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

Seminars in Cell & Developmental Biology 23 (2012) 574–582

Contents lists available at SciVerse ScienceDirect

Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

Review

In the beginning: The establishment of the mammary lineage during embryogenesis Beatrice A. Howard ∗ Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK

a r t i c l e

i n f o

Article history: Available online 15 March 2012 Keywords: Embryonic mammary morphogenesis Foetal breast development Mesenchymal–epithelial communication Mammary cell fate Mammary lineage establishment Mammary progenitor biology

a b s t r a c t The mammary primordium is comprised of an aggregate of immature, undifferentiated mammary epithelial cells and its associated mammary mesenchyme, a specialised tissue which harbours mammaryinductive capacity. The mammary primordium forms during embryogenesis as a result of inductive interactions between its two component tissues, the mammary mesenchyme and epithelium. These two tissues constitute a signalling centre that directs the formation of the mammary gland through a series of reciprocal mesenchymal–epithelial interactions. A rudimentary mammary ductal tree and stroma is formed prior to birth as a result of these interactions. The subsequent mammary outgrowths that arise upon hormonal stimulation during puberty originate from this rudimentary tissue. The initial appearance of the embryonic mammary primordium during embryogenesis represents the earliest morphological evidence of commitment to the mammary lineage. Classic tissue recombination studies of mouse mammary primordial cells have demonstrated that the epithelial cells are already functionally determined as mammary at the embryonic mammary bud stage. Recent studies have determined the molecular identity of the embryonic mammary cells by transcriptomic profiling and these have provided new insights into signalling components that mediate early embryonic mammary inductive signalling and lineage commitment. This review highlights what is currently known about the morphogenesis, function, and behaviour of embryonic mammary cells and examine current knowledge of the genetics underlying mammary cell fate and establishment of the mammary lineage during embryogenesis. © 2012 Published by Elsevier Ltd.

Contents 1. 2.

3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview of embryonic mouse mammary morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mammary line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mammary placode and bud stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Later invasive embryonic mammary stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classic functional studies reveal insights into developmental processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic factors required for normal mammary formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic studies of mouse models have identified factors that can induce ectopic mammary formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptomic profiling of the embryonic mammary cell populations reveal novel insights into pathways and network components that are signalling as the mammary lineage is established . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

574 575 575 575 576 576 579 579 580 580 580 580

1. Introduction Abbreviations: EMT, epithelial–mesenchymal transition; ER, estrogen receptor; K14, Keratin 14; Krt, keratin; SMA, smooth muscle actin; TnC, Tenascin C. ∗ Tel.: +44 2071535177; fax: +44 2071535430. E-mail address: [email protected] 1084-9521/$ – see front matter © 2012 Published by Elsevier Ltd. doi:10.1016/j.semcdb.2012.03.011

Mammary glands are one of the defining features of the Class Mammalia, meaning literally “of the breast”. The term mammal was first used by Carl Linnaeus in his tenth edition of Systema Naturae who recognised the mammary gland as an important distinguishing

B.A. Howard / Seminars in Cell & Developmental Biology 23 (2012) 574–582

feature for the classification of animal types [1]. The fully differentiated mammary gland functions to produce and distribute milk and other substances to provide nutrition and immune support to neonates and young offspring. The functional potential of the mammary gland is achieved only after pregnancy and parturition but the initial formation of the gland occurs much earlier in the timescale of months in rodents and decades in humans. The mammary organ forms during embryogenesis and is first evident at E11.5 or Theiler stage 19 in the mouse [2]. In humans, by twelve weeks of gestation, the breast bud has formed [3]. A few notable studies using embryonic rabbit, rat, and human mammary tissues have been published and will be briefly discussed here, but the majority of developmental studies of the embryonic mammary gland have been performed in the mouse and thus form the basis of most data discussed in this review. 2. Overview of embryonic mouse mammary morphogenesis 2.1. Mammary line Raised epidermal ridges known as “mammary lines” have been reported in rabbit, rat and human embryos and extend from the axilla to the groin along the lateral wall of the trunk [4]. The mammary line is obvious upon whole-mount analysis of the rabbit embryo and appears to fragment as the individual primordium form (Fig. 1) and [5]. The mammary lines span between and extend just beyond the limb buds and encompass the regions where the mammary primordium will subsequently form. Analysis of the rabbit mammary line using scanning electron micrography (SEM) detected cells with filopodia and lamellopodia, features characteristic of motile cells [5]. Analysis of mouse embryos with SEM failed to detect similar morphological structures in equivalent developmental stages [6]. The earliest stages of mouse mammary organ formation are characterised by the appearance of a pseudostratified epithelium which is detected upon histological sectioning or using confocal analysis (Fig. 2) of the presumptive mammary forming region between E10.5 and E11.0, stages at which no obvious localised thickenings on the ventral flank are detected [7,8].

575

Studies using reporter models and in situ hybridisation with probes, such as Wnt10b, have detected localised expression along an analogous region to the rabbit mammary line so that a molecular mammary line exists in the mouse [9]. The Wnt reporter mouse model, BAT-GAL which detects Wnt expression, and the Src homology 2 domain-containing inositol-5-phosphatase-reporter model, s-SHIP-GFP, which detects s-SHIP expression, both show expression along the mouse mammary line at sites where the mammary primordia will subsequently form from E10.5, suggesting that inductive events occur at a prior stage during mouse embryogenesis [10,11]. 2.2. Mammary placode and bud stages An elliptically shaped structure termed the mammary placode, comprised of an aggregate of epithelial cells, is the first morphological stage that is apparent upon light microscopy of unstained mouse embryos at a stage between E11.0 and E11.5 (Fig. 2B). This stage is followed by an early bud stage in which the primordium assumes a spherical shape (Fig. 2C). The mammary bud then increases in size until E14.5 (Fig. 2C–E). Results from calculating mitotic indices of the mammary-forming regions in both mouse and rabbit suggested that very little proliferation occurs at early bud stages of mammary formation and localised cell movements therefore are thought to largely contribute to the observed increase in cell numbers [8,12]. We have also observed a limited amount of cell proliferation within the embryonic mammary epithelium at E11.0–E14.5 stages when assessed using Ki67 staining (Kogata, Wansbury, and Howard, unpublished). Male mouse mammary buds undergo an androgen-mediated destruction when the mammary mesenchyme condenses around the neck of the mammary bud, which occurs at E14.5 [13–17]. This event is a notable distinction between rodent and human mammary development where the mammary bud is retained and proceeds to form a small ductal outgrowth connected to the nipple. Some mouse strains and genetically modified mouse models retain the nipple in males, but how mammary bud destruction is avoided is not understood. Benign breast anomalies, such as Gynecomastia, an enlargement of the glandular component, may occur in males.

