Abstract
The pubertal mammary gland is an ideal model for experimental morphogenesis. The primary glandular branching morphogenesis occurs at this time, integrating epithelial cell proliferation, differentiation, and apoptosis. Between birth and puberty, the mammary gland exists in a relatively quiescent state. At the onset of puberty, rapid expansion of a pre-existing rudimentary mammary epithelium generates an extensive ductal network by a process of branch initiation, elongation, and invasion of the mammary mesenchyme. It is this branching morphogenesis that characterizes pubertal mammary gland growth. Tissue-specific molecular networks interpret signals from local cytokines/growth factors in both the epithelial and stromal microenvironments. This is largely orchestrated by secreted ovarian and pituitary hormones. Here, we review the major molecular regulators of pubertal mammary gland development.
| Abbreviations | ||
| 3D | = | three-dimensional |
| Adam17 | = | ADAM metallopeptidase domain 17 |
| Areg | = | amphiregulin |
| CITED1 | = | Cbp/p300-interacting transactivator 1 |
| CSF | = | colony-stimulating factor |
| E | = | estrogen |
| ECM | = | extracellular matrix |
| EGF | = | epidermal growth factor |
| EGFR | = | epidermal growth factor receptor |
| EMT | = | epithelial-to-mesenchymal transition |
| ERα | = | estrogen receptor α |
| FGF | = | fibroblast growth factor |
| FGF1/aFGF | = | fibroblast growth factor/acidic fibroblast growth factor |
| FGF2/bFGF | = | fibroblast growth factor/basic fibroblast growth factor |
| FGF7/KGF | = | fibroblast growth factor 7/ keratinocyte growth factor |
| FGFR2 | = | fibroblast growth factor receptor 2 |
| GH | = | growth hormone |
| GHR | = | growth hormone receptor |
| GHR-BP | = | growth hormone receptor-binding protein |
| HELU | = | hyperplastic enlarged lobular unit |
| HGF/SF | = | hepatocyte growth factor/scatter factor |
| hGH | = | human growth hormone |
| HMEC | = | human mammary epithelial cell |
| IGF | = | insulin-like growth factor |
| IGFBP | = | insulin-like growth factor-binding protein |
| KGFR | = | keratinocyte growth factor receptor |
| LRC | = | label-retaining cell |
| MAPK | = | mitogen-activated protein kinase |
| MEC | = | mammary epithelial cell |
| Med | = | Mediator |
| MMP | = | matrix metalloproteinase |
| MMTV | = | mouse mammary tumor virus |
| P | = | progesterone |
| PMME | = | primary mouse mammary epithelial cell |
| PR | = | progesterone receptor |
| Prl | = | prolactin |
| PrlR | = | prolactin receptor |
| RANKL | = | receptor activator of nuclear factor-kappaB ligand |
| SRC | = | steroid receptor co-activator |
| STAT | = | signal transducer and activator of transcription |
| TACE | = | tumor necrosis factor-α-converting enzyme |
| TDLU | = | terminal duct lobular unit |
| TEB | = | terminal end-bud |
| TGFβ | = | transforming growth factor β |
| Timp | = | tissue inhibitor of metalloproteinases |
| TU | = | transitional unit |
| WT | = | wild-type |
| ZO1-3 | = | zonula occludens 1–3 |
Key messages
A hierarchy of regulation exists in the pubertal mammary gland such that systemic hormones modulate the activity of local growth factors to execute the progression of ductal morphogenesis.
There is a balance between regulators that promote proliferation and branching, and those that restrict and sculpt the epithelial network.
During pubertal mammary gland development, a stem cell niche is operative within the cap cell layer of the terminal end-bud and contributes to pubertal ductal morphogenesis.
Introduction
Core regulators of pubertal mammary gland development
At birth, the mammary gland contains a simple ductal network that fills a fraction of the mammary fat pad. The major difference between human and mouse mammary glands at birth is that several minor ductal networks are joined at the nipple in humans while a single network is found in mice (Citation1). Growth of this network keeps pace with normal body growth (is isomorphic) until puberty. At this point, drive from ovarian hormones stimulates the rapid proliferation and expansion of these simple structures (branching morphogenesis). This expansion is driven by terminal end-buds (TEBs) in mice and terminal duct lobular units (TDLUs) in humans; these are clover-shaped structures that encapsulate the tips of primary ducts. In mice, for instance, as primary ducts elongate, bifurcation (or primary branching) of the TEBs generates additional primary ducts. These in turn are subjected to lateral secondary branching (Citation2). This network can further support tertiary lateral branches which will occur at each diestrus and during pregnancy (Citation2,Citation3). When the extremities of the fat pad are reached, the end-buds shrink in size and become mitotically inactive, then the pubertal growth phase is complete (Citation4,Citation5) (, and ).
Figure 1. Morphology of the pubertal mouse mammary gland; developmental analysis of ERα−/− and amphiregulin−/− mammary glands. (A) Diagrammatic representation of a developing mammary tree and mammary gland duct and TEB. The cap cell layer of the TEB drives invasion through the fat pad at puberty and inner body cells eventually die away to form a hollow lumen. (B) H&E staining of mammary gland sections from pubertal (6.5 week old) CD-1 mice showing a duct and a TEB. Confocal images of transverse and longitudinal sections through a duct and a TEB. Section cartoon adapted from Howlin J, et al. Oncogene 2006;25(10);1532–42. (C) Whole-mounted mammary glands of WT and Erα−/− females at different developmental stages showing high magnification of the ductal tree. (Scale bars: 1 mm.), adapted, with permission, from PNAS 2006;103;2196–2201. The process of experimental transplantation is also shown. (D) Fluorescent images of mammary glands from recipients. Preparations were derived from virgin recipients engrafted with ER−/− or WT epithelium. Transplanted ERα−/− cells (GFP-positive) fail to generate a mammary tree adapted from PNAS 2006;103;2196–2201. (E) (A–D) Wholemount micrographs of inguinal glands from amphiregulin−/− (A, C) and WT (B, D) females were analyzed at the following developmental stages: day 24 (A and B), 6 weeks (C and D). Adapted, with permission, from Ciarloni L, et al. PNAS 2007;104:5455–5460.
Figure 2. Pattern of epithelial-associated gene expression during pubertal mammary gland development. A. Diagrammatic representation of normal mammary gland development from pre-puberty to post-puberty. After birth, the mammary gland consists of a fat pad with a lymph node in the centre and has a primitive epithelial network extending from the nipple. TEB's appear at the onset of puberty and they drive the expansion of the tree and invasion of the fat pad by the ductal network. Post-puberty, the TEB's have regressed and the ductal network extends to the extremities of the fat pad. Tertiary side branching can be seen in the mature adult. B. A time-line of mammary development in mouse versus human. C. Gene expression time-course for epithelial expansion in the pubertal mammary gland. Microarray expression data extracted from the NCBI-Geo dataset record GDS2721, which contains expression microarray data from pre-pubertal to post-pubertal mouse mammary glands, http://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS2721. Here we show a time course for increased gene expression of genes associated with epithelial expansion that occurs in the mammary gland at puberty.
Pubertal mammary gland development is initiated and maintained by steroid hormones and pituitary hormones, and local growth factors and cytokines (Citation6). Ovarian steroids and pituitary hormones are not only necessary for ductal expansion in the pubertal mammary gland but also act as mediators of mammary stem cell fate decisions. Here, we separate out the influence of each regulator on the morphological elements in the developing gland, namely ducts, branches, and on the driver of pubertal development, the TEB. The effect of regulators of pubertal mammary gland development on stem cell compartments and breast cancer progression is remarked on (reviewed in (Citation7)). This review is supported by three tables: lists the transgenic and gene knock-out (KO) mouse models with which key discoveries have been made; lists a subset of the many reviews written on this area of study in the past 10 years; and lists the regulators and their core influence on pubertal mammary gland development. These tables also refer to the roles played by immune cells and mitogen-activated protein kinase (MAPK) signaling on pubertal mammary gland development; we feel these are important but are beyond the scope of this review.
