REVIEW

Mammary gland development: cell fate specification, stem cellsand the microenvironmentJamie L. Inman*, Claire Robertson*, Joni D. Mott and Mina J. Bissell‡

ABSTRACTThe development of the mammary gland is unique: the final stages ofdevelopment occur postnatally at puberty under the influence ofhormonal cues. Furthermore, during the life of the female, themammary gland can undergo many rounds of expansion andproliferation. The mammary gland thus provides an excellent modelfor studying the ‘stem/progenitor’ cells that allow this repeatedexpansion and renewal. In this Review, we provide an overview ofthe different cell types that constitute the mammary gland, anddiscuss how these cell types arise and differentiate. As cellulardifferentiation cannot occur without proper signals, we also describehow the tissue microenvironment influences mammary glanddevelopment.

KEY WORDS: MMPs, Mammary gland, Microenvironment,Progenitor cells, Stem cells

IntroductionThe mammary gland, which distinguishes mammals from all otheranimals, functions to produce and secrete milk in order to nourishoffspring. It is also a unique glandular organ in that it reaches fulldevelopment only after birth. As such, the mammary gland providesa unique model for biologists to study development and organspecificity. The embryonic rudiment of the gland, the anlage, ispresent at birth and, in response to hormonal cues, begins branchinginto the fat pad as the female reaches puberty. During the lifetime ofthe female, the mammary gland undergoes many changes instructure and function, including cyclic expansions correspondingto the hormonal changes induced by the estrous/menstrual cycle, aswell as the dramatic changes that occur during pregnancy, lactationand involution. During these different stages, the cells of themammary gland proliferate, differentiate or apoptose in response tostimuli, giving rise to significant remodeling of the glandular tissuearchitecture.Indeed, studies of mammary gland development have offered

unique insights into the mechanisms regulating cell fatespecification, cell and tissue polarity, branching morphogenesisand the involution of a functional organ. Moreover, manydysregulated pathways and processes observed in breast cancerprogression mimic those observed during normal mammary glanddevelopment and tissue remodeling; these developmental programsare thus of interest also to cancer biologists. Here, we provide anoverview of mammary gland development, highlighting thedifferent cell types that make up the murine mammary gland, witha particular focus on mammary gland ‘stem/progenitor cells’. Wealso discuss how external factors in the mammary gland

microenvironment, such as extracellular matrix (ECM) andcell-cell interactions, influence cell fate and function.

An overview of embryonic mammary gland developmentIn mice, embryonic mammary gland development occurs betweenembryonic day (E) 10.5 and E18.5 (Hens and Wysolmerski, 2005;Sakakura, 1987; Veltmaat et al., 2003). It begins when the single-layered ectoderm enlarges to form the mammary lines on E10.5.These lines of cells extend from the anterior limb bud to the posteriorlimb bud. It is thought that mammary line cells then migrate to thelocation of the future mammary buds (five pairs in mice) (Hens andWysolmerski, 2005; Propper, 1978; Robinson, 2007). At E11.5,lens-shaped multilayered ectodermal structures called placodes areobserved, rising slightly above the surrounding ectoderm. Themammary placodes then become bulbs of epithelial cells that aredistinct from the surrounding epidermis. These buds are elevatedknob-like structures at E12-E13.5 but sink into the underlyingdermis at around E13.5 (Sakakura, 1987; Watson and Khaled,2008). Mesenchymal cells around the sunken bud condense andbecome the mammary mesenchyme. Androgen receptor activationin the mesenchyme of male embryos between E13.5 and E15.5signals for the degradation of the mammary buds (Sakakura, 1987).A second mesenchyme, the fat pad precursor, differentiates beyondthe mammary mesenchyme at E14.5 (Sakakura, 1987). Femalemammary gland development continues at E15.5, with epithelialcell proliferation and elongation in the bud leading to the formationof a sprout that invades the fat pad precursor. The nipple is formedfrom epidermal cells overlying the bud, and a lumen is formed in thesprout at E16.5 (Hogg et al., 1983). The sprout then branches intothe fat pad, giving rise to the rudimentary ductal tree by E18.5(Sakakura, 1987).

An overview of postnatal mammary gland developmentFrom birth to puberty, the mammary epithelium originating at thenipple remains quiescent (Fig. 1A). During puberty, and underthe control of hormones and other factors, the ductal epithelium of themammary anlage invades into the mammary fat pad (Fig. 1B) in aprocess referred to as branchingmorphogenesis (Lyons, 1958; Nandi,1958). Highly proliferative terminal end buds, which contain an outerlayer of cap epithelial cells surrounding multilayered body epithelialcells located at the invading front of the branch, lead the way(Silberstein and Daniel, 1982; Williams and Daniel, 1983). Theinvading epithelial cells display some characteristics of mesenchymalcells, suggesting that some degree of epithelial-to-mesenchymaltransition (EMT) occurs at the end bud (Kouros-Mehr and Werb,2006; Nelson et al., 2006). However, unlike tumor cells, EMT genesemployed during branching morphogenesis are highly regulated.Indeed, recent work has shown that the transcription factor Ovol2, amaster negative regulator of EMT, is required duringmammary glandmorphogenesis to regulate the expression of EMT genes (Watanabeet al., 2014). The mechanism by which the branching process stops

Life Sciences Division, Lawrence Berkeley National Laboratory, University ofCalifornia, Berkeley, CA 94720, USA.*These authors contributed equally to this work

‡Author for correspondence (mjbissell@lbl.gov)

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once the fat pad is filled involves the production and activation ofendogenous TGFβ, and is most likely regulated by mechanical andlocal cues from the gland architecture (Nelson et al., 2006), althoughthe exact steps remain an intriguing puzzle for both cell anddevelopmental biologists.The final arboreal, bilayered ductal structure is composed of

apically oriented luminal epithelial cells that are surrounded onthe basal side by contractile myoepithelial cells (Fig. 1C). In thegland of virgin mice, the epithelium proliferates and apoptosesduring each estrus cycle (Fata et al., 2001). However, duringpregnancy, the alveolar epithelium proliferates rapidly in responseto circulating hormones, developing secretory alveoli that arecapable of producing milk (Fig. 1D). During lactation, the apicallyoriented luminal epithelial cells synthesize and secrete milkproteins into the lumen of the alveoli; the release of oxytocincaused by the suckling infant causes the contraction of thesurrounding myoepithelial cells, thus moving the milk through theductal tree and to the nipple. During the process of weaning, thestimuli for milk production are lost and the expanded epithelialcompartment apoptoses in an event referred to as ‘involution’. The

gland is remodeled by a number of proteases, of whichmetalloproteinase 3 (Mmp3) is the most prominent (Talhouket al., 1991), to return to the ‘resting’ state of the pre-pregnantgland (Fig. 1E). Thus, the epithelial component and thesurrounding tissue architecture go through a significant amountof remodeling during each pregnancy.

Cell types of the mammary gland and their specificationAs with most glandular tissues, the adult mammary gland iscomposed of multiple cell types, including epithelial, adipose,fibroblasts, immune, lymphatic and vascular cells, that worktogether to sculpt and maintain a functional organ. These differentcell types have been demonstrated to be of importance at specificstages of mammary gland development.

Epithelial cellsMultiple epithelial cell types can be found in themammary gland. Themammary bilayer found throughout the adult gland of virgin mice istraditionally described as being composed of apically orientedluminal epithelial cells that line the ducts and of basally orientedmyoepithelial cells in contact with the basement membrane (BM)(Fig. 1C, inset). Luminal cells express keratins 8 and 18, whereasmyoepithelial cells express keratins 5 and 14 aswell as smoothmuscleactin that mediates their contractile function. In addition tomyoepithelial cells, cell-sorting experiments have identified severalputative stem and progenitor cell types in the basal cell populationwhich wewill discuss in later sections (Shackleton et al., 2006; Stinglet al., 2006; Visvader and Stingl, 2014).