Fig. 1. Early stages of embryonic mammary development in rabbit. (A) The mammary line, a raised ridge of epidermal cells is visible between the fore and hind limbs spanning the regions where the five pairs of mammary primordia will subsequently form in a rabbit stage E14.0 embryo. (B) The mammary line appears to fragment so that individual mammary primordium form at sites along the former line in a rabbit stage E14.5 embryo. FL, forelimb; HL, hindlimb, MP2, mammary primordium 2.

576

B.A. Howard / Seminars in Cell & Developmental Biology 23 (2012) 574–582

Male breast tissue is also susceptible to malignant transformation and, although at low incidence, male breast cancer does occur. A second mesenchymal tissue, the fat pad precursor forms at a site distal to the mammary primordium and becomes mature adipose tissue only after birth. Between stages E13.5 and E14.5, preadipocytes appear at the site where the fat pad will form [18]. A requirement of Lef1 for fat pad formation was reported by Boras-Granic et al. [19]. Poorly developed fat pads are associated with the single mammary primordial pair that form in Fgf10−/− embryos [20]. Many of the Erbb signalling network component are expressed at the fat pad precursor site and are poised to mediate paracrine signals between the future fat pad and embryonic mammary epithelium and/or mammary mesenchyme [21]. Fatty substances begin to accumulate at E16.0 in the fat pad precursor and increase in concentration until birth; these are converted into adipose tissue within two days of birth [18]. Mice that lack white adipose tissue form fewer ductal branches when compared to wildtype E18.0 stage mammary primordial, suggesting a key role for adipocytes in the regulation of mesenchymal–epithelial interactions required for normal mammary gland development as well as comprising a profound component of the mammary microenvironment [22]. 2.3. Later invasive embryonic mammary stages After E14.5, the mammary bud increases in size and a neck forms at the top of the mammary primordium. The cell types within the neck of the bud are distinct from those within the bud proper [23]. At E15.5, the neck of the bud appears to invaginate and push the bud proper deeper into the underlying mesenchyme (Fig. 3A and B). The basement membrane remains intact [24]. Remodelling and downregulation of cell adhesion components occurs within the mammary bud cells while those within the surface epithelium remain intact [24]. The first indication of branching is apparent as a cleft at the base of the bud which occurs between E15.5 and E16.5 (Fig. 3B). The formation of the nipple sheath, an umbrellashaped epithelial thickening at the surface of the primordium, occurs between E15.5 and E17.5 stages (Fig. 3D and E). As further branching of the primordial epithelium occurs, cell adhesion components remain downregulated in the most distal regions of E17.0 stage epithelial outgrowths but are restored in the rest of the nascent ductal tree [24]. By E17.5 lumen formation within the ductal branches begins (Fig. 3E) and this process is completed prior to birth [23]. Once the small ductal tree comprised of about a dozen small branches has been established, the mammary gland appears to be fairly quiescent from E18.5 stage until puberty (Fig. 3F). Mammary mesenchymal cells surrounding mammary epithelium express ER␣ from E12.5 onwards. No ER␣ stain is observed surrounding the mammary epithelial cells that have invaded into the fat pad precursor tissue (Fig. 3D). Both TnC and ER␣ mark the mammary mesenchyme and distinguish it from the precursor to the fat pad which does not express either marker (Fig. 3D and E) [18]. We have not observed ER␣ staining of the embryonic mammary epithelial cells between E12.0 and E17.5 (Howard, unpublished data). Within three weeks of birth, postnatal mammary epithelial cells, and adjacent stromal cells, strongly express ER␣ [25]. Precisely when and by what mechanism expression of ER is acquired by mammary epithelial cells is not yet clear. Fig. 2. Early stages of embryonic mouse mammary development. (A) The future site of a mammary placode (bracketed), at E11.0, prior to stratification. (B) Mammary placode (bracketed) stage at E11.5. (C) Mammary bud stage at E12.5. (D) Mammary bud stage at E13.5. (E) Mammary bud stage at E14.5. A white dashed line denotes the epithelial–mesenchymal boundary. MM, mammary mesenchyme. Scale bar: 50 ␮m.