Table I. Animal models (mostly transgenic and gene KO mice) exhibiting a pubertal mammary gland phenotype.a
Table II. Pubertal mammary gland literature reviews from 2000 to 2010.
Table III. Sites and modes of action of the regulators of pubertal mammary gland development.
Ductal morphogenesis
Hormonal regulation
In the pubertal mammary gland, the initial drive for ductal morphogenesis comes from circulating ovarian and pituitary hormones (reviewed in (Citation7)). There are critical requirements for estrogen (E), progesterone (P), and growth hormone (GH).
Estrogen and estrogen receptor α
Ductal morphogenesis in the mammary gland requires estrogen (reviewed in (Citation4,Citation8)). The mammary glands of mice ovariectomized at 5 weeks of age fail to develop a ductal network. This effect is rescued upon implantation of slow-release estrogen pellets into the mammary gland; this estrogen stimulates ductal morphogenesis (Citation9,Citation10). The ductal network in pubertal mice deficient in estrogen receptor α (ERα) is severely stunted. A complete failure of the ductal network to invade the stoma is seen, and adult mammary glands resemble those of a new-born female (Citation11,Citation12): ERα−/− epithelial cells will not generate a mammary tree at puberty when transplanted into an ERα+/+ fat pad ( and ), but ERα+/+ epithelial cells will undergo ductal outgrowth when transplanted into an ERα−/− fat pad; thus there is a requirement for ERα in epithelium, not stroma. Thus it is hypothesized that in order for ductal morphogenesis to occur in the pubertal mammary gland, epithelial ERα is required to act in a paracrine fashion; it is demonstrated that ERα−/− epithelial cells will persist in a mammary tree when transplanted into an abnormal fat pad, mixed with ERα+/+ cells (Citation13).
As a nuclear hormone receptor, ERα must recruit co-activators and co-repressors to have its transcriptional effects on target genes (Citation14). Pubertal mice deficient in steroid receptor co-activator 1 (SRC-1) protein display an under-developed ductal network and highlight a requirement for SRC-1-associated ERα activity (Citation14,Citation15). Another co-regulator of ERα in the mammary gland is Cbp/p300-interacting transactivator 1 (CITED1). This was identified by expression microarray analysis as up-regulated in the pubertal gland () (Citation16). CITED1 is co-expressed in ductal luminal epithelial cells with ERα, and CITED1-null females exhibit a stunted ductal network and significantly fewer TEBs at puberty.
Members of the Eph family of receptor tyrosine kinases are also implicated in estrogen-driven pubertal development. At puberty, the Ephb4 receptor is expressed in ductal myoepithelial cells and its ligand ephrin-B2 is expressed in luminal epithelial cells (Citation17). Ovariectomy of pre-pubertal mice leads to ablation of receptor–ligand expression, an effect only restored by estrogen administration (Citation17). Transgenic mice with the EphB4 receptor under the control of the mouse mammary tumor virus (MMTV) promoter showed that deregulating the normal timing of EphB4 receptor expression led to delayed development of the mammary tree at puberty and disrupted cellular architecture (Citation18).
Amphiregulin (Areg) is an epithelial-derived epidermal growth factor receptor (EGFR) ligand, and its increasing levels (and the increasing expression of a range of epithelial markers) in the pubertal mammary gland parallels ductal morphogenesis (, reviewed in (Citation19)). Gene-KO studies show that Areg is essential for ERα-driven epithelial cell proliferation and for ductal elongation during pubertal mammary gland development. It acts in a paracrine fashion to mediate the ERα effect (Citation20). When Areg−/− mammary epithelial cells are transplanted with wild-type cells into mammary fat pads, Areg−/− cells survive by receiving proliferative signals from their wild-type (WT) neighbors (Citation20). ERα directly stimulates Areg mRNA production in epithelial cells, and Areg−/− mice phenocopy ERα-null mice in that pubertal ductal morphogenesis is severely stunted in both genotypes (Citation13,Citation20) ( and ). The role of Areg in modulating the effects of its receptor, EGFR, during pubertal mammary gland development is considered below (see ‘Growth factor regulation of ductal morphogenesis’).
Hyperplastic enlarged lobular units (HELUs) evolve from normal TDLUs in the human breast, and this is a first histological sign of disease development (Citation21). The conversion of a TDLU to a HELU is the most frequent growth abnormality in the human female breast, and its identification represents the earliest possible chance of cancer precursor detection (Citation19,Citation21). Differential gene expression analysis of HELUs compared to normal TDLUs shows that ERα and Areg levels are elevated in HELUs (Citation21). A switch in preference of EGFR ligands (from EGF to Areg) may be mediated by estrogen and represents an early event in the progression to hyperplasia (Citation21). The role of ERα and Areg in regulating mammary gland epithelial expansion suggests a potential for two axes to contribute to breast tumor progression, and Areg is found to be elevated in most ERα-positive breast tumors (Citation21,Citation22).
Progesterone and PR
The rise in gonadotrophin levels at puberty leads to ovarian progesterone secretion. It acts as a mitogen in the mammary gland (Citation23). There exists a tight interplay of expression between ERα, the progesterone receptor (PR), and the prolactin receptor (PrlR). All three receptors co-localize to the same epithelial cells but show a non-uniform expression pattern during puberty (Citation24). ERα/PR-positive cells make up 30% of the luminal duct epithelium population (Citation7,Citation24). There are two PR isoforms, A and B, and the mammary gland contains these in a 2:1 [A:B] ratio; this expression ratio is maintained in the mammary glands of normal, estradiol-treated, and ovariectomized mice (Citation25). Despite the requirement for twice as much PR-A, PR-B is absolutely required for ductal development in the pubertal mammary gland (Citation26). Interestingly, adult mice carrying a transgene for the PR-A isoforms, leading to ratio disturbance, develop ductal hyperplasia showing that the balance of PR-A:PR-B must be maintained (Citation23). Epithelial PR is said to act in a paracrine fashion driving pubertal ductal morphogenesis, and PR−/− epithelium (deficient in both isoforms) displays a stunted ductal network when transplanted into WT cleared fat pads (Citation27,Citation28). Steroid receptor co-activator 3 (SRC-3) regulates PR activity in the pubertal mammary gland (Citation14). PR transcriptional activity is detectable in the luminal epithelium of mouse mammary glands upon treatment with either estrogen or progesterone. However, this activity is not detected in the mammary glands of SRC-3−/− mice which are under-developed at puberty, phenocopying the PR-null glands (Citation14).
Growth hormone (GH) and prolactin (Prl)
Experiments in the 1930s and 1940s demonstrated that the pituitary gland was required for mammary gland development and that estrogen-driven ductal development fails in hypophysectomized rodents (Citation29,Citation30). The two pituitary hormones required are GH and Prl; both act by receptor-mediated Jak2 phosphorylation and downstream Stat activation (Citation31).
GH has been shown to be required for ductal morphogenesis in the pubertal mammary gland, and it signals through insulin-like growth factor (IGF)-I of hepatic and mammary origin (Citation32). The ‘Laron’ mouse was created by targeted disruption of the growth hormone receptor-binding protein (GHR-BP) gene (these are encoded by a single GHR-BP gene (Citation33,Citation34)). Homozygous-KO mice lacking GHR and GHR-BP are severely growth-retarded and have decreased serum IGF-I levels and increased serum GH levels (Citation34). Their mammary glands displayed impaired ductal outgrowth at puberty, with the ductal tree barely reaching the lymph node (Citation32). By 15 weeks, when the WT ductal network has successfully invaded the entire stroma, ducts in mutant mice are thinner than in WT. This growth and mammary morphogenesis defect arises due to failures in systemic GH signaling: ductal networks arising from GHR-null epithelium transplanted into the cleared fat pads of syngenic hosts develop as normal, proving that GHR is required in the stroma (Citation32). The role of locally produced IGF-I in mediating the GHR effect is expanded on below (see ‘Growth factor regulation of ductal morphogenesis’).