Throughout puberty and pregnancy, there appear to be uniquecell types undoubtedly relating to the mammary functions at thesedevelopmental phases. During puberty, for example, cap cells andbody cells, which are both specialized epithelial cells, arise in theend bud. Cap cells, so named because they line the end bud formingthe cap of the structure (Fig. 1B, inset), contact the surroundingstroma through a thin basal lamina and appear to differentiate intomyoepithelial cells that generate a thicker basal lamina (Daniel andSilberstein, 2000; Williams and Daniel, 1983). The body cells, bycontrast, fill the interior of the end bud. The central body cells thenapoptose to form the lumen, and the remaining body cellsdifferentiate into luminal epithelial cells, giving rise to the ductalepithelium of the adult breast (Fig. 1C, inset) (Hennighausen andRobinson, 2005). During pregnancy, luminal epithelial cells rapidlyexpand, forming alveoli that are lined with cells primed to secretemilk at parturition (Fig. 1D, inset).

AdipocytesFat-filled adipocytes comprise a large proportion of the stromal fatpad in the adult and non-lactating gland. The dense fat padprecursor, which is observable at E14, develops throughoutembryogenesis, yet conversion to typical white fat tissue is notobserved until two to three days after birth (Sakakura, 1987).During pregnancy and lactation, adipocytes with reduced lipidcontent are observed, suggesting that this reservoir of fat isnecessary for the metabolically demanding process of milkproduction (Gregor et al., 2013; Hovey and Aimo, 2010).Adipocytes also serve an endocrine function in the gland: theyare thought to regulate epithelial growth and mammary epitheliumfunction, as well as to communicate with other cell types in themammary gland (Bartley et al., 1981; Hovey and Aimo, 2010). Asan example, adipocytes secrete vascular endothelial growth factor(VEGF) and probably regulate angiogenesis in the mammarygland (Hovey et al., 2001).

Birt

hP

uber

tyLa

ctat

ion

Invo

lutio

nVi

rgin

A

B

C

D

E

Alveolar cells Epithelial cap cells

Epithelial body cells Luminal epithelial cells

Key

Myoepithelial cells

Fig. 1. The mammary gland development is multistage and occurs afterbirth. (A) The mammary anlage is present at birth and remains quiescent untilpuberty. (B) Due to puberty hormones, the epithelial ductal cells expand intothe mammary fat pad, led by highly proliferative multilayered terminal endbuds (TEBs; inset). The TEBs have an outer layer of epithelial cap cells thatsurround multilayered epithelial body cells in rodents. (C) The mammary glandof adult virgin mice is filled with epithelial branching structures. The ducts ofthis structure (inset) contain an outer layer of myoepithelial cells and an innerlayer of luminal epithelial cells. (D) Pregnancy is accompanied by differenthormonal changes that signal a large expansion of alveolar cells that mature tomilk-secreting acini/alveoli during lactation. The alveoli (inset) expand outfrom the ducts filling the majority of the fat pad. (E) Upon weaning, involutionproceeds through cell death and ECM remodeling, giving rise to a statethat resembles the resting adult mammary gland. Schematic reproducedwith permission from Chuong et al. (2014) and Hennighausen andRobinson (2005).

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FibroblastsStromal fibroblasts are embedded within the fat pad and are oftenfound in close proximity to the basal side of the epithelial branchingtree (Muschler and Streuli, 2010; Sakakura, 1987). Fibroblastsserve many functions, one of which is bi-directional communicationwith the epithelium during branching morphogenesis, providinginstruction in the formof growth factors, proteases and other elements(Howard and Lu, 2014). In vivo and in vitro studies suggest thatfibroblasts play an important role in supporting both epithelial cellsurvival and morphogenesis in the fat pad (Liu et al., 2012;Makaremet al., 2013; Wang and Kaplan, 2012). Furthermore, fibroblasts arebelieved to be the chief stewards of the mammary ECM: theysynthesize a number of ECM components, such as collagens,proteoglycans and fibronectin. Additionally, fibroblasts synthesizemany enzymes, such asmatrix metalloproteinases, that are capable ofnot only degrading the ECM but also of releasing the growth factorsand cytokines harbored or embedded within the ECM that influencecellular and tissue function (Simian et al., 2001;Wiseman andWerb,2002). As such, these cells can regulate epithelial cell features and theepithelial cancer phenotype by altering ECM composition or density(Lühr et al., 2012). It is important to note, however, thatmyoepithelialcells produce copious amounts of laminin-111, which influencesmany aspects of mammary gland development and function,including tissue polarity (Gudjonsson et al., 2002) and survival(Boudreau et al., 1995) of luminal cells.

Vascular and immune cellsThe mammary gland is intercalated with extensive vascular andlymphatic networks present throughout the fat pad. Duringpubertal mammary gland morphogenesis, the lymphatic networkdevelops in close association with the mammary epithelial tree andblood vasculature (Betterman et al., 2012). Lymphangiogenesis inthe mammary gland is probably driven by myoepithelial-derivedVEGF-C (Vegfc – Mouse Genome Informatics Database) and/orVEGF-D (Vegfd – Mouse Genome Informatics Database)(Betterman et al., 2012). Immune cells, such as macrophages andeosinophils, are also required for branching morphogenesis, andthey are recruited to the branching tips of the epithelium to mediateinvasion into the fat pad (Gouon-Evans et al., 2000). Macrophagesare also required for epithelial cell death and adipocyterepopulation during involution (O’Brien et al., 2012). Throughactivation of their serine proteases and degranulation, mast cells areinvolved in normal mammary branching during puberty, and theyaccumulate, and possibly activate, plasma kallikrein, thusactivating the plasminogen cascade in involution (Lilla et al.,2009; Lilla and Werb, 2010).Although it is evident that the many cell types of the mammary

gland contribute to its structure, development and ultimatefunction in a dynamic and reciprocal fashion, the vast majorityof research has focused on the epithelium alone. This perhaps canbe explained because it is the epithelial cells that show dramaticalterations in function and structure in pregnancy and lactation,and because mammary tumors predominantly arise in theepithelial compartment. In an effort to better understandepithelial behavior, recent research efforts have focused onelucidating the hierarchy of epithelial cell differentiation, as wellas identifying factors that can influence mammary gland function.

Mammary gland stem and progenitor cells: dramaticregenerative potentialThe mammary epithelium displays dramatic regenerative potentialand the ability to undergo many cycles of growth and involution,

suggesting that the mammary gland epithelial compartmentcontains mammary epithelial stem cells, that is, single epithelialcells capable of generating the entire epithelial architecture. Theremarkable regenerative capacity of mammary epithelium wasfirst demonstrated in the late 1950s and early 1960s bytransplanting small numbers of epithelial cells into mammaryfat pads that had been cleared of their epithelial rudiments (Daniel,1975; Faulkin and Deome, 1960). These cells were able togenerate branching epithelia that filled the whole fat pad (i.e.reconstitute the glandular epithelium), and cell populations fromthese epithelia were able to reconstitute the gland again insubsequent cleared fat pads, demonstrating strong stem cell-likebehavior. More recently, it was shown that an entire functionalmammary gland can be derived from the progeny of a single cell(Kordon and Smith, 1998; Shackleton et al., 2006; Stingl et al.,2006), supporting the notion that the mammary epitheliumcontains a stem cell population.

Since these demonstrations, multiple studies have focused onidentifying and isolating mammary stem cell (MaSC)populations and defining the differentiation potential ofdifferent mammary epithelial populations (Plaks et al., 2013;Shackleton et al., 2006; Spike et al., 2012; Stingl et al., 2006).Many of these studies have used flow cytometry, or fluorescence-activated cell sorting (FACS), to select different epithelial cellpopulations, which are then tested for reconstitution efficiencyby injecting them into cleared mammary fat pads at limitingdilutions (Plaks et al., 2013; Shackleton et al., 2006; Spike et al.,2012; Stingl et al., 2006; Zeng and Nusse, 2010). However,confusion plagues this field of study, as several differentepithelial cell populations with stem cell properties have beenidentified, some of which might act as stem cells during the non-physiological process of cell transplantation but not duringnormal development and differentiation. To ensure clarity, wewill hereafter refer to cells with the ability to reconstitute theglandular epithelium on transplant as mammary gland-reconstituting units (MRUs). These are distinct from cells that,during normal development, give rise to both luminal andmyoepithelial cells, which we will refer to as bipotent MaSCs,and cells that give rise to just a single lineage during normaldevelopment, which we will refer to as unipotent mammaryepithelial progenitors.