3. Classic functional studies reveal insights into developmental processes Mammary glands are derivatives of the embryonic ectoderm. Interactions between the epithelium and mesenchyme occur in a

B.A. Howard / Seminars in Cell & Developmental Biology 23 (2012) 574–582

577

Fig. 3. Later stages of embryonic mammary development. (A) Histological section of E15.5 mammary primordium stained with p63. A neck (N) is present at the top of the bud, thought to arise as a result of invagination of the epithelium. (B) E16.5 mammary primordium whole-mount stained with Tenascin C (TnC) which marks the mammary mesenchyme. The first sign of branching is apparent at the small cleft (arrow). (C) E17.0 mammary primordium stained with CD24 showing the ductal system produced from further branching. (D) E17.5 mammary primordium stained with ER␣ showing different mesenchymal cells associated with the ductal system; mammary mesenchymal (MM) cells expresses ER␣ whilst the fat pad precursor (FPP) cells do not. (E) H&E staining of E18.5 mammary primordium showing nipple sheath (NS) and histological features of the mammary mesenchyme (MM) and fat pad precursor (FPP). A small lumen (arrow) is evident; a continuous lumen within the ductal system forms prior to birth. (F) H&E staining of three week postnatal mammary gland. A rudimentary ductal tree is embedded in a fat pad comprised of mature adipocytes as well as a variety of other cell types including fibroblasts, blood vessels and nerves. Scale bar: 50 ␮m.

reciprocal manner and have been reviewed in detail previously by Parmar and Cunha [26], but some notable features are highlighted here. Functional studies of the mammary mesenchyme have demonstrated its profound role in directing and maintaining normal mammary tissue differentiation and three-dimensional architecture. Initial results from a series of tissue recombination experiments suggested that the mammary mesenchyme induces mammary primordial formation [26,27]. Cunha et al. definitively demonstrated that mammary mesenchyme can induce mammary development from simple dorsal or ventral epithelium [28]. This

inductive function is conserved as recombinants of mammary mesenchymal and epithelial tissues isolated from different species, and even different classes, can elicit mammary development, with the remarkable outcome of rabbit mammary mesenchyme having the capacity to induce formation of mammary epithelium from chick epithelium [29]. The precursor to the fat pad, is a condensed mesenchymal tissue distinct from the mammary mesenchyme in both its composition and functional capacity [18]. Processes regulated by the primary mammary mesenchyme include promoting mammary

578

B.A. Howard / Seminars in Cell & Developmental Biology 23 (2012) 574–582

differentiation and lineage commitment, organotypic threedimensional structure, invasion, initial clefting and branching. The early fat pad precursor supports outgrowth patterns typical of embryonic mammary epithelia. Both E14.0 and E17.0 stage fat pad precursor tissues support organotypic mammary growth of E17.0 stage embryonic mammary epithelium resulting in the formation of a normal mammary ductal tree when tissue recombinants are syngrafted under the kidney capsule for three weeks [18]. E14.0 stage embryonic fat pad precursor tissue can also support organotypic development of many other types of embryonic epithelia including those isolated from salivary gland, stomach, intestine, colon, pituitary gland, and pancreas, when tissue recombinants are syngrafted under the kidney capsule [30]. These tissue recombinants produced outgrowths typical of their innate epithelial organ-type and not outgrowth patterns reflecting a mammary organotypic developmental programme [30]. These studies also suggested that the fat pad precursor lacks mammary inductive potential. The supportive capacity of the fat pad precursor to promote organotypic growth of non-mammary epithelia diminishes in later E17.0–E18.0 stages and is lost completely in postnatal stages. Mature mammary epithelium retains the ability to respond to signals arising from the mammary mesenchyme. Tissue recombinants composed of stage E14.0 through E17.0 mammary mesenchyme and E17.0 mammary epithelium resulted in the formation of highly branched solid nodular masses [18]. Hence, the fat pad precursor tissue is thought to mediate the formation of the normal branching structure of the mammary epithelium. Mammary mesenchyme isolated from E12.0 stage primordia can restore some features of differentiated tissue to mouse mammary tumours when grown together for two weeks in culture, separated by a Millipore filter. This suggests that mammary mesenchyme secretes soluble differentiation-promoting factors [31]. Mammary mesenchyme can accelerate the formation of mammary tumours locally when transplanted into mammary glands in mice strains harbouring the mammary tumour virus whilst no tumours formed in similarly treated mammary glands in non-virus-bearing strains [32]. Other studies of transplants of mammary mesenchyme into normal postnatal mammary glands have observed that the epithelium adjacent to the implanted mesenchyme forms structures resembling embryonic branches [33]. Coordination of developmental signals is likely to be required to attain the various roles that both types of embryonic mammary mesenchymal tissues regulate in vivo. Paracrine signals arising from the mammary mesenchyme and fat pad precursor may prove to be a considerable resource for exploring factors that have either the capacity to direct and influence mammary epithelial cell behaviour to promote stable differentiated mammary phenotypes, normal types of organotypic outgrowth and provide clues to the microenvironmental factors influencing early mammary progenitor cells. In addition, the mammary mesenchyme may contain factors that promote cell behaviours associated with invasion, and hyperplastic and nodular growth patterns of pre-neoplastic tissues. Mesenchymal cells have a tremendous ability to influence the behaviour of associated embryonic epithelial cells and provide a microenvironment that is distinct from that provided by the postnatal mammary stroma. Previous studies of embryonic mouse mammary epithelium indicated that by mid-gestation, these cells are determined to a mammary cell fate. When E13.0 mammary primordial epithelium and salivary mesenchymal tissue are co-cultured under the kidney capsule, they produce a ductal tree that has morphological features of salivary ducts, but is capable of secreting milk when the host is treated with lactogenic hormones [34]. These results suggest that by E13.0, mammary epithelium has been stably determined as mammary. When E14.5 mammary epithelium is recombined with prostate mesenchymal tissues and syngrafted under the