A range of Prl and PrlR gene deletion strategies in mice lead to infertile females (pregnancies in some mice can be maintained through treatment with progesterone) (Citation35,Citation36). A study in 1997 described a normally developed ductal tree in pubertal PRL−/− mice at 6 weeks (Citation35). In contrast, mammary glands from PrlR+/− females were reported to be smaller than WT, and their mammary ductal network was not as extensive as that of WT females. The defect is exacerbated in KO glands (Citation37). The Brisken group later extended these findings, showing that development was normal up to puberty in PrlR-null mice. The subsequent pubertal ductal defect could be rescued if the PrlR−/− mammary epithelium was transplanted into fat pads of WT hosts (Citation36). Thus, there is a requirement for the PrlR in the pubertal mammary gland. Interestingly, a KO mouse for one of its intercellular signaling targets, Stat5a (also a target of GH), has recently been shown to have deficient pubertal mammary gland development (see ‘Ductal morphogenesis – the process of branching’) (Citation32,Citation38).
Growth factor regulation of ductal morphogenesis
The ErbB family of growth factors
The ErbB family of growth factor receptors comprises EGFR (ErbB1), ErbB2, ErbB3, and ErbB4. Activation of EGFR, for instance, requires ligand binding which leads to receptor dimerization with either another monomer of EGFR or a monomer of ErbB2, ErbB3, or ErbB4. It has been proposed that pubertal mammary gland development may require formation of EGFR–ErbB2 heterodimers in the stroma (Citation39). Certainly, ductal morphogenesis requires functional EGFR and ErbB2 (reviewed in (Citation15)), and both receptors are tyrosine-phosphorylated (auto-activated) in the pubertal mammary glands (Citation39).
Using slow-release pellets delivering EGF, Coleman et al., in 1988, showed that ductal development could be restored in ovariectomized mice (Citation40): this exogenous EGF release in the gland resulted in luminal epithelial cell DNA synthesis and ducts with an increased diameter. It was hypothesized that such factors derived from the mammary epithelium induce activation of stromal EGFR family receptors. Ligand–receptor binding analysis showed that this binding was predominantly localized to the myoepithelium (Citation40). EGFR kinase activity is reduced in Wave-2 mice, and pubertal mammary glands are under-developed (Citation39). Mammary glands from new-born EGFR-KO mice transplanted under the renal capsules of WT nude mice show virtually no ductal growth (Citation39). However, when epithelium from EGFR-KO mouse mammary gland was transplanted into a WT fat pad, ductal morphogenesis was supported (Citation39). Thus, stromal EGFR is indispensable for normal pubertal mammary tree growth.
Areg has been proved to be the key paracrine mediator of estrogen-stimulated pubertal ductal morphogenesis. Areg is the only EGFR ligand whose expression has been shown to be increased at puberty, an effect that is transcriptionally regulated by ovarian estrogen (reviewed in (Citation4)). The mammary gland of ovariectomized mice can be forced from quiescence by treatment with recombinant Areg which leads to a resumption in epithelial cell proliferation (Citation41). Ductal morphogenesis is not supported in Areg-null mice (Citation42).
In luminal epithelial cells, Areg is membrane-anchored. It is released from the cell surface to bind stromal EGFR in the developing mammary gland, a reaction which is catalyzed by the Adam17(TACE) protease (reviewed in (Citation4)). Pubertal Adam17−/− mouse mammary glands phenocopy EGFR-KO mice: they die soon after birth, and mammary tissue transplanted under the kidney capsule fails to develop in WT hosts, whereas WT epithelium grows in Adam17−/− stroma (Citation43). However, soluble exogenous Areg relieves the former defect (Citation43). Taken together, these data emphasize that the stromal EGFR signaling drive for ductal morphogenesis requires epithelial Areg and Adam17.
There is also evidence to suggest that the non-ligand-binding ErbB receptor, ErbB2, is required for pubertal mammary ductal morphogenesis. There is delayed pubertal penetration by the ductal network of genetically rescued ErbB2−/− mammary tissue fragments that are transplanted into wild-type fat pads. The evidence suggests that ErbB2 signaling is required in the initiation stages of pubertal ductal morphogenesis (Citation44).
ErbB3 has no kinase activity and so for activity hetero-dimerizes with another ErbB family member upon binding of Neuregulin-1 (heregulin) or Neuregulin-2 (reviewed in (Citation45)). A role for ErbB3 in pubertal mouse mammary gland development was invoked in 1996 when slow-release elvax pellets containing heregulin-alpha were implanted into the mammary glands of pre-pubertal mice (Citation46). Heregulin induced branching even in the absence of estradiol and progesterone, suggesting that ErbB3 activation was sufficient to drive morphogenesis (Citation46). Since then, a mammary conditional ErbB3-KO has been generated. This showed a lower ductal density than wild-type litter-mates at puberty (Citation47). Most recently, mammary buds from ErbB3−/− embryos transplanted into wild-type hosts were shown to fail to fill the fat pad at puberty. This outcome differs from that seen with ErbB2−/− mice. In the latter the ductal deficit is maintained throughout adulthood, pregnancy, and early lactation (Citation48).
ErbB4 stands alone from the other members of the ErbB family in that it exerts pro-differentiation and anti-proliferative effects on mammary epithelial cells (Citation45). ErbB4 expression is lowest during periods of epithelial expansion (e.g. pubertal ductal morphogenesis), and this has been consistently observed in rat, mouse, and human mammary tissue/gland studies (reviewed in (Citation49)). Genetically rescued ErbB4-KO mice show no abnormalities in ductal morphogenesis at puberty (Citation50).
IGF family
In the pubertal mammary gland, there is a requirement for paracrine action by IGF-I in supporting ductal morphogenesis, an effect associated with apoptosis suppression (Citation51). Local production of IGF-I in the mammary gland is a core response to GH, and IGF-I is expressed in the mammary stroma during periods of ductal growth (Citation52) (IGF-I is also expressed in TEBs; see below). While IGF-I is expressed in the stroma, its receptor IGF-IR is expressed in the ductal epithelium in the pubertal mammary gland (Citation51). Extensive ductal outgrowth was seen when mammary glands from pre-pubertal C57/Bl6 mice (primed with estrogen and progesterone for 9 days) were removed and cultured with IGF-I and in the presence of nanomolar concentrations of insulin, suggesting an inductive role for IGF-I (Citation51).
Four major mouse models of defective IGF-I/IGF-R signaling highlight the postnatal requirement for IGF-I in normal ductal growth. Firstly, the Cre/loxP system has been used to generate IGF-I-null mice. These mice are born displaying a 35% growth retardation and never pass through puberty (Citation53). Also in 1999, independent generation of IGF-I-null mice showed reduced invasion of the mammary fat pad in the −/− mice, an effect alleviated by GH treatment (Citation54). Later, mice homozygous for the ‘midi’ allele showed a pubertal mammary branching defect associated with a severe IGF-I deficit due to an insertional deletion strategy (the ‘midi’ allele) (see below) (Citation55). KO-mice deficient in the IGF-I receptor (IGF-IR) also fail to support ductal outgrowth (reviewed in (Citation56)).
Over-expressing IGF-I leads to dysregulated mammary development as well as an increased incidence of mammary tumors. Where over-expression of IGF-I is targeted to the myoepithelial or basal cells (under control of the bovine keratin-5 promoter), mammary glands from pre-pubertal mice displayed increased ductal proliferation (Citation57).
It has been determined that stromal IGF-I induces expression of S and G2 phase cyclins in the epithelial compartment of the pubertal mammary gland (Citation58). There are also indications of cross-talk between IGF-I and Areg in regulating normal ductal morphogenesis (reviewed in (Citation59)). Of note, IGFs can modulate the action of the ERα; conversely, the ERα action has been shown to regulate the expression of IGF ligands, IGF receptors, and the IGF-binding proteins (Citation60–63).