Markers and features of mammary gland-reconstituting cellsUsing cell surface marker sorting (see Table 1) and the glandreconstitution assay, several different markers for MRUs havebeen identified. The murine mammary epithelium is typically firstisolated by selecting against markers of immune/hematopoietic(CD45), erythrocyte (Ter119) and endothelial (CD31) cells(commonly referred to as the lineage-marker negative, or Lin−

population). These epithelial cells are then sorted for expressionof moderate-to-high levels of CD24 (heat-stable antigen), highlevels of CD29 (β1-integrin) and/or high levels CD49f(α6-integrin) (Badders et al., 2009; dos Santos et al., 2013;Plaks et al., 2013; Shackleton et al., 2006; Stingl et al., 2006;Zeng and Nusse, 2010). The expression pattern of these markersappears to vary between studies, making quantitative comparisonsof the prevalence of different populations difficult (Fig. 2).Nonetheless, recent work suggests that the cells isolated via thesorting approach described above are all myoepithelial:myoepithelial cells expressing smooth muscle actin were theonly cells able to repopulate the gland in one recent study (Prateret al., 2014).

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Table 1. Mammary gland reconstitution studies and MRU markers

MaSCmarkerstudy Methods

Cell markersof MRU population

MRU frequencyor frequency of glandreconstitution

Glandreconstitution inother cells studied

Markers which didnot show glandreconstitution

Notes about MRUpopulations

Smith, 2005 DNA label retentionLabeled in 3-week-old mice withestradiol treatmentfrom week 3-8

DNA label retentionsuggests that theepithelium hasstem cells

Stingl et al.,2006

FACS and glandreconstitution from8-14-week-old virginmice (in 2% FBS)

Cd31−CD45−Ter119−

CD140a−CD24med

CD49fhi

1/62 CD24med

CD49fhi (FVB mice)1/91 CD24medCD49fhi

(C57BL/6 mice)

<1/230CD24hiCD49flow

1/3400CD24low

CD49flow

1/1400unsorted cells

Sca1hi- inducedby culture, noMaSC in Sca1hi

populationHoechst 3342-or Rhodamine123-effluxingcells 10% ofMaSC

Shackletonet al.,2006

FACS and glandreconstitution from8-week-old mice(injected in 50%FBS)

CD45−CD31−TER119−

CD24+CD29hi1/64 Lin−CD29hi

CD24+1/47,000 inCD29lowCD24−

<1/7200 inCD29low

CD24+

1/2900 inCD29hiCD24−

0/22 in Lin+

1/30,000Sca1hi (noCD29loCD24+

Sca1hi cellsobserved)Hoechst 33342exclusion – noenrichmentover Lin−

CD29hiCD24+

MRU location:terminal endbuds

Badderset al.,2009

FACS and glandreconstitution fromvirgin 12-14-week-old mice (injected in50% matrigel)

CD45−CD31−

Lrp5+Lrp6+1/485 Lrp5+

1/142 CD24+

CD49fhi

1/7760 in unsortedpopulation

1/100,000 Lrp5−

All Lrp5+ and Lrp6+

are K5+ or SMA+

basal cells butnot all are CD24+

CD49fhi. 40% ofLrp5+ are Vwf+

Zeng andNusse,2010

Transgenic Axin2reporter, FACS andglandreconstitution from8-12-week-old mice(in 50% matrigel orWnt3a)

CD31−CD45−Ter119−

CD24+CD29hiAxin2+11/16 glands Lin−

CD24+CD29hi

Axin2+ (50 cells)

5/16 glands Lin−

CD24+CD29hi

Axin2− (50 cells)

5% of Lin−CD24+

CD29hi areAxin2+

Spike et al.,2012

FACS and glandreconstitution fromfetal (E18.5) oradult (ageunspecified) mice(±50% matrigel)

CD31−CD45−Ter119−

CD24medCD49fhi1/14 fetal CD24med

CD49fhi +matrigel1/50 adult CD24med

CD49fhi +matrigel

1/400 fetalCD24med

CD49f–matrigel1/800 adultCD24med

CD49fhi–matrigel

dos Santoset al.,2013

Dox-induced DNAlabel retention+FACS and glandreconstitution in 6-10-week-old virginmice (injected in50%matrigel)

CD31−CD45−Ter119−

(MACS depleted)CD24+

CD29hiCD49fhiCD1d+

1/8 Lin−CD24+CD29hi

CD1d+

1/33 DNA-retainingcells

1/44 Lin−CD24+

CD29hiCD49fhi

1/149 unlabeledDNA

CD1d+ cells makeup 1% of basalcells

Machadoet al.,2013

FACS and glandreconstitution from8-12-week-old mice(in 50% matrigel)

CD3e−CD11b−CD45R−

Ly6G−Ly6C−TER119−

CD31−CD24+CD29hi

large (<10 μmdiameter)

1/66 Lin−CD24+

CD29hi large1/132 Lin−

CD24+CD29hi

1/237 Lin− large

Continued

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The reported likelihood of a single cell from this MRU-enrichedpopulation in a limiting dilution giving rise to a full glandularepithelium is ∼1/40-1/800, depending on the study (Badders et al.,2009; dos Santos et al., 2013; Plaks et al., 2013; Shackleton et al.,2006; Stingl et al., 2006; Zeng and Nusse, 2010). This can becompared with 1/1400 to 1/7600 in the unsorted Lin− epithelialpopulation (Badders et al., 2009; Shackleton et al., 2006) or lessthan 1/3000 in the Lin−CD29− or Lin−CD24− MRU-depletedepithelial populations (Shackleton et al., 2006). However,Lin−CD24med-hiCD29highCD49fhigh populations are not pureMRU populations; rather, they are MRU-enriched populations,and the complement populations are mostly, but not completely,MRU-depleted (Shackleton et al., 2006).

Refining MRU surface markers by isolating DNA label-retaining cellsSeveral subsequent studies have since attempted to refine the surfacemarkers of MRUs and have identified several disparate populationswith gland-reconstituting capacity. The adult mammary glandcontains cells that retain their parental DNA strand, suggesting thatthese cells divide infrequently and via asymmetric cell division,which are properties of stem and progenitor cells (Smith, 2005).These DNA label-retaining cells were indeed demonstrated to havefivefold higher gland-reconstituting capacity compared with non-labeled and non-label-retaining epithelial cells in a recent study (dosSantos et al., 2013). Furthermore, the isolation and subsequentprofiling of DNA-retaining CD29highCD49fhighCD24+ cells hasbeen used to identify improvedMRUmarkers. Through thiswork, anadditional cell surface marker, CD1d, a glycoprotein typically foundon antigen-presenting cells, was identified in the DNA label-retaining population (dos Santos et al., 2013). Adding this marker tothe current sorting technique led to a fivefold enrichment of MRUsin the Lin−CD29highCD49fhighCD24+CD1d+ population to 1/8.However, CD1d+ cells might not represent the entire stem cellpopulation, given the rarity of the CD1d+ population (1% ofCD29highCD49fhighCD24+) compared with the frequency of MRUsobserved in this study (1/44 Lin−CD24+CD29hi versus 1/8Lin−CD24+CD29hiCD1d+), suggesting that CD1d+ cells are asubset of MRUs. This study also did not determine whether theLin−CD24+CD29hiCD1d− population was depleted of stem cellactivity.

