilization that induces programmed In the past decade, numerous signaling pathways cell death at the earliest stages of involution without have been identified that regulate the developmental the involvement of the classical apoptotic pathways. switch from lactation to involution, but the STAT fam- Taken together, these data illustrate that specific acti- ily of proteins were found to play particularly impor- vation of STAT3 or STAT5 alone is sufficient to tant roles in this transition, by regulating the flow promote or suppress apoptosis, respectively, and that of survival signals through the PI3-kinase/AKT path- these transcription factors elicit some of their many way. STAT5A and STAT5B positively regulate AKT actions by regulating the AKT pathway. signaling, whereas STAT3 inhibits it. Within 3–6 h of milk stasis, a change in the phosphorylation status of The Second Phase of Involution STAT5 proteins alters their activity state, inactivat- is Controlled by Circulating Factors 116 ing STAT5A and STAT5B, but activating STAT3. The second phase of involution is accompanied by This toggle-like nature of STAT proteins in the lacta- dramatic changes in mammary gland architecture, tion–involution switch was demonstrated in a series including the remodeling of the basement membrane, of gain-of-function and loss-of-function experiments. collapse of alveoli, and differentiation of adipocytes For example, overexpressing either Stat5a or a consti- (Figure 6). Important regulators of these processes are tutively activated form of Akt1 delays mammary gland members of the serine protease and MMP families that 122,123 involution, while knocking out these genes has are responsible for activating plasminogen and break- 107,124 the opposite effect and accelerates involution. ing down the ECM, respectively. The expression of STAT5 binds to consensus sequences within the these factors in the mammary gland stroma begins dur- Akt1 locus in a PRL-dependent manner and initi- ing the first phase of involution when serine proteases ates transcription of Akt1 from a mammary specific are activated in order to mediate epithelial apopto- promoter, generating a strong cell survival signal.122 sis. At the same time, MMPs are kept in check by In contrast, STAT3 antagonizes pro-survival STAT5 the coordinate expression of their negative regulators, signaling and promotes pro-apoptotic pathways,125 tissue inhibitors of metalloproteinases (TIMPS). as evidenced by conditional deletion of Stat3 in the Theserineprotease,plasminogen, is synthesized mammary gland, resulting in decreased apoptosis in the liver and circulates as a zymogen through blood. and a dramatic delay in involution.126 The primary Its activation to plasmin is controlled locally in the activator of STAT3 in the gland is the cytokine, mammary gland by the serine protease kallikrein 1 leukemia inhibitory factor (LIF), and in its absence (KLK1). Mammary glands lacking plasminogen fail to Stat3 is dephosphorylated, resulting in delayed invo- involute after weaning and are marked by the accumu- lution, similar to the delay observed in the Stat3−/− lation of fibrotic stroma and the abnormal differentia- glands.127 Once activated, STAT3 directly regulates tion of adipocytes.131 A similar phenotype is observed

© 2012 Wiley Periodicals, Inc. Advanced Review wires.wiley.com/devbio by inhibiting KLK1 through intraperitoneal injection In contrast to involution that occurs post- of, ecotin, an inhibitor engineered to be highly specific lactationally, age-related lobular involution is a dis- for this enzyme.132 During the first phase of involu- tinct process that involves the replacement of glandu- tion, plasmin contributes to the apoptotic process by lar epithelium and interlobular connective tissues with disrupting interactions between cells and the ECM. fat. Eventually in the aged breast, all that remains In the second phase of involution, plasmin is joined are a few acini and ducts embedded in thin strands by the upregulation and activation of MMPs, such of collagen. This reduction in epithelial tissue has as MMP3, whose overexpression disrupts the basal beneficial consequences for the diagnosis and devel- lamina and causes premature involution.133 Simi- opment of breast cancer. First, the increased ratio larly, premature activation of MMP2, which occurs of fat to epithelium increases the effectiveness of in TIMP3-deficient glands, results in an accelerated mammography.141 Second, this reduction in epithe- involution that is irreversible, even during the first lial tissue may physiologically protect the breast from phase.134 These studies demonstrate that balanced cancer, as evidenced by a recent clinical study that protease activity, controlled by both regulated activa- correlated lobular involution with reduced breast can- tion and direct inhibition, is required for the gland to cer risk.142 Premenopausal women whose breasts properly transition through the phases of involution. had undergone partial or complete lobular involu- Moreover, these enzymes influence the differentiation tion were found to have significantly decreased risk of of adipocytes that is required to replace tissue loss due breast cancer, while postmenopausal women, whose to apoptosis of epithelial cells. One of the ways they breast exhibited delayed lobular involution, had an contribute to adipogenesis is by releasing growth and elevated breast cancer risk. These results suggest that differentiation factors from the cell surface and ECM. a fundamental protective event occurs with lobular Thus, by giving the gland the ability to remodel in involution. Recent studies suggest this protection may response to fluctuating local and hormonal environ- be due, at least in part, to the depletion of mammary ments, MMPs and serine proteases play critical roles stem cells in the aging breast. in mammary gland biology. While their importance in tissue restructuring during involution is highlighted here, they also participate in other processes such as MAMMARY STEM CELLS 135 milk production during pregnancy. and branch- Stem cells are specialized cells with the capacity to 136 ing morphogenesis during puberty, underscoring both self-renew and generate daughter cells that can their central position in regulating mammary gland differentiate down several lineages to produce all cell development. types found in mature tissue. Over 50 years ago, researchers postulated the existence of multipotent mammary stem cells (MaSCs) following the obser- Involution and Its Connection to Cancer vation that mammary epithelium is able to undergo There is a great deal of interest in understand- multiple rounds of proliferation, differentiation, and ing the signaling processes involved in postlacta- apoptosis with pregnancy. In vivo serial transplan- tional and age-related lobular involution, and the tation assays were designed to test this hypothesis. connection these processes share with cancer. Post- In this assay, a small tissue fragment is transplanted lactational involution is a process subject to much into precleared fat pads and allowed to grow into a regulation; transcriptional profiling studies show that mature mammary tree, before another small fragment this rapid and extensive period of tissue remodeling is harvested and transplanted. This can be performed mimics wound healing and pathological conditions serially for up to seven generations until senescence, such as tumorigenesis.137 This raises the possibility which is thought to occur as a result of depletion of the that involution creates a tumor microenvironment original stem cells contained in the initial fragment.143 that promotes further transformation of preneoplas- Later experiments revealed that neither the develop- tic mammary cells, providing an explanation for mental state nor reproductive history of the gland had the transient increase in breast cancer risk observed a significant impact on the longevity of the mammary following weaning.138 This possibility has been val- transplants.144,145 It was not until 2006, however, idated by experiments in which ECM was isolated that self-renewing multipotent stem cells were isolated from postlactational mammary glands and shown from mouse mammary gland by fluorescent activated to contain tumorigenic ECM fragments that facili- cell sorting (FACS), which defects cell surface mark- tate the invasiveness of breast cancer cells in culture ers CD24 (heat stable integrin) and either CD29 and increase metastasis in animal models of breast (β1-integrin) or CD49f (α6-integrin).146,147 Similarly, cancer.139,140 a mammary stem cell has also been identified in