Homologous to both IGF-I and insulin is the IGF-II protein (Citation64). In-situ hybridization studies using mammary glands from pubertal C57/Bl6 mice detects IGF-II mRNA in the ductal epithelium, which contrasts with IGF-I (Citation51). Its expression pattern correlates with that of rapidly proliferating cells. Thus, IGF-II has been described as a paracrine or autocrine mitogen for ductal epithelial cells in the pubertal mammary gland (Citation51). Whereas GH induces expression of IGF-I, IGF-II is expressed in response to Prl signaling; IGF-II and Prl co-localize in mammary epithelium (Citation65). Hovey et al. also report increased IGF-II mRNA and increased PrlR expression in the mouse mammary gland at puberty (Citation66). The cascade of events in cultured primary mammary epithelial cells (MECs) is that Prl induces IGF-II mRNA (and protein) production which then induces cyclinD1 expression (Citation65). Both Prl and IGF-II are over-expressed in a range of breast cancers.
The IGF-I signaling axis is further complicated by the presence of secreted, high-affinity IGF-binding proteins (IGFBPs). These proteins regulate IGF signaling, but an in-depth analysis of the action of these proteins is beyond the scope of this review. The expression of IGFBP-1 to 5 is detected in the mammary gland at puberty suggesting that these proteins do have a role to play in mammary ductal morphogenesis; IGFBP-3 and -5 mRNA localize to developing epithelial structures and rare stromal cells during puberty, whereas IGFBP-2 and −4 are mainly detected in the stroma (reviewed in (Citation67)).
Fibroblast growth factor family
The fibroblast growth factor (FGF) system, comprising 22 ligands and various receptors, has been implicated in hormonally driven local control of postnatal mammary development. Members of the ligand family have been shown to be stromally active morphogens and to exhibit specific temporal and spatial patterns of expression in the mammary gland, suggesting that the level of redundancy between members is not particularly high (reviewed in (Citation68)).
FGF1, FGF2, FGF7, and FGF10 are expressed in the mammary gland during periods of ductal growth such as during puberty (Citation69). FGF1 expression localizes to the luminal epithelium (Citation69). Imagawa et al. showed that FGF1 is a mitogen for parenchymal MECs grown on collagen gels (Citation70). Addition of FGF1 to serum-free media in the presence of heparin was enough to stimulate proliferation of three-dimensional (3D) colonies on the gel matrix (Citation70).
Stromal FGF2 (basic FGF, bFGF) is a ligand for FGFR2 which is expressed on mammary epithelial cells (Citation43). Mammary epithelium induces stromal FGF2 expression, and FGF2 is also found in the myoepithelial layer of cells (Citation69). In an in-vitro model of morphogenesis using primary mouse mammary organoids, exogenous FGF2 induces initiation and elongation of ducts in a process dependent on Rac1 and myosin light chain kinase (Citation71). It has been suggested that FGF signaling in the pubertal mammary gland may act in concert with a signaling drive from EGFR (EGFR signaling in ducal morphogenesis is considered below). In vitro, growth and branching of EGFR−/− mammary organoids was rescued by treatment with either bFGF of FGF7 (Citation43).
Despite evidence for a role in mammary gland development, FGF7-null mice display no defect in the structure or function of the mammary gland (Citation72). The FGF7 receptor (KGFR) is a splice variant of FGFR2 (FGFR2-IIIb) (Citation72,Citation73). KGFR mRNA was detected in human mammary epithelial cells (HMECs), but not in stromal cells (Citation74). The level of KGFR mRNA in mammary tissue strongly increased during the postnatal period to a maximum in adult mice. KGFR expression in the mammary gland is inhibited by E2 but is increased by progesterone (Citation75).
FGF10 is a stromally derived ligand of KGFR and shows similar receptor binding specificity and affinity to FGF7, but it also binds the FGFR1 receptor isoforms (Citation73). Changes in FGF10 mirror changes in the ratio of stroma to epithelium. FGF10 is highly expressed in the mouse mammary gland, maximum level at puberty. FGF10 mRNA is 15-fold higher than FGF7. FGF10−/− mice die at birth due to failed lung development because of disrupted pulmonary branching morphogenesis (Citation76). As such, a predicted consequence of the loss of FGF10 in the pubertal mammary gland may be a severe branching defect.
A sculpting role for TGFβ1
The transforming growth factor β (TGFβ) family of cytokines has long been established as regulators of cell proliferation, apoptosis, and differentiation in the developing mammary gland (Citation77). Seminal experiments performed in the 1980s using slow-release elvax pellets highlighted the ability of TGFβ to locally inhibit ductal growth in the mouse mammary gland, an effect which was both reversible and specific (Citation78,Citation79).
All three isoforms of mammalian TGFβ (TGFβ1–3) are found in the epithelium at all stages of mammary gland development (Citation80,Citation81). However, over-expressing latent TGFβ does not generate a mammary phenotype (Citation81). Transgenic mouse models have allowed the investigation of where and when active TGFβ is exerting its effect.
At puberty, active TGFβ1 is restricted to the luminal epithelial cells (Citation82). It is thought that sub-populations of epithelial cells are differentially regulated by TGFβ1 such that the proliferation drive from ovarian hormones during puberty is targeted to non-TGFβ-expressing cells. Using TGFβ-KO mice, it was shown that ductal development was accelerated in the heterozygotes (TGFβ+/−) which have <10% wild-type TGFβ levels (Citation82). Although ducts from TGFβ-deficient mice were morphologically normal, the proliferation index in proliferating tissue was increased 4-fold in transplants from pubertal TGFβ+/− glands, and this effect was shown to be a property of the epithelium (Citation82).
TGFβ-null mice can now be maintained to adulthood on an immune-deficient background, and the gross morphology of mammary glands from these mice appears relatively normal (slightly fewer TEBs) (Citation2). Transplantation/tissue recombination studies reveal that the outgrowth from TGFβ-deficient epithelium is increased, compared to WT tissue (reviewed in (Citation2)). In contrast, female mice harboring a constitutively active form of TGFβ (simian TGFβ1S223/225) under the control of the MMTV promoter have an under-developed ductal network at 7 weeks (Citation83). Current knowledge suggests two distinct and contrasting actions of TGFβ in normal mammary gland development. Epithelial ductal morphogenesis is stimulated by TGFβ in an endocrine or paracrine fashion, whereas TGFβ restricts epithelial proliferation in an autocrine fashion (TGFβ1 action in the pubertal mammary gland is considered further below).
The terminal end-bud —the driver of ductal morphogenesis
Cap cells, generation of the myoepithelial lineage, stromal environment
Local availability of growth factors is required for the success of the invading TEB. TGFα mRNA is localized to the cap cells of the advancing TEB and in stromal cells at the base of the TEB (Citation84). EGF and TGFα-release pellets implanted into regressed glands of ovariectomized mice are sufficient to induce the appearance of TEBs (Citation84). Slow release of exogenous EGF in the glands of ovariectomized mice led to the re-emergence of a cap cell layer and new end-buds that were morphologically normal (Citation40). Further analysis showed that the cap cell layer displayed high EGFR ligand-binding activity. The primary EGFR ligand, Areg, has also been localized to luminal epithelial cells and cap cells in the advancing TEBs of pubertal C57/Bl6 mouse mammary glands (Citation85) (TEB diagram is shown in ).
As stated previously, epithelial Adam17 is required to cleave membrane-bound epithelial Areg so that it can activate stromal EGFR in the pubertal mammary gland. Tissue inhibitor of metalloproteinases (Timp)-3 is the only known inhibitor of Adam17 activity in vivo. Its expression is relatively low in invading TEBs (Citation43). One other metalloproteinase inhibitor, Timp1, displays the opposite expression profile of Timp3, suggesting that a [Timp1 on–Timp3 off] switch is required from stromal EGFR to drive cell proliferation in the TEB. ErbB2 is also required for normal TEB assembly: ErbB2-deficient pubertal mammary glands have abnormal TEBs with increased numbers of cap-like cells (Citation44).
Decreased numbers of TEBs have been detected in pubertal TGFβ1-null mice (Citation86), but overall mammary gland morphology appears normal. Active TGFβ is localized to luminal epithelial cells (Citation82). As early as 1987, Daniel and Silberstein showed that TGFβ1 induces restraint of ductal growth by termination of DNA synthesis in populations of cells that are undergoing rapid proliferation, such as the TEB cap cell layer (Citation79). Stromal cells encapsulating the TEBs are unaffected by this TGFβ activity. Endogenous TGFβ is lost from the immediate environment of ducts that are actively forming new buds and undergoing branching events, whereas mature ducts sit within an TGFβ-enriched extracellular matrix (ECM) (Citation87).