Refining MRU surface markers by interrogating the Wnt signalingpathwayAnother fruitful approach for isolating MRUs has been to identifyelements of the Wnt signaling pathway that are active in mammarydevelopment, given the importance of Wnt signaling in stem cellbehavior (Reya and Clevers, 2005) and the high activation of theWnt/β-catenin pathway in DNA label-retaining cells (dos Santoset al., 2013). Indeed, a downstream target of Wnt, the G-protein-

Table 1. Continued

MaSCmarkerstudy Methods

Cell markersof MRU population

MRU frequencyor frequency of glandreconstitution

Glandreconstitution inother cells studied

Markers which didnot show glandreconstitution

Notes about MRUpopulations

Plaks et al.,2013

FACS and glandreconstitution oncells from 7-9-week-old mice (in 50%matrigel+FGF2)

CD31−CD45−Ter119−

CD24+CD49fhi

Ck14+Lgr5+

5/16 glands Lin−

CD24+CD49fhi

Ck14+Lgr5+

(10 cells)

0/16 glands Lin−

CD24+CD49fhi

Ck14+Lgr5− (10cells)

Lgr5+ cells are 6%of K14+ basalcells

Prater et al.,2014

FACS, in vitro cultureand glandreconstitution oncells from 10-14-week-old mice (in25% matrigel)

CD31−CD45−Ter119−

CD49fhiEpCAMhi orSMA+

1/57 EpCAMhi

1/79 cultured CD49fhi

cells15/18 culturedEpCAMhi colonies

1/93 SMA+

1/300 EpCAMlow

1/205 non-culturedCD49fhi

11/18 culturedEpCAMlow

colonies>1/300 SMA−

Wang et al.,2015

FACS and glandreconstitution oncells from 8-12-week-old mice (in50% matrigel, 20%FBS)

Lin−CD24+CD29hi

ProcR+1/12 CD24+CD29hi

ProcR+

1/15 CD24+

CD29hiProcR+

Lgr5−

1/70 CD24+

CD29hi

1/2000 CD24+

CD29hiProcR–

1/160 ProcR–

Lgr5+

0/16 glandsCD24+ CD29hi

ProcR–Lgr5−

Abbreviations: MRU, mammary reconstituting unit; FACS, fluorescence-activated cell sorting; MACS, magnetic-activated cell sorting; Vwf, VonWillebrand factor;SMA, smooth muscle actin; Ck, cytokeratin; FBS, fetal bovine serum; CD31−CD45−Ter119− (Lin−), lineage-marker cocktail negative.

CD29

CD

24

A B

Fig. 2. Flow cytometry-based studies of MRUs: discordance betweenstudies. Cell-surface marker expression levels in luminal cells (circled in blue)and myoepithelial cells (circled in red) from adult (8-12-week old) murinemammary glands show discordance between studies using similarmethodologies. (A) In one study (Shackleton et al., 2006), similar levels ofCD24 were reported in luminal and myoepithelial cells, with CD29 serving todistinguish the two cell types. (B) A second study (Zeng and Nusse, 2010)showed higher CD24 levels in luminal compared with myoepithelial cells.Images shown are reproduced with permission from Nature Publishing Group(Shackleton et al., 2006) and Elsevier (Zeng and Nusse, 2010).

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coupled receptor Lgr5, appears to act as a marker of stem cells inseveral other organ systems (Barker et al., 2010, 2007; Jaks et al.,2008). In the mammary gland, Lgr5+ cells are a subset of the keratin14+ (K14) basal Lin−CD24+CD49fhigh MRU-enriched populationand are superior to their parent population in regenerating functionalmammary glands (de Visser et al., 2012; Plaks et al., 2013).Supporting a role for Lgr5 in not just gland reconstitution but normaldevelopment, loss-of-function and deletion experiments show thatLgr5 and its principal ligand, R-spondin, are necessary for normalpostnatal mammary gland organogenesis (Chadi et al., 2009; deVisser et al., 2012; Plaks et al., 2013). The Lin−CD24+CD49fhigh

Lgr5−-depleted population showed no gland-reconstituting ability inone study (Plaks et al., 2013), whereas Lgr5− cells showed rarerepopulating activity in another (Rios et al., 2014). However, a thirdstudy found the opposite, such that Lgr5− cells that also expressedprotein C receptor (ProcR) showed stronger MRU behavior thanLgr5+ cells (Wang et al., 2015).Other Wnt pathway elements, including the canonical Wnt

pathway receptors Lrp5 and Lrp6 (Goel et al., 2012), also appear tomark MaSC-enriched populations (Badders et al., 2009; Lindvallet al., 2009) compared with epithelial cells as a whole. Accordingly,selection for Lrp5, an LDL receptor-related protein, increases glandreconstitution efficiency compared with unsorted epithelial cells(Badders et al., 2009), and Lrp6 is necessary for normal mammarybranching invasion in vivo (Lindvall et al., 2009). A very recentstudy reported that ProcR, which is a Wnt3A target, is also a markerof MRUs: CD24+CD29hiProcR+ cells showed much higher glandreconstitution potential than their parent or ProcR-depletedpopulations (Wang et al., 2015). Furthermore, anthrax toxinreceptor 1 (Antxr1), which also acts in the Wnt pathway, mightact as a stem cell marker based on in vitro colony-forming assays(Chen et al., 2013). It will be of interest to investigate whether thecell populations characterized by these various Wnt signalingcomponents overlap with the CD1d+ MaSC-enriched populations.

Issues with MRU studiesAlthough flow cytometry-based studies have been useful foridentifying various populations of MRUs and the markers thatthey express, variations in FACS results and the frequency ofMRU detection are observed from study to study (Fig. 2). Thesemight partially be explained by methodological variations,including those in donor age and transplant conditions. As miceundergo puberty and massive changes in epithelial architecturearound week 6-8, followed by estrous cycles every 4 days, 6-week-old murine mammary glands might show a very different cellularcomposition to that from 12-week-old mice. The estrus cycle canalso dramatically alter gland reconstitution efficiency, resulting intenfold higher MRU numbers during progesterone-high lutealphases (Joshi et al., 2010). Likewise, we recently demonstrated thatvery subtle differences in cell isolation can result in discordance inFACS data (Hines et al., 2014). Furthermore, transplant conditionscan dramatically affect reconstitution efficiency, leading to a tenfoldincrease in gland reconstitution in one study (Spike et al., 2012).For example, the use of matrigel to prevent anoikis, and theinjection of epithelia in their native configuration with attachedstroma, both strongly increased gland reconstitution efficiency(Spike et al., 2012). Likewise, the culture of myoepithelial cells forone week in vitrowas sufficient to massively increase MRU activity(Prater et al., 2014). Given these many potential variables, aconsensus technical paper, comparing leading separation methodsand providing guidance for future studies, has been published(Smalley et al., 2012).

It should also be noted that, although gland reconstitutionstudies have demonstrated the remarkable regenerative capacityof MRUs, it is unclear whether normal mammary developmentrelies on the same cellular populations. Gland reconstitutionlikewise involves injection site wounding, which might inducedifferent fate decisions than those occurring during normalgland development (Plaks et al., 2013; Shackleton et al., 2006;Van Keymeulen et al., 2011).

Mammary gland stem and progenitor cells: insights fromlineage tracingGiven some of the issues with FACS-based approaches, lineagetracing of cell populations in transgenic animals is increasingly beingemployed to define and better understand the stem and progenitor cellpopulations in the mammary gland. However, such studies have alsoyielded some conflicting results on the nature and potency ofmammary stem and progenitor cells during pubertal development,pregnancy and involution (summarized in Figs 3 and 4).