© 2012 Wiley Periodicals, Inc. WIREs Developmental Biology Mammary gland development human breast, although different markers were used of tissue fragments from any part of the tree can result for its identification.148,149 These studies show that in mammary outgrowth.156 Yet, tissue fragments har- most, if not all, stem cells express α6andβ1inte- vested from the periphery of ductal outgrowths result grins, positioning them in a basal position in the in earlier senescence compared to tissue transplanted gland, possibly corresponding with a basal-positioned from the center of the outgrowth.157 These studies small light cell (SLC) that was identified by electron suggest that peripheral transplants contain MaSCs microscopy and occurs at a frequency of 1–3% within that have been aged by having undergone more cell the epithelium.150,151 Other estimates of stem cell divisions than their counterparts in the center. Addi- frequency are derived from calculating the number tionally, there may also be a difference in MaSC of mammary repopulating units (MRUs) in freshly concentration in different locations of the gland as a dissociated mammary cells using a limiting dilution recent multiscale in situ analysis has shown that puta- transplantation assay. These studies have generated tive MaSCs are distributed in a graded manner with a wide range of estimates from 1 MRU in 100 total more toward the nipple and fewer at the opposite end cells to <1 in 4900 total cells, a discrepancy that is of the mammary gland, with large ducts containing presumably due to the differences in techniques used a reservoir of these cells.158 Signaling pathways, such to dissociate and transplant cells.146,147,152 as the Notch,159 Hedgehog,160 and Wnt pathways,161 This range of estimated stem cell frequencies regulate these proliferative behaviors and niche char- illustrates how difficult it has been for researchers acteristics and appear to be similar to those pathways to achieve an understanding of mammary stem cell directing stem cells in other systems, although many biology. There are many impediments including the details concerning the control of these individual sig- technical complexity of dissociating solid tissue into naling pathways and their crosstalk remain unknown. single cell suspensions for FACS and the lack of This ongoing research on mammary gland devel- markers to specifically label these cells. Nevertheless, opment and stem cells is significantly contributing there is currently a tremendous effort to delineate to our understanding of breast cancer. Breast can- the stem cell hierarchy of the gland (Figure 7). The cers have been divided into six distinct subtypes current model is that an MaSC gives rise to committed on the basis of gene expression profiling: luminal progenitors, which proceed to differentiate along the A, luminal B, basal-like, claudin-low, Her2/ErbB2- myoepithelial and luminal epithelial lineages. It seems overexpressing, and normal-breast-like subtypes.162 likely that there is at least one intermediate cell, a bipo- In 2003, breast cancer stem cells were identified in tent progenitor, which lies between the MaSC and the patient samples,163 echoing the identification over a committed progenitor, but its identity remains elusive. decade ago of stem cells in the hematopoietic sys- The identification of the myoepithelial progenitor also tem that are the source of acute myeloid leukemia remains elusive, hindered by the fact that the MaSCs (AML).164 These and other findings have led to themselves express basal markers. A luminal progen- the cancer stem cell (CSC) hypothesis, positing that itor population, however, has been identified,153 and at least some tumors are initiated by one or more there is evidence that it gives rise to cells committed self-renewing CSCs that differentiate into large popu- to either ductal or alveolar cell fates154 (Figure 7). lations of non-self-renewing, yet rapidly dividing, cells How an MaSC gives rise to progenitor cells, responsible for generating the tumor mass.165 In the while regenerating itself, is also a subject of much hematopoietic system, there is strong evidence that investigation. In invertebrate organisms and some resident stem cells are the likely target of oncogenic mammalian organs, stem cells undergo asymmetric mutation in some diseases,164 but in solid tumors the cell division in which the cell reproduces itself and origin of the CSC remains unresolved. They could generates a daughter cell with a different cell fate.155 arise from mammary stem/progenitor cells or be gen- However, asymmetric cell divisions have not been erated de novo as part of the multi-step mutational definitively demonstrated in the mammary gland. An process that gives rise to a tumor. At least for some alternate mechanism of stem cell renewal and division breast tumors, evidence is growing that tissue-resident is symmetric cell division, whereby an MaSC simply stem/progenitor cells are the likely targets of trans- divides to give rise to two progenitors or divides to formation because some of the breast tumor subtypes replenish itself. Whether these symmetric and asym- share molecular profiles with stem/progenitor cells.166 metric cell divisions occur and how they are controlled This finding supports the notion that mutations in raises additional questions; where do stem cells reside stem/progenitor cells generate a self-renewing CSC and what is the nature of the stem cell niche? Studies capable of producing cells that constitute the tumor have shown that MRUs are distributed throughout mass. This self-renewal could occur at the same time the mammary epithelial tree, because transplantation that a subpopulation of CSCs maintain a quiescent