Elvax pellets releasing blocking antibodies against either laminin-1 or β1-integrin, implanted into the pubertal mouse mammary gland, lead to decreased numbers of TEB structures (Citation88). This highlights the requirement for integrin–ECM interactions within the immediate environment of the TEB (Citation88). Regulated expression of adhesion molecules also supports the maintenance of TEB structure/function. P-cadherin is expressed in the cap cell layer and its progeny, the myoepithelial cells (Citation89). Implanted slow-releasing pellets of function-blocking antibody against P-cadherin disrupts the cap cell layer (Citation89). P-cadherin-deficient mice highlighted its role in negatively regulating development of the pubertal tree (Citation90). TEBs from P-cadherin-null females resemble alveoli from females in early pregnancy, and with age the mammary epithelium becomes hyperplastic with dysregulated infiltration of lymphocytes into the fat pad (Citation90).
More recently, axonal guidance molecules have been shown to play a role in the structural development of the TEB in the pubertal mammary gland. The cap cells express neogenin, a receptor for netrin-1. This ‘guidance’ ligand is detectable in TEB body cells during puberty (Citation91). Failure of these two proteins to interact, by loss of either one, leads to a loss in adhesion between the cap cell layer and the body cells of the TEB (Citation4,Citation15,Citation91). Other axonal guidance molecules expressed in the TEB are semaphorins and their receptors, plexins and neuropilins. Their expression patterns implicate them in ductal morphogenesis, but their exact roles are unclear at present (Citation92).
The PrlR-KO mouse has highlighted a substantial requirement for Prl-induced mitogenesis in the development of the pubertal mammary gland (Citation36). TEB-like structures were found to persist in the mammary glands of adult female PrlR−/− mice. Brisken et al. (Citation36) suggest that these PrlR-deficient TEBs are dormant and perhaps represent an intermediate structure en route to differentiation. They are similar to WT TEBs in that there is direct contact between stromal fat cells and the epithelium of the TEB, but there is no definitive cap cell layer and fewer body cell layers overall, such that these structures can never go on to form alveolar buds after puberty (Citation36).
Body cells—proliferation
A role for hepatocyte growth factor/scatter factor (HGF/SF) in TEB formation and function has also been established: primary mouse mammary epithelial (PMME) cells over-expressing HGF/SF and transplanted into a cleared pre-pubertal mammary fat pad generate a mammary tree with TEBs of increased size and number (Citation93). Milder effects of HGF/SF over-expression in vivo included clover-shaped end-buds (perhaps reflecting a role in primary branching)—all identifying HGF/SF as a mitogen/morphogen which targets the TEB (Citation93).
3H-thymidine labeling experiments have also identified FGF7 as a regulator of body cell proliferation (Citation70). TEBs isolated from virgin mammary glands and grown in matrigel are as sensitive to FGF7 as they are to EGF at its maximum stimulatory dose. Oophorectomized mice, administered with FGF7 systemically, display mammary gland epithelial cell proliferation; however, pretreatment of these mice with estrogen and progesterone leads to the appearance of TEBs. However, this treatment causes the appearance of hyperplastic epithelium in the TEBs (Citation94). FGF7 is a stromally derived ligand for FGFR2 which is also required for body cell proliferation and invasion of TEBs. A genetic mosaic analysis that allows comparison of FGFR2-null and FGFR2-heterozygous cells within the same gland shows that FGFR2-null cells are out-competed by neighboring heterozygous cells (Citation95).
Genetically rescued ErbB2−/− mammary glands transplanted into WT hosts exhibit delayed pubertal ductal penetration, and this is a defect caused by aberrant TEB formation such that the body cell number is diminished (Citation44). Conditional-KO of ErbB3, where mammary ErbB3 expression is suppressed, results in decreased numbers of TEBs in the pubertal mouse (Citation47). Ligands of the ErbB signaling cascade are also implicated in supporting body cell proliferation during pubertal development. EGF mRNA has been detected in the inner body cell layers of the TEB, and Areg-null mice demonstrate the absolute requirement for Areg-mediated ERα-driven proliferation for normal TEB formation (Citation13,Citation84). CITED1, the ER co-regulator, is co-expressed with ERα in the same body cells of the TEB (except in the inner dying cells, see below). The ductal network in pubertal CITED1-null mammary glands is retarded, and they have fewer TEBs, presumably due to a failure of estrogen-mediated Areg expression, Areg-driven luminal epithelial cell proliferation, TEB formation, and body cell proliferation (Citation16).
GH, signaling through locally produced IGF-I, is required for mammary tree expansion very early during puberty. In GH deficiency, TEBs fail to form (reviewed in (Citation96)). It is an expectation that IGF, IGF-R ligands, and IGF-IR are also essential for TEB formation. In-situ hybridization studies of mammary glands from pubertal C57/Bl6 mice detected IGF-I, IGF-II, and IGF-IR mRNA in the TEBs (Citation51); IGF-I was also detected in the surrounding stroma (Citation51). As early as 1992, IGF-I was found to mimic the actions of GH in initiating mammary tree expansion. Using implanted elvax P40 release pellets, delivery of IGF-I protein into the immature mammary glands of hypophysectomized, castrated, and E2-treated male rats led to dose-dependent generation of TEB structures, to an extent similar to that of GH (Citation97). At the receptor level, IGF-IR-null glands have reduced proliferation rate in TEBs (Citation98). With respect to the binding proteins, in-situ hybridization studies have shown IGFBP-1 to 5 transcripts to be present in the pubertal mammary gland (Citation99). Body cells of the TEB express IGFBP-5, whereas IGFBP-3 is only found in cap cells. IGFBP-2 and -4 are detected in the stroma, but IGFBP-2 protein can also be found in the neck of the TEB (Citation99).
Transgenic mice that over-express the A-form of the PR display abnormal TEB structures, and these structures also persist in mice that have undergone pre-pubertal ovariectomy (Citation23).
‘Gata-3 is a transcription factor required to allow estrogen-driven mammary tree growth and is detected in the body cells of TEBs (Citation100, Citation101). TEBs will not develop at all in the absence of GATA3 and this phenotype correlates with the absolute requirement for GATA3 to interpret estrogen signals. Similarly, a complete ERα-KO leads to a complete failure of TEBs to form. TEB-associated GATA-3 and ERα activities are considered in detail in the next section which relates to pubertal mammary stem cells, as both factors play a role in regulating cell-fate determination. The ERα co-regulator CITED1, which co-expresses with ERα in the proliferative fraction of body cells, also displays decreased TEB numbers in KO mice [16].
While the outer cap cell layer of the TEBs express the adhesion molecule P-cadherin, the body cells express E-cadherin (Citation89). Pellets releasing an anti- E-cadherin monoclonal antibody implanted in the pubertal mammary gland lead to a disrupted body cell epithelium and the appearance of epithelial cells floating in the lumen. This outcome was correlated with a drastic decrease in DNA synthesis in the TEB (Citation89).
Body cells—cell death and lumen formation
The second role of the body cells within the TEB of the pubertal mammary gland is in fact to contribute to lumen formation by dying away at the right time. Death of internal layers of body cells is required for ductal lumen formation. Apoptosis is detected in the TEB and is one way in which body cells are lost and so facilitate lumen formation within the growing duct (Citation102). Programmed cell death in the mammary gland is relatively most intense in the TEB, and apoptosis regulators Bcl-x, Bax, and Bcl-2 are all highly expressed in body cells (Citation102). Over-expression of Bcl-2 reduces rates of body cell apoptosis (Citation77). The pro-apoptotic protein Bim is also required for lumen formation in the TEB (Citation103). Mammary glands from 5-week-old Bim−/− mice show TEBs with a filled lumen. Cells within these lumen-filled structures eventually undergo squamous cell differentiation and clear by a caspase-independent mechanism (Citation103). ErbB3 may also be a regulator of lumen formation in this context. A study using embryonic buds from ErbB3−/− mice transplanted into cleared fat pads show a ductal deficit associated with a decrease in the size of TEBs but an increase in the number of TEBs and the area of luminal space (Citation48). Moreover, ErbB3−/− TEBs had increased rates of apoptosis, thought to reflect the aberrant Akt signaling in these structures (Citation48).