kidney capsule for four weeks, only a partial and incomplete response is achieved with respect to altering lineage specification, since both prostatic and mammary markers are expressed by the epithelium produced from the tissue recombinant indicative of only a partial transdifferentiation [35]. Precisely how plastic the embryonic mammary cells are when removed from their native microenvironment remains to be explored. Mammary primordia from different embryonic stages have been implanted into fat pads cleared of mammary epithelium permitting assessment of their repopulating capacity, which provides a measure of stem cell activity within a population. Mammary primordia from E12.5 onwards have been analysed and found to be capable of forming extensive arborised ducts and has been routinely used to evaluate postnatal mammary phenotypes in mice with genetic mutations which exhibit embryonic lethal phenotypes [36]. Mammary epithelium, isolated from mammary primordia using enzymatic tissue separation and independent of mammary mesenchyme, spanning E13.0 and E17.0 stages, can give rise to all mammary epithelial cell types when evaluated in this assay, and can even induce the formation of milk-secreting mammary alveoli in fat pads of pregnant females [37]. These observations suggest that tissue-specific stem cells reside within the primordial mammary epithelium by mid-gestation and that these cells have the ability to respond to the postnatal microenvironment in an appropriate fashion. Recently, Src homology 2 domain-containing inositol-5phosphatase (s-SHIP) promoter expression has been shown to mark activated stem cells in postnatal mammary tissue [38]. It is, therefore, notable that mammary primordial epithelial cells express GFP driven by the s-SHIP promoter [11], which indicates that the mammary primordial epithelial cells may represent a population of activated primitive mammary progenitor cells. From E11.0 through E14.5, most of the mammary primordial epithelial cells appear to express s-SHIP-GFP [11]. At E15.5, most s-SHIP-GFP expression is found in the neck of the mammary bud, whilst little expression is observed in the mammary bud proper. At E16.5 s-SHIP-GFP stain is observed in subsets of suprabasal and basal mammary epithelial cells and the entire nipple sheath. By E18.5, s-SHIP-GFP-expressing cells are found predominantly in the basally located embryonic mammary cells and nipple sheath. The pattern of expression of s-SHIP-GFP expression during embryonic mammary morphogenesis is dynamic and may represent a means of distinguishing distinct embryonic mammary progenitor cell types. Specific cytokeratin expression is associated with distinct postnatal mammary lineages. In the postnatal mammary epithelium, keratins become predominantly segregated by expression within cells of either the myoepithelial (Krt5, Krt14, Krt17) or luminal lineages (Krt8, Krt18). Postnatal mammary cell subpopulations are distinctly localised within the mammary epithelium with myoepithelial cells located at the basal position and the luminal cells lining the ducts. This polarity has not been fully assumed in E12.5–E18.5 embryonic stages or in neonates [39,40]. Embryonic mammary cells expressing both myoepithelial and luminal markers are observed throughout embryonic mammary development [39]. Recent lineage tracing studies labelled K14+ embryonic mammary cells with YFP at stage E17.5 using a doxycycline-inducible transgenic mouse model, K14-rtTA/TetO-Cre/Rosa-YFP [41]. The majority of postnatal mammary epithelial cells analysed at five weeks of age were marked, including both myoepithelial cells and luminal cells, demonstrating that E17.5 stage K14+ progenitors give rise to all mammary epithelial cell lineages [41]. It is unclear how K14+ cells present in earlier mammary primordial stages contribute to the postnatal mammary lineage. What role K14− embryonic mammary epithelial cells play in mammary lineage establishment is also not yet clear.

B.A. Howard / Seminars in Cell & Developmental Biology 23 (2012) 574–582

4. Genetic factors required for normal mammary formation Genetic studies have established roles for several paracrine signalling components in mediating the epithelial–mesenchymal interactions required for normal embryonic mammary morphogenesis. One of the most thoroughly investigated signalling axis required for normal embryonic mammary cell fate decisions is Pthlh which encodes a ligand, Pthrp, and Prhr which encodes its cognate receptor. Absence of either gene, results in developmental arrest of mammary primordial formation at the late bud stage and neither branching of the mammary primordium, nor nipple formation occur. Epithelial Pthlh to mesenchymal Pthr signalling is required for the formation of the mammary mesenchyme and the induction of Tnc expression [42]. When Pthlh is mis-expressed throughout the basal layer of the epidermis using the K14-promoter, the ventral surface transgenic mice is transformed into nipple skin, implicating Pthlh signalling as a critical mediator of nipple cell fate [43]. Pthrp is secreted by mammary epithelial cells and makes mammary mesenchymal cells responsive to Bmp signals by upregulating the expression of Bmpr1A in the mammary mesenchyme. Bmp4 can restore sprout formation and branching morphogenesis when added to Pthlh-null mammary primordia cultured ex vivo [44]. In this study, Noggin, a Bmp inhibitor, partially inhibited mammary bud sprouting of cultured wild-type primordia. Further studies should elucidate the precise mechanisms by which Pthrp/Pthr paracrine signals ultimately regulate a cascade other signals required for these key aspects of mammary lineage regulation. Factors known to regulate mesenchymal–epithelial communications in other developmental systems are also required for mammary primordial development. Activation of Fgfr2b in the surface ectoderm by somatically expressed Fgf10 is required for thoracic mammary primordial two and three formation; presumably another Fgf10 source mediates receptor activation for primordia one and five, which are not closely associated with the somites [45]. Primordium four does form in the absence of Fgf10, but does not progress developmentally [20]. Positive Hedgehog (HH) signalling is absent throughout mammary development. HH downregulation is required for normal mammary primordial development. The repressor function of Gli3 is needed during early stages of mammary primordial development [46]. K14driven Dkk1 expression blocks mammary primordial formation in a transgenic mouse model, indicating an absolute requirement for canonical WNT signalling in early mammary primordial development [47]. Genetic ablation of Lef1 completely impedes formation of mammary primordium two and three; mammary primordium one, four and five form but the transition of Wnt signalling from mesenchyme to the mammary epithelia is blocked and these primordia disappear by 15.5 highlighting the distinct requirement of various primordia for Lef1 dependent Wnt signals at specific development stages [19]. Mammary primordia from embryos deficient for the Wnt co-receptor Lrp5 or Lrp6 are significantly smaller than wild-type primordial and exhibit substantially impaired canonical Wnt signalling [48,49]. Pygo2deficienct mammary primordia are also smaller [50]. Rho GTPase activating protein-encoding gene, Arhgap5 (p190-B), has been shown to regulate epithelial–mesenchymal interactions necessary to sustain mammary bud morphogenesis; mammary primordia lacking Arhgap5 are hypoplastic and their mammary mesenchymal cells appear disorganised and lack expression of Androgen Receptor [51]. The Igf1 pathway components, Irs1, Irs2, and Igf1r are also required for normal development of the mammary primordium [51]. Several transcription factors are also required for normal embryonic mammary formation including p63 and Tbx3 [52–54]. Mammary primordial development fails during the initial stages