Ductal cell Luminal progenitor

CD24+ CD29hi CD49fhi ER- (mouse) Alveolar Alveolar cell progenitor

Stem cell

Bipotent progenitor

Myoepithelial cell

Myoepithelial progenitor

FIGURE 7| A schematic representation of the stem cell hierarchy within the mammary gland. A stem cell divides symmetrically or asymmetrically to generate a bipotent progenitor cell that gives rise to both a luminal and myoepithelial cell progenitor. Studies suggest that luminal progenitors differentiate into cells that are restricted to either ductal or alveolar lineages and as yet unidentiﬁed intermediate cells may be required for terminal differentiation. In contrast, myoepithelial progenitors are thought to differentiate directly into myoepithelial cells. This illustration represents one interpretation of available data, but the true hierarchy is likely to be much more complex. For details, see text.

program, allowing them to escape traditional thera- many developmental processes and are responsible for pies such as radiation and chemotherapy that target determining the location of the gland and controlling rapidly dividing cells.167 Current challenges are in cell fate, such that by birth the tissue compartments developing strategies to identify all the cell types in are precisely positioned and the nascent structure is the mammary hierarchy with specific markers and in correctly established. Epithelial–mesenchymal inter- defining the mutations that transform stem/progenitor actions also govern development during postnatal cells into CSCs. With knowledge of the parent cells stages of mammary gland morphogenesis, but after and the transformation processes, novel therapies, puberty, regulation by hormones and growth factors such as the use of small interfering (si)RNAs, can be profoundly changes the nature of these interactions. further developed to eradicate cancers by targeting Hormones generate complex signaling networks that the cells from which they arise. influence epithelial–mesenchymal interactions by regulating the production of secondary signaling pathways, which drive crosstalk between and within com- CONCLUSION partments. In all these complex processes, however, Tremendous progress has been made in delineating the one message is clear: Mother Nature is parsimo- signaling pathways controlling the three distinct stages nious and the same pathways are deployed over and constituting mammary gland development: embry- over again. This is evident as STAT proteins are onic, pubertal, and reproductive. These stages have involved in branching, lactation, and involution; FGFs many unique characteristics as well as many common in embryogenesis and branching; MMPs in branching attributes. Occurring over the lifespan of an individ- and involution; the list goes on. These same path- ual, each stage is separated temporally, as well as by ways are also hijacked in cancer cells and our growing hormonal requirement. Embryonic development pro- understanding of them has fueled tremendous progress ceeds in the absence of hormonal regulation, whereas in breast cancer research. These gains in our under- the pubertal and reproductive stages are governed standing have ushered in exciting times, and as this by customized hormonal inputs that prepare for and knowledge is translated into molecule-based diagnos- then result in milk production. During embryogen- tics and therapies, we are currently witnessing the esis, local epithelial–mesenchymal interactions direct tangible benefits basic research has brought to society.

ACKNOWLEDGMENTS We apologize to authors whose work was not cited due to space limitations. We thank Drs Jennifer Comp- ton and Gary Silberstein for comments. We acknowledge the NIH (RO1 CA-128902), Congressionally Directed Medical Research Program (W81XWH-08-1-0380), Santa Cruz Cancer Beneﬁt Group, Initiative for Maximizing Student Diversity (NIH GM058903) (HM) and Center for Biomolecular Science and Engineering (5P41HG002371-09) (HM).

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