Pubertal development might rely on bipotent MaSCsTwo single-color lineage-tracing studies concluded thatmammary development during puberty predominantly occursthrough unipotent progenitor cells, such that all basal cells arisefrom basal progenitor cells alone, and all luminal cells fromluminal progenitors (Fig. 4C) (Van Keymeulen et al., 2011; vanAmerongen et al., 2012). By contrast, recent, extensive lineage-tracing studies demonstrated that the pubertal gland containsseveral different stem and progenitor cell populations, includingbipotent stem cells that give rise to both the luminal andmyoepithelial cells of the duct (Rios et al., 2014) (Figs 3 and 4).This work used multicolor lineage tracing to mark single cellsand their clonal progeny, and confocal microscopy on thicksections to analyze large regions of the ductal tree, therebyallowing for more sensitive detection of rare cell types.

In this study, keratin 5 (K5)-expressing cells labeled at pubertywere all found in the basal compartment at the time of labeling, butgave rise to contiguous patches of both luminal and basal cells,suggesting bipotency (Rios et al., 2014). At the initial labeling timepoint, all K5-labeled cells were in the basal compartment, asdetected both by FACS analysis (91% CD29hi, CD24+ and lessthan 1% luminal CD29lo, CD24+) and by histopathology. At1 week after K5 induction, no single-colored luminal patches wereobserved, and 65% of clonal patches were solely myoepithelial.After an 8-week chase, 61% of contiguous, single-color patcheshad both luminal and myoepithelial cells, a result unlikely to bedue to adjacent, commonly colored stem or progenitor cells givingrise to single-color patches (Fig. 4A′). Indeed, cells labeled foreither K5, K14 or Lgr5 in the prepubertal gland all appeared to giverise to clonal patches of cells from both lineages when assessedeither by confocal microscopy or by FACS (Rios et al., 2014). Asmall subset of dividing K5+ cells at puberty were also observed toexpress markers of multiple lineages, including the basal markerK14, the luminal marker Elf5 and the putative stem cell markerLgr5, suggesting that this cell population represents a progenitorpopulation.

In addition to these bipotent stem cell populations, Elf5+ luminalcells labeled before puberty gave rise to luminal and alveolar cellsalone, suggesting that Elf5+ cells contain a luminal progenitor pool(Fig. 4D) (Rios et al., 2014). This result is mirrored by the earlierfinding that keratin 8+ (K8) and keratin 18+ (K18) luminal cells inthe prepubertal gland give rise to luminal and alveolar cells alone(Van Keymeulen et al., 2011).

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Remodeling in the adult mammary gland of virgin miceWhen using lineage tracing in a postpubertal mammary gland ofvirgin adult mice, contiguous patches of labeled cells were observedin multiple studies (Rios et al., 2014; Van Keymeulen et al., 2011),suggesting that, even in the quiescent gland, extensive cell turnoverand remodeling occurs. Using lineage tracing of Elf5+ luminal cellsto assess adult animals, large patches of luminal cells derived fromthe same progenitor were observed at 8 weeks, but fewer clonalpatches were observed at 20 weeks, consistent with the expansionand slow depletion of luminal progenitors over time (Rios et al.,2014). When using lineage tracing of K5+ basal cells in the adultgland, patches of both luminal and myoepithelial cells wereobserved, including some physically coupled cell pairs,suggesting recent asymmetric division (Rios et al., 2014). Overlong periods of time, fewer, larger patches, containing both luminaland basal labeled cells were observed, suggesting massive clonal

expansion of these K5+ bipotent progenitors during glandularremodeling (Rios et al., 2014). Additionally, over long chases, ashift from mixed or myoepithelial-only clonal patches topredominantly luminal clonal patches was observed with thislabeling scheme, suggesting that bipotent progenitors give rise to apopulation of luminal progenitors that then expands for luminalductal maintenance (Fig. 4B) (Rios et al., 2014).

Conflicting evidence for stem/progenitor cells in pregnancy andinvolutionDuring pregnancy, the glands of virgin adult mice develop anextensive network of secretory alveoli lined by specialized luminalcells. These alveolar luminal cells are believed to be derived fromthe ductal luminal cells seen in the adult virgin mice, based onlineage-tracing experiments using luminal lineage markers, such asElf5, K8 or K18 (Rios et al., 2014; Van Keymeulen et al., 2011). In

Fig. 3. An overview of lineage tracing invivo. Different labeling-basedexperiments, in which the fate of Elf5-,K5-, K8-, K14-, K18-, Lgr5- or Axin2-expressing cells were followed, arerepresented by arrows, starting at thedevelopmental time of labeling andending at the time of harvest and analysis.The location of labeled cells at the onsetof the experiment is listed (whenavailable) and the cell types derived fromthe labeled cells in each case are listed atthe endpoint. Gray boxes indicate singlestudies [from top to bottom: Rios et al.(2014); van Amerongen et al. (2012); VanKeymeulen et al. (2011)]. It is instantlyapparent that K5, Lgr5 and K14 labeling isnot consistent across studies.

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multicolor labeling approaches, most alveoli were shown to expressone to four distinct colors, suggesting that, on average, two luminalprogenitors give rise to each alveolus (Fig. 4D) (Rios et al., 2014).When tracing the bipotent K5+, K14+ or Lgr5+ populations, bothlabeled luminal alveolar and myoepithelial cells are observed.However, multicolor experiments suggest that contiguous alveolarand myoepithelial cells do not arise from the same progenitor. Thissuggests that, although bipotent progenitors in the adult gland cangive rise to alveolar cells, these pass through a luminal progenitorstate first (Rios et al., 2014). However, a separate study usinglineage tracing of Axin2, which is expressed exclusively in basalcells in the gland of virgin mice, showed that the induction ofpregnancy resulted in both basal cells and luminal alveolar cellswithin the same alveoli being labeled, suggesting that alveolar cellsarise from a bipotent stem cell instead of a luminal progenitor (vanAmerongen et al., 2012). Additionally, a separate, rare, populationof Notch2-expressing luminal cells appears necessary for tertiarybranching and formation of alveoli during pregnancy, although thelineage and properties of this cell type have not been fully explored(Šale et al., 2013).The fate of alveolar cells during involution remains unclear.

Using the luminal progenitor cell marker Elf5, one study showedthat a new pool of Elf5-labeled luminal progenitors gave rise toalveolar luminal cells in pregnancy, but that this labeled populationthen died off during involution, to be replaced with a new pool ofprogenitors for subsequent rounds of pregnancy (Rios et al., 2014).However, a different study showed that descendants of K8- or K18-labeled luminal progenitor cells persist through multiple cycles ofpregnancy (Van Keymeulen et al., 2011). A different study found

that progeny of secretory alveolar cells, marked according to wheyacidic protein promoter (Wap) expression, persist through multiplecycles of pregnancy and involution. These parity-induced epithelialcells are found in the lumens between pregnancy and in thesecretory alveoli during pregnancy; they express markers of luminalcells, including Elf5, and appear to not express hormone receptors(Chang et al., 2014).

Potential causes of discrepancies in lineage-tracing studiesThe discrepancies between these various lineage-tracing studies canbe explained partially by differences in gene-labeling techniques,labeling times and induction agents (Rios et al., 2014). Some studieschoose to label fewer total cells by inducing with lower drugconcentrations (Van Keymeulen et al., 2011), which aids in theidentification of clonal patches, whereas others use strongerinduction techniques to label all potential cells of a given lineage(Rios et al., 2014), which compromises some ability to identifyclones. In addition, tamoxifen, which is commonly used for cre-loxrecombination, is in fact an estrogen blocker, and the doses of thisdrug used in floxing appear to alter mammary epithelial cell fatedecisions (Rios et al., 2014; Visvader and Stingl, 2014).