579

of formation when either gene is functionally ablated in mouse models. Studies of mice with compound Msx1/Msx2 genetic deficiencies have demonstrated roles for both factors in embryonic mammary development as although mammary primordial form, no developmental progression beyond the placode stages occurs [55]. Functional redundancy is apparent of these factors since Msx2−/− mutants exhibit defective mammary development and display developmental arrest the late bud stage while no defects have been reported for Msx1−/− mutants. Ablating Msx2 genetically can restore hair follicle formation in the ventral skin of K14-Pthlh mice [44]. Pthrp and Bmp4 can induce Msx2 expression in a synergistic manner in cultured mesenchymal cells and are thought to act together to induce Msx2 expression in the mammary mesenchyme [44]. Interactions between Bmp4 and Tbx3 appear to regulate the positioning of the mammary primordia along the dorsal–ventral axis [56]. The location of Lef1-expressing cells (used as a marker to indicate the localisation of mammary primordial cells) were shifted and expanded in their positions along the mammary line when Tbx3 expression constructs were electroporated into E10.5 stage mouse embryos prior to ex vivo culture [56]. Noggin-soaked beads (a Bmp4 antagonist) locally inhibited Lef1 expression along the mammary line in explanted cultured embryos [56]. Whether any of these various factors can induce ectopic mammary formation in vivo or ex vivo has not yet been established. Conditional deletion of Gata3 from K14-expressing cells results in formation of hypoplastic mammary primordia or a complete loss of mammary primordial formation [57]. At E12.5, a heterogeneous population of both strongly K14-positive and K14-negative or weakly K14-positive cells exists within the mammary bud epithelium [40], which indicates that a sub-population of nontargeted cells is likely to remain in this mouse model which would account for the observed hypoplastic mammary primordia at late E11 stages. Conditional deletion of Gata3 from K14-expressing cells is sufficient to ablate both nipple sheath formation and mammary ductal outgrowth potential suggesting that a population of K14+, Gata3+ cells is required to mediate both processes and that any residual cells remaining in the hypoplastic mammary primordia are insufficient to accomplish either developmental process. 5. Genetic studies of mouse models have identified factors that can induce ectopic mammary formation Several studies on the very early stages of mammary primordial development have provided substantial insights in determining how undifferentiated tissue is directed to form the mammary primordium during embryogenesis. The scaramanga (ska) mouse mutant displays mammary gland phenotypes indicative of defective mammary specification. Mice harbouring the ska mutation, a hypomorphic allele of Neuregulin3 (Nrg3), often fail to form mammary primordium three [58]. Unstratified epithelium was observed at the future site of primordium three at a high frequency, consistent with the absence of expression of mammary primordial markers at this site. Nrg3 encodes a growth factor, which binds and activates Erbb4 [59], a receptor tyrosine kinase that regulates cell proliferation, differentiation, adhesion and migration [60,61]. During mouse embryogenesis, Nrg3 is expressed in dermal mesenchyme underlying the sites where the mammary placodes will subsequently form, suggesting an inductive role for Nrg3. Formation of mammary placode three was restored after culture of ska embryos implanted with beads soaked in recombinant Nrg3 (rNrg3). Mammary primordium formed, epithelial multilayering and increased expression of Wnt signals occurred adjacent to rNrg3-soaked beads implanted along the mammary line (at sites where mammary primordium would not usually form) in wild-type mouse embryo cultures explanted at stages prior to mammary morphogenesis. Nrg3 appears to function as a mesenchymal paracrine

580

B.A. Howard / Seminars in Cell & Developmental Biology 23 (2012) 574–582

signal to regulate epithelial stratification and formation of epithelial aggregations at the sites that mammary primordia form. A study of a transgenic mouse model that expresses Nrg3 throughout the basal layer (progenitor/stem cell compartment) confirmed that ectopic Nrg3 expression promotes the initiation of mammary primordial formation since K14-Nrg3 female founders formed ectopic nipples along and adjacent to the mammary line and is consistent with Nrg3 promoting early mammary morphogenesis [62]. Recently clinical studies including molecular characterisation of recurrent deletions of a region of 10q22q23 that spans NRG3 have identified individuals in which congenital breast aplasia are among the reported developmental anomalies, suggesting a possible inductive role for NRG3 in human breast development [63]. Ectopic placodes are often observed adjacent to the site of mammary primordium four in ska mutants. It is unclear how altered Nrg3 signalling affects the region adjacent to mammary primordium four to induce a small, ectopic mammary-forming field but this unusual phenotype is likely due to distinct genetic regulation for each mammary primordium [6]. Another growth factor, EctodysplasinA1 (Eda-A1) can also elicit the formation of ectopic mammary primordium in vivo. K14-EdaA1 mice, mis-express EctodysplasinA1, the ligand for Edar, in the developing epidermis and form ectopic mammary primordia along the mammary line during embryogenesis [64,65]. These primordia continue to develop and appear to function in the mature lactating gland. Mice with genetic deficiencies of EctodysplasinA1 form normal mammary primordia [66]. Results from several studies have suggested that placodal fate is regulated by the balance of global regulators (with Wnts and Edar being particularly strong activators of placodal fate) and regional factors are likely to confer organ identity [67–70]. How all of the various factors that have been implicated in regulating the formation of embryonic mammary primordium act in concert to achieve the precise morphogenesis observed remains to be clarified.