Ductal morphogenesis—the process of branching
The process of lateral secondary branching within the pubertal mammary gland receives less attention than ductal morphogenesis. Key regulators direct this epithelial-to-mesenchymal transition (EMT)-like event which requires tight control; sufficient space must remain in the post-pubertal gland to support lobuloalveolar development in the adult and during pregnancy. This is achieved by restricting the number of branching events.
The role of morphogens
Inhibitory morphogens, such as active TGFβ1, act to control the spatial geometry of branching morphogenesis (Citation104). Using computer modeling and an in-vitro 3D branching morphogenesis assay, Nelson et al. have shown that branching events are most likely to occur at sites where there is the lowest local gradient of inhibitory morphogens, and this is linked to the proximity of the nearest neighboring branch site. Paracrine morphogens are required for this process of lateral branching (Citation104). In vivo, transgenic mice over-expressing a dominant-negative TGFβ type 2 receptor (DNIIR) display increased lateral branching (Citation105), again highlighting the restraining role of TGFβ on the space occupied by the invading ductal network in the mammary fat pad. Increased lateral branching in these DNIIR-expressing glands is thought to be due to reduced sensitivity to stromal TGFβ (Citation105). TGFβ has been found to complex with ECM molecules in areas where budding is ‘inhibited’, and in this way it prevents excessive branching and invasion of the fat pad by the advancing ductal network. TGFβ is also reported to regulate epithelial cell ECM synthesis: in those areas where morphogenesis is restricted there are concentrated chondroitin sulfate and type 1 collagen deposits (Citation5).
Hepatocyte growth factor
The cytokine HGF/SF is a positive regulator of branching morphogenesis and drives proliferation and EMT-like events in the mammary gland. In both human and mouse mammary glands, the source of HGF/SF is the fibroblast population of cells (Citation106,Citation107). More specifically, HGF/SF is a stromal-derived paracrine mediator which promotes morphogenesis in vitro and in vivo and signals through the Ras pathway (Citation108,Citation109). The effects of TGFβ signaling are thought to compound the role played by HGF/SF, with both cytokines regulating the limits of branch spacing in the mammary gland.
In transgenic virgin female mice over-expressing a dominant-negative TGFβ type II receptor (DNIIR), HGF/SF mRNA levels were found to be increased along with an increase in epithelial branching, suggesting a role for HGF in opposing the effect of TGFβ (Citation105). Constitutive over-expression of HGF/SF in primary mouse mammary epithelial cells transplanted into cleared mammary fat pads has been shown to stimulate secondary branching and so also contributes to patterning in the mammary gland (Citation93).
In addition, the dose-dependent morphogenic/branching effect of HGF/SF has been established and analyzed in vitro using HMEC (1-7HB2) cells and clonal mouse MECs cultured in 3D collagen matrices (Citation108,Citation110,Citation111). Conditioned medium from fibroblasts (which contains HGF/SF) was also shown to stimulate branching morphogenesis in murine TAC-2 cells and in human 1-7HB2 cells. The ability of HGF/SF to induce lumen formation in branches from TAC-2 cells is exacerbated upon the addition of hydrocortisone (Citation110).
In experiments carried out on 1-7HB2 cells, Berdichevsky et al. highlighted that α2β1-integrin not only modulates the strength or weakness of cell–cell contacts but is also required for growth of HB2 cells on collagen. Additionally, they suggest that morphogenesis requires diminished activity of α3β1, as addition of an antibody against P1β5 (an α3 subunit) to control medium stimulated branching in a similar manner to that seen upon addition of HGF/SF itself. Later work with HB2 cells showed that the cellular response to HGF/SF was abrogated upon addition of an ‘activating’ β1-integrin antibody or ‘non-blocking’ α2 antibodies. The effect of these additions was to strengthen the integrin–collagen interaction such that branching was significantly impaired (Citation112). Using an in-vitro cell culture model of ductal morphogenesis, the Streuli group have also shown that HGF and β1-integrins co-operate during HGF-induced tubulogenesis (Citation88).
Retroviral delivery methods have also been employed to transduce primary mouse mammary epithelial cells with resultant constitutive over-expression of HGF/SF (Citation93). Based on such studies, Yant et al. conclude that HGF/SF is a mitogen, morphogen, and a motogen and show that over-expression of HGF leads to an accelerated growth rate and increased branching in collagen gels.
The role of mitogens
Signal from the PR is also required for ductal lateral branching in the pubertal mammary gland (Citation113). PR−/− epithelium transplanted into WT fat pads gave rise to a ductal network that was not as extensive as WT (Citation27). The presence of PR in the epithelium surrounding the transplant allows for normal branch development, but stromal PR does not (Citation27). Supporting these findings is the observation that treatment of mammary glands from pre-pubertal mice with exogenous progesterone leads to increased branch numbers. Interestingly, cells of the secondary ducts are also much more responsive to the mitogenic effects of progesterone than estrogen, and PR within these pubertal mammary glands is relatively highly localized to branch point sites (Citation3). Transgenic mice which over-express the A-isoform of the PR show increased lateral branching (Citation23). In contrast, mice deficient in co-activators, SRC-1 or SRC-3, display reduced lateral branching at puberty. As previously indicated, these co-activators are required for PR (and ER) function in the pubertal mammary gland, with SRC-3 deemed the principal PR activator (Citation14). Other possible mediators of paracrine PR-induced lateral branching in the mammary gland are members of the Wnt family. Transplantation studies have shown that Wnt1 and Wnt4 can alleviate a mammary PR deficit and allow ductal lateral branching to occur (Citation113).
Pituitary hormones—Prl and GH
Pubertal mammary glands from PrlR-null females have very large ducts existing within a reduced ductal network with very limited numbers of branch points (Citation37). A similar phenotype was observed in a second PrlR-KO mouse which was characterized by diminished frequency of branch points compared to WT; this situation lasted until adulthood (Citation36). Recently, a Stat5a-null mouse has been generated which has defective lateral and secondary branching in the pubertal gland (Citation38). And this year, it has been shown that Prl-induced activation of Stat5 leads to the production of a unique Akt1 mammary-specific transcript which is growth-factor regulated and promotes survival of MECs (Citation114). Mammary glands from GHR-null mice show severely stunted ductal growth and reduced lateral branching at puberty. This phenotype is not maintained in the same way as the PrlR-KO mice, and the fat pad is seen to be occupied by a functional ductal network by 15 weeks (Citation32). Richards et al. showed a branching defect in the mammary glands of adult IGF-Im/m mutant mice, with ducts extending to the extremities of the fat pad but the number of branching points being reduced by 50%. These mice have 30% of the normal levels of mammary and hepatic IGF-I; the IGF-Im or ‘midi’ allele was produced during efforts to create an IGF-I-null mouse (Citation115).
ErbBs, ER, and CITED1
Paracrine EGFR activation, inducing pubertal mammary branching morphogenesis, is a necessary response to ERα signaling (reviewed in (Citation4)). Tyrosine phosphorylation of EGFR and ErbB2 is enhanced in extracts from Balb/c mouse mammary gland upon treatment with EGF or E2 (Citation39). Secondary branching occurs in the mammary glands of ER-KO mice when treated with exogenous TGFα or heregulin (both EGFR ligands) (Citation116). Thus, EGFR ligands can in fact overcome defects in estrogen signaling and restore the branching effect. Over-expression of Areg in MECs transplanted into cleared fat pads leads to the generation of hyperplastic ducts with increased lateral branching (Citation41). In pubertal C57/Bl6 mouse mammary glands, Areg is localized to stromal cells, myoepithelial cells, and luminal epithelial cells of branching ducts (Citation85). The ER co-regulator CITED1, which is required to drive Areg expression, is also required for normal lateral branching in the pubertal mammary gland. CITED1-null mice display dilated ductal structures which do not branch in a spatially aware and regulated manner (Citation16). TGFβ-induced Smad4-modulated transcriptional activity is also disturbed in CITED1-null mammary glands, and this may contribute to the fact that branching architecture is not informed by the geometry of neighboring ducts and branches (Citation15,Citation16,Citation117). This is thought to confirm a previously proposed model of co-ordinated and reciprocal estrogen/TGFβ drive in the pubertal mammary gland such that estrogen-driven proliferation must be met with a TGFβ-driven constraint on stromal invasion (Citation16,Citation82,Citation118). Mammary ErbB2 is required for elongation of ducts, such that ErbB2-deficient glands display a stunted branched epithelium at puberty (Citation119). Conditional ErbB3-KO mice also show decreased numbers of branches in the pubertal mammary gland (Citation47). Other studies also implicate matrix metalloproteinase (MMP)-2 and MMP-3 in branching morphogenesis; MMP-2 represses lateral branching during puberty whereas MMP-3 promotes secondary and tertiary lateral branching of ducts at this time (Citation120).