When a lineage is traced with a single-color label, it is verydifficult to determine whether adjacent, labeled cells are derivedfrom a single precursor or from two different ones (Van Keymeulenet al., 2011). The more recent use of multi-color labeling schemesmakes this less likely, as the probability of two cells being labeledwith the same color is related to the probability of labeling squaredtimes the probability of the two having the same color (1/16 for four-color labeling), but it is still possible that some single-color patches

Fig. 4. Mammary gland stem cells in normal development: models of stem cell behavior. (A) During puberty, MaSCs can be found, among other places, inthe terminal end bud of the murine mammary gland. Accordingly, confetti lineage tracing (A′) shows that a few such K5+ cells in the terminal end bud (inset) giverise to all the cells in mature ducts (Rios et al., 2014). (B) Other evidence from the adult virgin mouse gland suggests that bipotent stem cells can give rise to bothluminal and myoepithelial cells. In line with this, K14-based lineage tracing (B′) shows that both luminal and myoepithelial cells can be derived from K14+ (green)cells (Rios et al., 2014). (C) Some lines of evidence suggest that unipotent progenitor cells contribute to ductal maintenance in the adult gland. (C′) In a separatestudy, cells traced by labeling K14 (green) in puberty only give rise to or remain K14+ myoepithelial cells (red) 10 weeks after labeling (Van Keymeulen et al.,2011). (D) Finally, during pregnancy, alveolar progenitor cells in the luminal compartment give rise to the secretory alveolar cells. (D′) Luminal cells are oftenlabeled with a single color in the alveoli of K5-rtTA/TetO-cre/R26R-Confetti mice, indicating that alveoli are derived from one stem cell (Rios et al., 2014). Imagesshown in A′,B′,C′,D′ are reproduced with permission from Nature Publishing Group (Rios et al., 2014; Van Keymeulen et al., 2011).

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were not clonal. Although lineage tracing studies appear to besuperior for identifying the normal developmental potential ofvarious mammary gland stem and progenitor cell populations,further studies are needed to reconcile the relationship between theK5+, K8+, K14+, Lgr5+, Axin2+, CD1d+ and parity-inducedmammary epithelial cell populations (Chang et al., 2014; dosSantos et al., 2013; Plaks et al., 2013; Šale et al., 2013; VanKeymeulen et al., 2011; van Amerongen et al., 2012; Wagner et al.,2002).

The differentiation of mammary gland cell typesLineage tracing-based studies have thus revealed that, duringmammary gland development and homeostasis, bipotent stem cellsin the prepubertal gland give rise to both luminal and myoepithelialcells, possibly by passing through a unipotent progenitor state. Themechanisms governing the differentiation of mammary progenitorsand/or their progeny into luminal, alveolar or myoepithelial cells arenot well understood, but recent transcriptional profiling studiescoupled with mouse knockout models are very useful in providingnew insights into these cellular differentiation pathways. Here, wesummarize the factors known to act on epithelial cell differentiationin the murine mammary gland.

Luminal progenitor cell differentiationDuring pregnancy, luminal cells or luminal progenitors give rise toalveolar epithelial cells, and this differentiation is driven by anumber of factors. The transcription factor Gata-3, for example, isknown to be a crucial regulator necessary for luminal epithelialdifferentiation from luminal progenitors (Asselin-Labat et al., 2007;Kouros-Mehr et al., 2006). It has been demonstrated thatβ3-integrin/CD61 is a marker of luminal progenitor cells (Asselin-Labat et al., 2007, 2011); using transgenic models it was shown thatGata-3 expression is necessary for the differentiation of the CD61+

population into luminal cells and for alveolar development inpregnancy (Asselin-Labat et al., 2007). Furthermore, induction ofGata-3 in a MaSC-enriched population drove differentiation to analveolar luminal phenotype, defined by Wap and β-casein (Csn2 –Mouse Genome Informatics Database) expression (Asselin-Labatet al., 2007). These studies indicate that Gata-3 is a crucial regulatorof commitment and maturation of the luminal epithelial lineage.The massive hormonal changes in puberty, menstruation and

pregnancy profoundly alter breast structure, yet luminal progenitorcells themselves are believed to be hormone insensitive (Asselin-Labat et al., 2010; Joshi et al., 2010). The effects of hormones onMaSCs and progenitors are thus believed to occur via paracrinesignaling from adjacent cells. Indeed, recent studies have shownthat, in pregnancy, progesterone induces mature luminal cells tosignal to MaSCs via RANKLigand (Tnfsf11) in a Stat5a-dependentmanner (Obr et al., 2013). In line with this, using a floxed Stat5a/ballele and mammary-specific Cre expression, Yamaji et al. (2009)showed that Stat5a/b was required for alveolar differentiation but notfor ductal branching. It was shown also that RANK Ligand inducesexpression of the transcription factor Elf5 in CD61+ luminalprogenitors (Lee et al., 2013). When Elf5 expression is blocked, thepopulation of K8+K14+p63+ MaSCs expands and overaccumulatesin the mammary gland of pregnant mice, and the mammary glandsof these animals fail to lactate (Chakrabarti et al., 2012; Lee et al.,2013; Oakes et al., 2008). Accordingly, Elf5 conditional knockoutsexhibit a complete block in alveolar differentiation (Choi et al.,2009).Stat5a signaling induced by myoepithelial cell-derived

neuregulin 1 (Nrg1) is also necessary for MaSC activity

(Vafaizadeh et al., 2010; Forster et al., 2014). Nrg1 is expressedin myoepithelial cells in a p63-dependent manner and is detected byluminal cells via the Erbb4 receptor. This signaling induces theexpression of the Stat5a targets Elf5 and cyclin D1, necessary forluminal and luminal progenitor cell function (Forster et al., 2014).The expansion of progenitor cells also requires Myc, suggesting thatthis proto-oncogene has a physiological role in mammarydevelopment (Moumen et al., 2012). Finally, Lrg5 appears to actas a receptor for an alveologenic signal in pregnancy (de Visseret al., 2012; Plaks et al., 2013; Rios et al., 2014), and when eitherLgr5 or its ligand, R-spondin (de Lau et al., 2011), are absent,secretory alveoli fail to develop (Chadi et al., 2009; Chakrabartiet al., 2012).

Functional mammary differentiation is also mediated by contactof mammary epithelial cells with the ECM via laminin-111-ligationof integrins. Luminal cells express β-casein in response to laminin-111 signaling, independent of cell-cell contact (Streuli et al., 1991,1995). It was further demonstrated that there is a transcriptionalenhancer (BCE1) in the bovine β-casein promoter that is responsiveto prolactin and ECM signaling (Schmidhauser et al., 1992), andlaminin-111 was shown to be the specific protein mediating theECM signaling (Streuli et al., 1995). Molecular characterization ofthe BCE1 enhancer revealed two essential regions that bind C/EBP-β (Cebpb –Mouse Genome Informatics Database) and Stat5 (Myerset al., 1998). Importantly, the enhancer had to be integrated into thegenome in order to be activated by laminin-111 and prolactin,suggesting that chromatin structure probably plays an essential rolein activation of this element (Myers et al., 1998). In line with thisfinding, later studies determined that sustained activation of Stat5mediated by laminin-111 is essential for chromatin remodeling andβ-casein transcription (Xu et al., 2009).

Myoepithelial cell differentiationMammary myoepithelial cells are identified by the expression ofspecific proteins, including keratin isoforms and contractileproteins [reviewed by Moumen et al. (2011)]. However, thetranscription factors that mediate the differentiation of a basalprogenitor into a myoepithelial cell in vivo are not well understood.In the human breast, p63 has been identified as a potential mediatorof the basal phenotype (Yalcin-Ozuysal et al., 2010). Thedifferentiation of myoepithelial cells is dependent on serumresponse factor (Srf ), as demonstrated by studies of the knockoutmouse model of the Srf coactivator myocardin-relatedtranscription factor A (MRTF-A; Mkl1 – Mouse GenomeInformatics Database), which reveal that MRTF-A is required formyoepithelial differentiation upon lactation (Li et al., 2006; Sunet al., 2006). Additional transcription factors, such as Slug andSmad3, as well as Notch signaling, have been implicated in thebasal phenotype [reviewed by Moumen et al. (2011)]; however, itis clear that still more studies are required to understand themechanisms underlying myoepithelial differentiation.