6. Transcriptomic profiling of the embryonic mammary cell populations reveal novel insights into pathways and network components that are signalling as the mammary lineage is established Recent transcriptomic profiling studies of embryonic mammary primordia have substantially increased our knowledge of the molecular signals that are likely to be mediating embryonic mammary morphogenesis [40,71]. Importantly, both studies profiled intact tissues that were isolated in manners that are known to retain their biological properties in tissue recombinant or repopulating experiments and should therefore represent underlying biologically relevant signalling. When compared to postnatal datasets profiling microdissected terminal end bud, ductal, and stromal tissues described by Kouros-Mehr and Werb [72], a number of genes were found to be expressed at two key stages of development, embryonic bud formation and branching morphogenesis at puberty and these represent candidate master regulators of mouse mammary development. Embryonic mammary cells largely exhibit distinct transcriptomic features when compared to their mature postnatal descendants at the sub-population level, likely reflecting their unique microenvironment and immature state [40]. The immature status of the embryonic mammary epithelial populations is further supported by the absence of expression of differentiation markers, such as ER␣ and SMA, and the lack expression of c-Kit and CD24 by the majority of cells. Gene set enrichment analysis of embryonic mammary epithelial cells compared to postnatal cell sub-populations indicated that the embryonic cells are enriched for genes encoding hematopoietic progenitors when compared to both mature luminal and luminal

progenitor cells profiled by [73], suggesting that common pathways and networks could be used by the embryonic mammary epithelial and hematopoietic progenitor cells, with Gata3 being a striking example [57,74,75]. Genes expressed by embryonic mammary cells and postnatal mammary progenitor cells are likely to mediate signals from mammary lineage inception onward and thus may represent key mammary progenitor cell regulators. Among these were several genes shown to regulate mammary cell fate and development previously including Gata3 and Sox6. A large number of novel candidate mammary lineage regulators were also identified with many poised to mediate cell–cell matrix interactions and reciprocal mesenchymal–epithelial signalling. Gene expression profiles of both embryonic and postnatal mammary cells suggest that many normal mouse mammary epithelial cells express molecules that have been attributed to epithelial–mesenchymal transition (EMT). Studies of human foetal breast tissues indicate that expression of Vimentin an intermediate filament protein, and commonly used marker of EMT, is also a feature of normal immature breast cells [76], demonstrating the usefulness of basic studies of normal mammary and breast cells with respect to our understanding of their inherent biological features.

7. Conclusions The pathways implicated in cell fate decisions are clearly relevant to transformation as most forms of breast cancer involve deregulation of signalling pathways (Egf, Fgf, Notch, Wnt) that also determine cell identity when they are initially specified from undifferentiated precursor cells [77]. The processes that are deregulated in cancer (cell adhesion, migration and proliferation) to detrimental effect are in large part those that regulate organ development during embryogenesis. The study of early embryonic development is providing information that is key to attaining a fundamental understanding of the native functions of these cellular processes at a time when they are first establishing an environment in which the mammary primordial cells may differentiate and perpetuate amidst a variety of other cell types and signals. The most primitive mammary cell populations arise during development of the embryonic mammary gland and studies of their cellular behaviours should provide deeper knowledge of the fundamental links between developmental genetics, stem cell biology, and breast cancer biology.

Acknowledgements Breakthrough Breast Cancer funds the author’s research and NHS funding to the NIHR Biomedical Research Centre is acknowledged.

References [1] Linnaeus C. Systema naturæ per regna tria naturæ, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis 1758–1759. [2] Theiler K. The house mouse. Berlin, Heidelberg, New York: Springer; 1972. [3] Jolicoeur F. Intrauterine breast development and the mammary myoepithelial lineage. J Mammary Gland Biol Neoplasia 2005;10:199–210. [4] Howard BA, Gusterson BA. Human breast development. J Mammary Gland Biol Neoplasia 2000;5:119–37. [5] Propper AY. Wandering epithelial cells in the rabbit embryo milk line. A preliminary scanning electron microscope study. Dev Biol 1978;67:225–31. [6] Veltmaat JM, Mailleux AA, Thiery JP, Bellusci S. Mouse embryonic mammogenesis as a model for the molecular regulation of pattern formation. Differentiation 2003;71:1–17. [7] Panchal H, Wansbury O, Howard BA. Embryonic mammary anlagen analysis using immunolabelling of whole mounts. Methods Mol Biol 2010;585:261–70. [8] Balinsky BI. On the prenatal growth of the mammary gland rudiment in the mouse. J Anat 1950;84:227–35. [9] Veltmaat JM, Van Veelen W, Thiery JP, Bellusci S. Identification of the mammary line in mouse by Wnt10b expression. Dev Dyn 2004;229:349–56.