Thus, signals from growth factors, cytokines, and matrix components, orchestrated by ovarian and pituitary hormones, are tightly co-ordinated, such that the bordering mammary mesenchyme is met by the invading epithelium and this is counterbalanced by the requirement for free space which will go on to support pregnancy-induced alveologenesis.
Stem cells in the pubertal mammary gland
The existence of mammary stem cells has been predicted since the late 1950s: in early experiments limiting cell dilution transplantation was used to generate mammary outgrowths from a ‘clonal’ precursor (Citation121). Limiting dilution/transplantation studies further described three distinct multipotent progenitors (duct-limited, lobule-limited, and fully competent progenitor cells) within a clonally derived epithelial population, and on this basis the authors hypothesized the existence of a master stem cell from which these multipotent progenitor cells all originate (Citation122–124). A single stem cell can indeed account for the renewal of a complete mammary epithelium over several transplant generations (Citation123,Citation125–127). For instance, Zeps et al. describe the existence of proliferative units within the mammary epithelium which consist of a single stem cell surrounded by its progeny (Citation128). These units are long-lived and have no requirement for estrogen in order to replicate. Cell surface marker signatures can aid in enriching for mammary stem cells, and it has been shown that a functional mammary gland can be generated from a single cell with either a CD24 + CD49fhigh (α6-integrin) or a CD24 + CD29high (β1-integrin) signature (Citation127,Citation129).
The focus of this review is mammary gland development during the pubertal growth phase. We therefore concentrate on presenting the evidence for the important role played by stem cells in pubertal development. Mammary stem cells in the pubertal gland primarily reside in the TEB but are also distributed throughout the ductal network (Citation77). The outer layer of cap cells acts as precursors for myoepithelial cells, while the body cells will differentiate into luminal epithelial cells (Citation130,Citation131). Exciting research from the past 10 years suggests the role of key modulators of the activity of these progenitor cells at puberty.
Pea3 Ets
Mouse Pea3 Ets was the first member of the Ets family of transcription factors to be described. The family consists of more than 30 genes encoding proteins which are generally involved in transcriptional activation of target promoters, induced by mitogen-activated pathways (Citation132). Aberrant Ets transcription has been associated with mammary oncogenesis, and Pea3 Ets transcripts are found at elevated levels in 93% of human breast tumors over-expressing ErbB2 (Her2/Neu) (Citation133). In the mammary gland, Pea3 transcripts peak at times of ductal elongation and branching (Citation132). Pea3 is detected in all cells of the outer cap cell layer, also in a small number of body cells within the TEB at puberty, and in the mature myoepithelial cells in mature virgin glands. An in-vitro strategy for culturing of primary MECs as non-adherent mammospheres (a technique which enriches for stem cells) showed the preferential expression of Pea3 in ‘undifferentiated’ mammospheres rather than in subsequently differentiated cells (Citation132). Whole mounts from Pea3-null mice show that loss of Pea3 leads to a more than doubling in number of TEB structures within the pubertal mammary gland (5 weeks) and also a failure of these structures to regress after week 8. This coincides with an increase in the number of proliferating progenitor cells. Reciprocal transplant studies determined that these effects are intrinsic to the mammary epithelium. All signs point to a role for Pea3 in regulating proliferation and determining the fate of undifferentiated cells within the pubertal mammary gland.
Mediator
Mediator is a transcriptional co-activator complex that ‘bridges the gap’ between transcriptional regulators and RNA pol II. It is comprised of many subunits, one of which is the Med1 subunit that contains LxxLL motifs required for docking with nuclear receptors. Transgenic mice expressing Med1 with mutated LxxLL motifs display a severe puberty-associated mammary gland developmental defect (Citation134). Paraffin-embedded sections from 8-week-old mice show that Med1KI/KI (mutant Med1 knock-in) mammary glands contain a large population of CK14+(basal) and CK18+ (luminal) double-positive cells whereas WT mammary glands have defined sub-populations of either CK14+ or CK18+cells. Mutant glands are also enriched for a CD29low CD24high cell surface marker signature and a CD24+Sca1− (stem cell antigen 1) signature, both of which are signals for luminal progenitor cells (Citation127,Citation134). Not only does Mediator seem to regulate progenitor cell selection but it also appears to ‘co-operate’ with ERα signaling (both Med1 and ERα are expressed in luminal epithelial cells: the mammary glands of Med1KI/KI mice are non-responsive to estrogen-induced pubertal growth (Citation134)).
GATA3 and CDK4/6 inhibitor p18Ink4c
The CDK4/6 inhibitor p18Ink4c (known as p18) has also emerged as a regulator of luminal cell differentiation in the pubertal mammary gland (Citation135). Epithelia from mammary glands of 8-week-old p18−/− mice harbor hyper-proliferative luminal cells, an effect that persists in the post-pubertal mammary gland. Pubertal p18−/− mammary glands have an expanded CD29lowCD24high cell population and an expanded label-retaining cell (LRC) population, 83% of which stain for CK8. This pubertal luminal cell expansion in p18−/− mice leads to a high incidence (87%) of spontaneous, ERα-positive luminal mammary tumors, most of which are non-invasive DCIS. This pubertal luminal cell expansion in p18−/− mice leads to a high incidence (87%) of spontaneous, ERα-positive luminal mammary tumors, most of which are non-invasive DCIS (ductal carcinoma in situ).
The link with GATA3
GATA3 is a member of the zinc-finger family of transcription factors and is preferentially expressed in pubertal mouse MECs (Citation100,Citation136). Mammary glands from GATA3-deficient mice phenocopy ERα-null glands in that TEBs do not form, and ductal morphogenesis fails (Citation100,Citation101). ERα activation induces GATA3 transcription in breast cancer cell lines (Citation137). GATA3 regulates luminal cell differentiation, and expression analysis of 295 human breast cancer samples shows elevated levels of GATA3 transcript in luminal tumors (Citation138,Citation139). Up to 80% of breast tumors are of luminal origin and are ERα-positive, whereas basal tumors, which have virtually undetectable levels of GATA3, are generally ERα-negative (Citation140). The p18 gene promoter contains several GATA3 binding sites: GATA3 binds the p18 promoter and negatively regulates its transcription (Citation139). In MCF10A cells, over-expression of GATA3 leads to diminished p18 mRNA levels and increased cell proliferation (Citation139). In mammary luminal epithelium, GATA3 expression inversely correlates with p18 expression; this relationship is also found in human luminal A breast tumors, and high GATA3/low p18 expression levels predict good patient outcome (Citation138,Citation139).