The microenvironment as a regulator of mammary glanddevelopment and homeostasisThe maintenance and differentiation of the various mammarygland cell types is also dependent on the features and properties ofthe local tissue microenvironment, in particular those of thesurrounding ECM. The importance of the ECM and stroma inmammary gland development and function were proposed severaldecades ago [(Williams and Daniel, 1983), reviewed by Varnerand Nelson (2014)]. Chimeric recombination models [reviewed byNelson and Bissell (2006)] demonstrated the power of the stroma

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to influence the developing epithelium. When mammaryepithelium was recombined with mammary mesenchyme, notsurprisingly, the outcome was that of a typical mammary ductaltree. However, mammary epithelium recombined with salivarygland mesenchyme resulted in structures that resembled salivarygland epithelium (Sakakura et al., 1976). On the other hand,outgrowths of salivary epithelium in contact with mammarymesenchyme resembled a mammary gland ductal tree and couldeven become competent for lactation and respond to hormonalstimuli (Cunha et al., 1995). These and other studies revealed thatthe epithelial component is highly malleable and that cell fate andtissue function are strongly influenced by the stromal componentof the gland.More recent studies (Bruno and Smith, 2012; Bussard and Smith,

2012) demonstrated, amazingly, that non-mammary cells and evenhuman cancer cells can be reprogrammed and incorporated intomammary outgrowths that are capable of self-renewal upon furthertransplantation into a murine cleared mammary fat pad (Boulangeret al., 2013, 2012, 2007). In these studies, co-injection withmammary epithelial cells was required, indicating that a ‘stem cellniche’, which includes the microenvironment and ECMcomponents, is also necessary for signaling from bona fidemammary epithelial cells. How does the tissue microenvironmentsupport growth, cellular differentiation and development of themammary gland? As we discuss below, an intricate signalingnetwork exists between the epithelium and its microenvironment,and includes signaling in response to mechanical forces and cell-cellcontact, signaling from the ECMmolecules, stromal-derived growthfactors and cytokines, and the activities of proteolytic enzymes inthe microenvironment.

The role of cell contacts and mechanical forcesMammary cells are tightly connected both to each other and to theirsurrounding environment, and they require this connection fornormal function. Myoepithelial cells, for example, are anchored toother myoepithelial cells and luminal epithelial cells viadesmosomes, and to the BM via hemidesmosomes (Adrianceet al., 2005; Pandey et al., 2010). Similar properties, mediated by theintegrin α6β4 complex, are exhibited by mammary luminal cellswhen cultured in laminin-rich, three-dimensional (3D) ECM gels(Weaver et al., 2002). In vivo, the two layers of the duct expressdifferent cell-cell contact mediators (Chanson et al., 2011; Huebneret al., 2014; Mroue et al., 2014) and cell-ECM connections (Brizziet al., 2012); as a result, the layered structure of the duct self-organizes when luminal and myoepithelial cells are mixed in 3Dcultures (Chanson et al., 2011; Gudjonsson et al., 2002; Huebneret al., 2014; Runswick et al., 2001). Furthermore, these connectionsare necessary for normal cell function: mammary cells in thelactating gland require E-cadherin to survive (Boussadia et al.,2002). Together, these tight connections between mammary cellsallow the transmission of mechanical forces and biochemicalsignals throughout the growing epithelium and allow collective cellmotion (Gjorevski and Nelson, 2010), in which mechanically activecells push or pull more passive cells along with them (Rørth, 2012).

ECM-mediated control of mammary gland developmentThe ECM is a major regulator of epithelial architecture and function.Within the mammary gland, myoepithelial cells reside on aspecialized layer of ECM, termed the basement membrane (BM).Myoepithelial and stromal cells synthesize ECM components, suchas laminins, collagens, fibronectin and proteoglycans, which areincorporated into theECMaswell as theBM.Thesematrices provide

not only physical support for correct tissue architecture but are alsoessential biochemical signaling networks that guide cellular fate andfunction of the gland. The BM also anchors the myoepithelial cellsby ligating cell surfacemolecules into position and by organizing thebasal side of the myoepithelium, through myoepithelial-secretedlaminin-111 and hemidesmosomes (Inman et al., 2011).

Studies using microenvironmental protein microarrays havestarted to uncover the ECM molecules that influence cell fatedecisions in mammary progenitor cells (LaBarge et al., 2009; Linet al., 2012). These studies revealed that laminin-111 regulatesmammary progenitor self-renewal, whereas other combinations ofECM proteins result in growth, differentiation and apoptosis(LaBarge et al., 2009). As discussed above, myoepithelial cellsare a major source of laminin-111 of the BM, and it is this lamininthat is essentially responsible for establishing luminal epithelial cellapical-basal polarity (Gudjonsson et al., 2002; Weir et al., 2006).Furthermore, because of their position and function within themammary tissue, myoepithelial cells are thought to act as tumorsuppressors in the adult gland (Adriance et al., 2005; Bissell andLaBarge, 2005; Pandey et al., 2010; Sternlicht and Barsky, 1997;Sternlicht et al., 1997).

Other stromal ECM proteins are important for instructing thedevelopment of the mammary arboreal structure. For example, recentstudies show that collagen I fiber orientation in the mammary fat padis a patterning cue for mammary branch orientation duringdevelopment (Brownfield et al., 2013). These collagen I fibersappear to be oriented prior to mammary branching morphogenesis,indicating that epithelial architecture might be pre-patterned in thestroma of the pubertal mammary gland. Such structural components,which support tissue architecture, are often overlooked as regulatorsof morphogenesis, but we now understand that correct tissuearchitecture and stiffness of the ECM are essential components ofnormal development, differentiation and function within themammary gland (Bissell et al., 1982; Maller et al., 2013; Schedinand Keely, 2011). Future studies of the stroma in mammary glanddevelopment would undoubtedly provide additional clues into therole of stroma in breast cancer initiation and progression.

Proteases are essential regulators of mammary glandmorphogenesis and differentiationProteolytic action remodels the ECM and stroma, and releasessequestered growth factors and cytokines. Not surprisingly,proteases are essential for mammary gland development andfunction. For the epithelial rudiment to fill the fat pad uponhormonal signals during puberty it is essential for the cells to take onan invasive phenotype and for proteinases, in this case matrixmetalloproteinases (MMPs), to pave the way by remodeling theECM (Talhouk et al., 1991). To understand the role of proteinases inthe mammary gland, a number of transgenic mice in which aparticular proteinase (or proteinase inhibitor) is silenced oroverexpressed have been developed. Although compensation bydifferent MMPs makes it difficult to exclusively define the functionof a single proteinase in vivo, the transgenic mouse models haveprovided insight into the importance of proteinases duringmammary gland development and function. Additionally, 3Dphysiologically relevant organotypic culture models have shedlight on the importance of both the catalytic activity and the newlydiscovered functions of the non-catalytic domains of proteinases inmammary gland development (Correia et al., 2013; Kessenbrocket al., 2013; Mori et al., 2013).