B.A. Howard / Seminars in Cell & Developmental Biology 23 (2012) 574–582 [10] Chu EY, Hens J, Andl T, Kairo A, Yamaguchi TP, Brisken C, et al. Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis. Development 2004;131:4819–29. [11] Rohrschneider LR, Custodio JM, Anderson TA, Miller CP, Gu H. The intron 5/6 promoter region of the ship1 gene regulates expression in stem/progenitor cells of the mouse embryo. Dev Biol 2005;283:503–21. [12] Balinsky B. On the developmental processes in mammary glands and other epidermal structures. Trans Roy Soc Edinburgh 1949–1950;62:1–31. [13] Heuberger B, Fitzka I, Wasner G, Kratochwil K. Induction of androgen receptor formation by epithelium–mesenchyme interaction in embryonic mouse mammary gland. Proc Natl Acad Sci U S A 1982;79:2957–61. [14] Kratochwil K. Development and loss of androgen responsiveness in the embryonic rudiment of the mouse mammary gland. Dev Biol 1977;61:358–65. [15] Durnberger H, Kratochwil K. Specificity of tissue interaction and origin of mesenchymal cells in the androgen response of the embryonic mammary gland. Cell 1980;19:465–71. [16] Kratochwil K, Schwartz P. Tissue interaction in androgen response of embryonic mammary rudiment of mouse: identification of target tissue for testosterone. Proc Natl Acad Sci U S A 1976;73:4041–4. [17] Kratochwil K. Organ specificity in mesenchymal induction demonstrated in the embryonic development of the mammary gland of the mouse. Dev Biol 1969;20:46–71. [18] Sakakura T, Sakagami Y, Nishizuka Y. Dual origin of mesenchymal tissues participating in mouse mammary gland embryogenesis. Dev Biol 1982;91: 202–7. [19] Boras-Granic K, Chang H, Grosschedl R, Hamel PA. Lef1 is required for the transition of Wnt signaling from mesenchymal to epithelial cells in the mouse embryonic mammary gland. Dev Biol 2006;295:219–31. [20] Mailleux AA, Spencer-Dene B, Dillon C, Ndiaye D, Savona-Baron C, Itoh N, et al. Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. Development 2002;129:53–60. [21] Wansbury O, Panchal H, James M, Parry S, Ashworth A, Howard B. Dynamic expression of Erbb pathway members during early mammary gland morphogenesis. J Invest Dermatol 2008;128:1009–21. [22] Couldrey C, Moitra J, Vinson C, Anver M, Nagashima K, Green J. Adipose tissue: a vital in vivo role in mammary gland development but not differentiation. Dev Dyn 2002;223:459–68. [23] Hogg NA, Harrison CJ, Tickle C. Lumen formation in the developing mouse mammary gland. J Embryol Exp Morphol 1983;73:39–57. [24] Nanba D, Nakanishi Y, Hieda Y. Changes in adhesive properties of epithelial cells during early morphogenesis of the mammary gland. Dev Growth Differ 2001;43:535–44. [25] Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, et al. Estrogen receptors: how do they signal and what are their targets. Physiol Rev 2007;87:905–31. [26] Parmar H, Cunha GR. Epithelial–stromal interactions in the mouse and human mammary gland in vivo. Endocr Relat Cancer 2004;11:437–58. [27] Propper A, Gomot L. Tissue interactions during organogenesis of the mammary gland in the rabbit embryo. C R Acad Sci Hebd Seances Acad Sci D 1967;264:2573–5. [28] Cunha GR, Young P, Christov K, Guzman R, Nandi S, Talamantes F, et al. Mammary phenotypic expression induced in epidermal cells by embryonic mammary mesenchyme. Acta Anat (Basel) 1995;152:195–204. [29] Propper A, Gomot L. Control of chick epidermis differentiation by rabbit mammary mesenchyme. Experientia 1973;29:1543–4. [30] Sakakura T, Kusano I, Kusakabe M, Inaguma Y, Nishizuka Y. Biology of mammary fat pad in fetal mouse: capacity to support development of various fetal epithelia in vivo. Development 1987;100:421–30. [31] DeCosse JJ, Gossens CL, Kuzma JF, Unsworth BR. Breast cancer: induction of differentiation by embryonic tissue. Science 1973;181:1057–8. [32] Sakakura T, Sakagami Y, Nishizuka Y. Acceleration of mammary cancer development by grafting of fetal mammary mesenchymes in C3H mice. Gann 1979;70:459–66. [33] Sakakura T, Sakagami Y, Nishizuka Y. Persistence of responsiveness of adult mouse mammary gland to induction by embryonic mesenchyme. Dev Biol 1979;72:201–10. [34] Sakakura T, Nishizuka Y, Dawe CJ. Mesenchyme-dependent morphogenesis and epithelium-specific cytodifferentiation in mouse mammary gland. Science 1976;194:1439–41. [35] Taylor RA, Wang H, Wilkinson SE, Richards MG, Britt KL, Vaillant F, et al. Lineage enforcement by inductive mesenchyme on adult epithelial stem cells across developmental germ layers. Stem Cells 2009;27:3032–42. [36] Klinowska TC, Streuli CH. Analyzing cell–ECM interactions in adult mammary gland by transplantation of embryonic mammary tissue from knockout mice. Methods Mol Biol 2000;139:345–58. [37] Sakakura T, Nishizuka Y, Dawe CJ. Capacity of mammary fat pads of adult C3H/HeMs mice to interact morphogenetically with fetal mammary epithelium. J Natl Cancer Inst 1979;63:733–6. [38] Bai L, Rohrschneider LR. s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue. Genes Dev 2010;24:1882–92. [39] Sun P, Yuan Y, Li A, Li B, Dai X. Cytokeratin expression during mouse embryonic and early postnatal mammary gland development. Histochem Cell Biol 2010;133:213–21. [40] Wansbury O, Mackay A, Kogata N, Mitsopoulos C, Kendrick H, Davidson K, et al. Transcriptome analysis of embryonic mammary cells reveals insights into mammary lineage establishment. Breast Cancer Res 2011;13:R79.