The PolycombGroup protein Bmi1
PolycombGroup proteins such as Bmi1 are epigenetic regulators of gene transcription, and Bmi1 is a gene silencer known to be required for supporting cell fate decisions and the maintenance of self-renewal and proliferative capacity (Citation141). Bmi1 is over-expressed in invasive ductal breast cancer, and mice with a targeted deletion of Bmi1 are runted and are characterized by a progressive loss of neural and hematopoietic stem cells (Citation142–144). Bmi1 is expressed in all cells of the mammary gland but its expression is especially high in luminal cells (Citation145). There is a severe growth defect in mammary glands deficient in Bmi1 at 5 weeks, and the population and activity of mammary stem cells is reduced; this can be rescued either by co-deletion of the Ink4a/Arf locus (repression of the Ink4a/Arf locus depends on Bmi1 in mammary epithelium) or by pregnancy (Citation145). TEBs are effectively absent in the mammary glands of Bmi1-deficient mice, and, as such, the epithelial network remains primitive due to an inability to invade the fat pad. Bmi1−/− MECs injected into cleared fat pads and transplanted Bmi1−/− tissue both fail to induce growth in recipients (Citation145). The authors suggest an essential role for Bmi1 protein in postnatal mammary development.
Cell surface markers and the role of estrogen
Failure of mammary tree expansion in ERα-KO mice shows that epithelial proliferation and morphogenesis is estrogen-dependent, and transplantation studies show the potential of ERα-positive epithelial cells to signal to neighboring progenitor cells in the mammary gland to induce their proliferation (Citation13). A number of prominent research groups have proposed that undifferentiated ERα-positive (luminal) epithelial cells may arise (possibly via asymmetric division) from an ERα-negative (basal) progenitor cell; growth and division of neighboring ERα-negative cells will then be induced by local paracrine factors secreted by the ERα-positive cells (Citation19,Citation146). The CD24+CD29high population of cells is a basal progenitor population and is usually ERα-negative (Citation147). In fact, limiting dilution and transplantation studies have shown these ERα-negative basal cells (CD24low) to have greater stem cell activity than ERα-positive luminal cells (Citation148).
Responsiveness of mammary stem cells to steroid hormones
Neither human nor mouse mammary stem cells are ER- or PR-positive, but both respond to estrogen and progesterone signaling and are presumably regulated by both of these steroid hormones in a paracrine fashion through the action of locally produced growth factors or cytokines (see above) (Citation129,Citation147,Citation149). In vivo, outgrowth potential and mammary stem cell numbers are diminished upon ovariectomy (Citation149). Conversely, mice exposed to endogenous estrogen and progesterone display elevated mammary stem cell activity compared to controls. The number of repopulating cells in the mammary gland is decreased 4.3-fold in transplants of limiting numbers of mammary stem cell-enriched (CD29hiCD24+) cells from ovariectomized mice into cleared fat pads of recipients (Citation149). Treatment with the aromatase inhibitor, letrozole, for 3 weeks diminishes the pool of mammary stem cells. To further confirm the importance of estrogen signaling for mammary stem cells, Asselin-Labat et al. went on to show that the mammary glands of aromatase knock-out mice (ArKO; display reduced endogenous estrogen biosynthesis) only develop a primitive ductal network, have a diminished mammary stem cell-enriched population of cells, and have decreased numbers of mammary CD24+ cells (Citation149).
Luteal diestrus in the mouse, the period of highest progesterone production/secretion, coincides with a 14-fold increase in the number of mammary repopulating units relative to a quiescent phase (Citation150). Limiting dilution assays performed by transplanting total mammary epithelial (enriched) cells from diestrus or estrus glands into the cleared fat pads of 3-week-old pre-pubertal recipients show an amplification of stem cell-enriched CD49fhi cells from the diestrus population, thus suggesting a role for progesterone in expanding the mammary stem cell pool (Citation150). There is also an increase in solid colony formation in a colony-forming cell assay with glands from ovariectomized mice treated with 17-β-estradiol and progesterone. Solid colonies (as opposed to acinar colonies) arise from cells of basal/myoepithelial origin. And so, it is thought that a progesterone-enriched environment induces expansion of the population progenitors of a basal lineage. The mechanism of progesterone action and its target genes, in this regard, is unclear, but the authors suggest that luminal cells provide RANKL (receptor activator of nuclear factor-kappaB ligand) and Wnt4 signals to basal cells, and this is driven by progesterone (Citation150).
ZO1 and α-catenin
Tritiated-thymidine autoradiography, fluorescent tracker dye, and/or BrdU labeling of individual cells has facilitated the identification and successful localization of long-lived label-retaining mammary epithelial cells (LRCs) or stem cells, in vivo (Citation128,Citation151). Pulse-labeling of primary mammary epithelial cells transferred into cleared fat pads of syngenic juvenile mammary glands are maintained for 5 and 8 weeks to allow TEB and duct formation. LRCs are classed as stem cells, and their progeny are called transitional cells. Transitional cells are found grouped together in clusters at the tips of the TEB and in areas of the subtending duct (Citation151). Within transitional unit structures, there are cell–cell contacts between LRCs, and expression of the adhesion molecules zonula occludens 1 (ZO1) and α-catenin were detected in >75% of LRCs in the transitional units (TUs) (Citation151). The role of these adhesion molecules may be to allow cross-talk with cells outside of the transitional unit.
β1-Integrin in basal epithelial cells
The ECM receptor, β1-integrin, has also been shown to play a role in the maintenance of stem cell character within cells in the developing mammary gland. Mice lacking β1-integrin from the basal epithelium (K5Cre:itgb1F/F) do not survive until puberty, and so rudimentary glands from mutant pre-pubertal mice grafted into the cleared fat pads of pre-pubertal nude Balb/c mice were used to assess regenerative potential (Citation152). Ductal morphogenesis progressed more slowly in the mutant glands which also showed decreased regenerative potential and a disorganized ductal branching pattern with few side-branches (Citation152). And 89% of secondary grafts from mutant mice failed to develop beyond one or two small buds. Flow cytometric analysis confirms a reduction in the number of potential stem cells in the K5Cre:itgb1F/F epithelium, with the number of CD25+/β1-high cells very significantly less than WT (Citation152).
Areg
CDβgeo (COMMA-D-β-geo) cells are immortalized cells derived from mid-pregnant Balb/c mouse mammary tissue, and these cells have progenitor cell characteristics (Citation153,Citation154). A recent study by Booth et al. (Citation155) suggested a role for Areg in the self-renewal ability of CDβgeo cells in vitro. They show that Areg is required for the initiation of anchorage-independent mammosphere formation by CDβgeo cells and that these mammospheres actually release Areg into the media (Citation155).
Conclusion
The pubertal mouse mammary gland is an ideal experimental model system: it allows ready analysis of branching morphogenesis (development) of this internally branching epithelium, which is both of interest in itself but also has relevance/potential as a source of molecular therapeutic targets for treating breast cancer. It is also readily genetically manipulated and lends itself to cell–cell interaction studies by using transplantation technologies. Systemic estrogen and GH would seem the master drivers of pubertal mammary gland development, but many questions still remain concerning what hierarchy exists for other signaling nodes and for the integration of these signals by downstream effectors. A constant struggle to balance the interpretation of signals from developmental cues versus mediators of cancer progression exposes the mammary gland to the risk of disease. For this reason, the concept of functional excess and of redundant roles for differing regulators allows potentially aberrant development to be rapidly rescued. The pubertal mammary gland acts as the competent cellular precursor for the development in pregnancy of the gland in its final differentiation/functional state (the lactating gland), such that the leading functional requirement at puberty is to prepare a precursor suitable for eventual proliferation and fate decisions during pregnancy. However, post-puberty, successive rounds of estrus exert a pro-tumorigenic effect on the mammary gland, a condition which is dampened by parity. Some KO mice with diminished development of the mammary tree still have a sufficient base to generate a functional gland during pregnancy. And this begs the question to what degree does the pubertal mammary gland act as a repository of pluripotent stem cells because the post-pubertal gland which does not go on to support a pregnancy contains the highest levels of undifferentiated cells which must correlate with the highest level of cancer risk (Citation156). A major step forward will be to elucidate precisely how pathways required for development are maintained in tumor formation and which regulators are indispensible for this recapitulation.
Acknowledgements
There is a very large body of primary and review literature in this area. Thus all authors could not be cited. We apologize to those who we have not referenced.
Declaration of interest: Studies carried out in the authors’ laboratory were funded by Science Foundation Ireland, the Health Research Board, Ireland, and the European Union (FP5-RTN). The authors declare no conflicts of interest.
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