Several different classes of proteases are necessary for propermammary gland development and function. Proteinases provide

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local environmental signals to promote EMT and invasion. Forexample, we showed more than two decades ago that Mmp3 has acrucial role in remodeling the mammary gland in involution(Talhouk et al., 1991), and that its aberrant activity in 3D collagengels leads to EMT and a premalignant phenotype (Lochter et al.,1997). Others have shown that cathepsins play important rolesduring involution and apoptosis of the mammary gland (Sloane,2012; Watson and Kreuzaler, 2009). Serine proteases areimportant as well: mice deficient for the serine proteaseplasminogen have difficulties supporting lactation, due to adisruption in factors that control involution (Green et al., 2006).Other studies identified the serine protease plasminogen activatorkallikrein as being important for adipocyte differentiation in themammary gland. Kallikrein is thought to play a role in theplasminogen cascade of remodeling the fibrin-rich pre-adipocytestromal ECM (Selvarajan et al., 2001).Themostwidely studied enzymes in the context ofmammary gland

development and differentiation are metalloproteinases, whichinclude MMPs and the ‘a dysintegrin and metalloproteinases’(ADAMs). MMPs consist of a family of over 20 zinc-dependentproteinases synthesized as latent enzymes that must be activated post-translationally (Kessenbrock et al., 2010). Their activity is modulatedby endogenous inhibitors referred to as tissue inhibitors ofmetalloproteinases (TIMPs) (Murphy, 2011). Collectively, MMPscan degrade all protein components of the ECM, andwe now know ofa number of other proteins, including E-cadherin (Lochter et al.,1997). Over the past decades, many transgenic mouse models havebeen developed to investigate MMP function in development andcancer (Gill et al., 2010;Wiseman andWerb, 2002). Interestingly, theMmp14knockout is the onlysingleMMPgeneknockoutmodel that islethal. Overexpression of Mmp14 (Ha et al., 2001) or Mmp3(Sympson et al., 1994) in the mammary gland leads to excessive sidebranching and advanced alveolar morphogenesis (Fata et al., 2004).Increased levels of the active form of Mmp3 not only causessupernumerary branching in themammarygland but is responsible forcausing production of reactive oxygen species (ROS), changes insplicing of Rac1, EMTand genomic instability (Radisky et al., 2005),which precedes mammary tumor development (Sternlicht et al.,1999). Crossing the Mmp3-overexpressing mouse model with amouse overexpressing Timp1 decreases the detrimental effects ofMmp3 overexpression/activity. These data support the notion thatMmp3 activity in the mammary gland is important for branchingmorphogenesis, but indicate that unscheduled activity alters themammary tissuemicroenvironment. Furthermore, the fact thatMmp3activity could be tempered by Timp1 emphasizes the importance of abalance between enzymes and inhibitors for proper tissuedevelopment and homeostasis, and might explain why TIMPs arealso risk factors in mammary cancer.Other studies, using a mouse model in which Mmp3 has been

genetically suppressed, have revealed a role for Mmp3 in adipocytedifferentiation (Alexander et al., 2001). Timp1 overexpression givesrise to a phenotype similar to that seen following Mmp3 depletion,supporting the idea that MMP activity, in particular Mmp3 activity,is important for adipocyte differentiation during mammary glandinvolution. In studies in which slow-release pellets of Timp1, 2, 3 or4 were implanted into mouse mammary glands, it was shown thatTimp1, 3 and 4 inhibited ductal elongation, most likely viainhibition of MMP activity. However, Timp2 had an elongationpromoting effect (Hojilla et al., 2007). On the surface, the Timp2result seems contradictory, but increased levels of Timp2 mostlikely raise the levels of Mmp2 activation by increasing theformation of a tertiary complex (Mmp14-Timp2-Mmp2) that is

responsible for Mmp2 activation in vivo (Ellerbroek and Stack,1999; English et al., 2006).

To unravel the mechanisms underlying these observationsin vivo, cell culture studies have been most informative. Thesestudies have revealed that active Mmp3 increases ROS levels,leading to genetic instability and EMT (Radisky et al., 2005), whichcontribute to tumor development in the aging gland. More recently,non-proteolytic functions of Mmp3 (within the hemopexin domain)were found to be crucial for EMT and invasion during branchingmorphogenesis (Correia et al., 2013). Proteomic analysis of Mmp3hemopexin domain binding partners revealed, surprisingly, that thechaperone heat shock protein, 90β (Hsp90β) interacts specificallywith the hemopexin domain of Mmp3 in the extracellular space andthat this interaction is crucial for Mmp3 function. Other work hasimplicated the hemopexin domain of Mmp3 as a regulator of Wntsignaling andMaSC activity (Kessenbrock et al., 2013). In line withthis, mice deficient for Mmp3 exhibit decreased numbers of MaSCsand diminished mammary-reconstituting activity. Conversely, inthe same study it was shown that Mmp3 overexpression elevatedMaSC function. Together, these observations suggest that Mmp3activity is necessary for the maintenance of MaSCs.

Three-dimensional cell culture models have also been essentialfor elucidating the many functions of MMPs in normal andmalignant mammary glands (Barcellos-Hoff et al., 1989; Petersenet al., 1992). For example, investigations into the role of Mmp14 inmammary development were hampered due to the lethality of thisgene knockout. However, using micropatterned gels of collagen I, itwas revealed that the hemopexin domain of Mmp14 is important forsorting mammary epithelial cells to points of branching, thushighlighting that the non-proteolytic domains of Mmp14 also areessential for proper branching morphogenesis (Mori et al., 2009). Inaddition to the hemopexin domain, recent evidence indicates thatthe short intracellular domain of Mmp14 is crucial for epithelial cellinvasion (Mori et al., 2013). Using engineeredMmp14 constructs inwhich different domains were deleted, we discovered that only theshort intracellular domain of Mmp14 is needed to rescue branchingmorphogenesis in Mmp14-deficient cells, despite the fact that thissequence does not contain kinase activity. This deficiency iscompensated for by integrin β1, which interacts with the shortcytoplasmic domain of Mmp14. It is required for interaction withthe ECM, and for transducing the extracellular signals needed forepithelial cells to invade. These recent observations provide strongevidence that MMPs are important not only for ECM remodelingbut also for the microenvironmental signaling necessary formorphogenic programs within the mammary gland.

Combining mouse models with physiologically relevant 3Dmodels of human cells will allow further investigations into themechanisms of action of these proteinases. This information isimportant not only for understanding tissue development andmammary function, but also for identifying new targets for cancertherapy, as researchers focus on the new functions of the non-catalyticdomains of MMPs as regulators of tissue morphogenesis and tumordevelopment (Dufour and Overall, 2013; Rodríguez et al., 2010;Strongin, 2010). The non-catalytic domains are targetable usingantibodies or blocking peptides directed specifically against thosedomains (Basu et al., 2012). Such biological pharmaceuticals directedto domains other than the catalytic domains of these proteinases surelywill provide more specificity in therapy.

ConclusionsLike all other organs, the mammary gland is composed of manyspecialized cell types that carry out mammary functions, with

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interconnected signaling occurring between the different cellularcompartments. With its unique developmental mode occurringessentially after birth and its remarkable regenerative properties, themammary gland provides a superb model for investigatingdevelopmental programs, stem and progenitor cell properties, andthe stability of the differentiated state.Early serial transplant experiments and transgenic mouse models

have shed light on the identity and role of MaSCs and progenitorcells. More recently, lineage-tracing experiments have identifiedmultiple different, and somewhat conflicting, populations of stemand progenitor cells. Despite the discrepancies, these studies havebegun to fine-tune our understanding of MaSCs and how they drivedevelopment of the gland and maintain homeostasis of the resultingarboreal architecture. Additionally, the results of a number of recentstudies are revealing the role MaSCs play in the many cycles ofproliferation and apoptosis needed both to expand and maintain thegland form and functions during pregnancy and to return it to aquiescent state after involution.Many questions remain. Even the most fundamental puzzles – for

example, what signals drive cells down a particular lineage path;and why does this go wrong in cancer? – are not clearly delineated.Of course, interactions and signaling between cells are important,but a major driver of differentiation appears to be signaling from thetissue microenvironment, especially from the ECM in general andthe BM in particular. For decades, the ECM and BM were thoughtto be the inert ‘bricks and mortar’ of a tissue, simply providingphysical structure. We now know that correct tissue architecture,including the organization and stiffness of the ECM, together withthe reservoir of growth factors, cytokines and proteinases withinwhich the stem cell niche nestles, are essential for mammaryglandular tissue to develop and function properly.

Competing interestsThe authors declare no competing or financial interests.

FundingC.R. was supported by a fellowship from the Congressionally Directed MedicalResearch Programs of the US Department of Defense (DoD). M.J.B. was funded byCongressionally Directed Medical Research Programs of the DoD, The BreastCancer Research Foundation and The National Cancer Institute. This work was alsosupported by the US Department of Energy Office of Biological and EnvironmentalResearch and Low Dose Radiation Program.

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