581

[41] Van Keymeulen A, Rocha AS, Ousset M, Beck B, Bouvencourt G, Rock J, et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 2011;479:189–93. [42] Dunbar ME, Dann PR, Robinson GW, Hennighausen L, Zhang JP, Wysolmerski JJ. Parathyroid hormone-related protein signaling is necessary for sexual dimorphism during embryonic mammary development. Development 1999;126:3485–93. [43] Foley J, Dann P, Hong J, Cosgrove J, Dreyer B, Rimm D, et al. Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development. Development 2001;128:513–25. [44] Hens JR, Dann P, Zhang JP, Harris S, Robinson GW, Wysolmerski J. BMP4 and PTHrP interact to stimulate ductal outgrowth during embryonic mammary development and to inhibit hair follicle induction. Development 2007;134:1221–30. [45] Veltmaat JM, Relaix F, Le LT, Kratochwil K, Sala FG, van Veelen W, et al. Gli3mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes. Development 2006;133:2325–35. [46] Hatsell SJ, Cowin P. Gli3-mediated repression of Hedgehog targets is required for normal mammary development. Development 2006;133:3661–70. [47] Andl T, Reddy ST, Gaddapara T, Millar SE. WNT signals are required for the initiation of hair follicle development. Dev Cell 2002;2:643–53. [48] Lindvall C, Evans NC, Zylstra CR, Li Y, Alexander CM, Williams BO. The Wnt signaling receptor Lrp5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis. J Biol Chem 2006;281:35081–7. [49] Lindvall C, Zylstra CR, Evans N, West RA, Dykema K, Furge KA, et al. The Wnt co-receptor Lrp6 is required for normal mouse mammary gland development. PLoS One 2009;4:e5813. [50] Gu B, Sun P, Yuan Y, Moraes RC, Li A, Teng A, et al. Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation. J Cell Biol 2009;185:811–26. [51] Heckman BM, Chakravarty G, Vargo-Gogola T, Gonzales-Rimbau M, Hadsell DL, Lee AV, et al. Crosstalk between the p190-B RhoGAP and IGF signaling pathways is required for embryonic mammary bud development. Dev Biol 2007;309:137–49. [52] Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 1999;398:708–13. [53] Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999;398:714–8. [54] Davenport TG, Jerome-Majewska LA, Papaioannou VE. Mammary gland, limb and yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary syndrome. Development 2003;130:2263–73. [55] Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet 2000;24:391–5. [56] Cho KW, Kim JY, Song SJ, Farrell E, Eblaghie MC, Kim HJ, et al. Molecular interactions between Tbx3 and Bmp4 and a model for dorsoventral positioning of mammary gland development. Proc Natl Acad Sci U S A 2006;103:16788–93. [57] Asselin-Labat ML, Sutherland KD, Barker H, Thomas R, Shackleton M, Forrest NC, et al. Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol 2007;9:201–9. [58] Howard B, Panchal H, McCarthy A, Ashworth A. Identification of the scaramanga gene implicates Neuregulin3 in mammary gland specification. Genes Dev 2005;19:2078–90. [59] Zhang D, Sliwkowski MX, Mark M, Frantz G, Akita R, Sun Y, et al. Neuregulin-3 (NRG3): a novel neural tissue-enriched protein that binds and activates ErbB4. Proc Natl Acad Sci U S A 1997;94:9562–7. [60] Sartor CI, Zhou H, Kozlowska E, Guttridge K, Kawata E, Caskey L, et al. Her4 mediates ligand-dependent antiproliferative and differentiation responses in human breast cancer cells. Mol Cell Biol 2001;21:4265–75. [61] Nie S, Chang C. Regulation of Xenopus gastrulation by ErbB signaling. Dev Biol 2007;303:93–107. [62] Panchal H, Wansbury O, Parry S, Ashworth A, Howard B. Neuregulin3 alters cell fate in the epidermis and mammary gland. BMC Dev Biol 2007;7:105. [63] van Bon BW, Balciuniene J, Fruhman G, Nagamani SC, Broome DL, Cameron E, et al. The phenotype of recurrent 10q22q23 deletions and duplications. Eur J Hum Genet 2011;19:400–8. [64] Mustonen T, Pispa J, Mikkola ML, Pummila M, Kangas AT, Pakkasjarvi L, et al. Stimulation of ectodermal organ development by Ectodysplasin-A1. Dev Biol 2003;259:123–36. [65] Mustonen T, Ilmonen M, Pummila M, Kangas AT, Laurikkala J, Jaatinen R, et al. Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages. Development 2004;131:4907–19. [66] Mikkola ML. Genetic basis of skin appendage development. Semin Cell Dev Biol 2007;18:225–36. [67] Zhang Y, Andl T, Yang SH, Teta M, Liu F, Seykora JT, et al. Activation of beta-catenin signaling programs embryonic epidermis to hair follicle fate. Development 2008;135:2161–72. [68] Narhi K, Jarvinen E, Birchmeier W, Taketo MM, Mikkola ML, Thesleff I. Sustained epithelial beta-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation. Development 2008;135:1019–28. [69] Mou C, Jackson B, Schneider P, Overbeek PA, Headon DJ. Generation of the primary hair follicle pattern. Proc Natl Acad Sci U S A 2006;103:9075–80.

582

B.A. Howard / Seminars in Cell & Developmental Biology 23 (2012) 574–582

[70] Rinn JL, Wang JK, Liu H, Montgomery K, van de Rijn M, Chang HY. A systems biology approach to anatomic diversity of skin. J Invest Dermatol 2008;128:776–82. [71] Hens J, Dann P, Hiremath M, Pan TC, Chodosh L, Wysolmerski J. Analysis of gene expression in PTHrP−/− mammary buds supports a role for BMP signaling and MMP2 in the initiation of ductal morphogenesis. Dev Dyn 2009;238:2713–24. [72] Kouros-Mehr H, Werb Z. Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis. Dev Dyn 2006;235:3404–12. [73] Kendrick H, Regan JL, Magnay FA, Grigoriadis A, Mitsopoulos C, Zvelebil M, et al. Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate. BMC Genomics 2008;9:591.

[74] Ting CN, Olson MC, Barton KP, Leiden JM. Transcription factor GATA3 is required for development of the T-cell lineage. Nature 1996;384: 474–8. [75] Kouros-Mehr H, Slorach EM, Sternlicht MD, Werb Z. GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell 2006;127:1041–55. [76] Jolicoeur F, Gaboury LA, Oligny LL. Basal cells of second trimester fetal breasts: immunohistochemical study of myoepithelial precursors. Pediatr Dev Pathol 2003;6:398–413. [77] Howard B, Ashworth A. Signalling pathways implicated in early mammary gland morphogenesis and breast cancer. PLoS Genet 2006;2:e112.