© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
REVIEW
Mammary gland development: cell fate specification, stem cells
and the microenvironment
Jamie L. Inman*, Claire Robertson*, Joni D. Mott and Mina J. Bissell‡
The development of the mammary gland is unique: the final stages of
development occur postnatally at puberty under the influence of
hormonal cues. Furthermore, during the life of the female, the
mammary gland can undergo many rounds of expansion and
proliferation. The mammary gland thus provides an excellent model
for studying the ‘stem/progenitor’ cells that allow this repeated
expansion and renewal. In this Review, we provide an overview of
the different cell types that constitute the mammary gland, and
discuss how these cell types arise and differentiate. As cellular
differentiation cannot occur without proper signals, we also describe
how the tissue microenvironment influences mammary gland
development.
KEY WORDS: MMPs, Mammary gland, Microenvironment,
Progenitor cells, Stem cells
Introduction
The mammary gland, which distinguishes mammals from all other
animals, functions to produce and secrete milk in order to nourish
offspring. It is also a unique glandular organ in that it reaches full
development only after birth. As such, the mammary gland provides
a unique model for biologists to study development and organ
specificity. The embryonic rudiment of the gland, the anlage, is
present at birth and, in response to hormonal cues, begins branching
into the fat pad as the female reaches puberty. During the lifetime of
the female, the mammary gland undergoes many changes in
structure and function, including cyclic expansions corresponding
to the hormonal changes induced by the estrous/menstrual cycle, as
well as the dramatic changes that occur during pregnancy, lactation
and involution. During these different stages, the cells of the
mammary gland proliferate, differentiate or apoptose in response to
stimuli, giving rise to significant remodeling of the glandular tissue
architecture.
Indeed, studies of mammary gland development have offered
unique insights into the mechanisms regulating cell fate
specification, cell and tissue polarity, branching morphogenesis
and the involution of a functional organ. Moreover, many
dysregulated pathways and processes observed in breast cancer
progression mimic those observed during normal mammary gland
development and tissue remodeling; these developmental programs
are thus of interest also to cancer biologists. Here, we provide an
overview of mammary gland development, highlighting the
different cell types that make up the murine mammary gland, with
a particular focus on mammary gland ‘stem/progenitor cells’. We
also discuss how external factors in the mammary gland
Life Sciences Division, Lawrence Berkeley National Laboratory, University of
California, Berkeley, CA 94720, USA.
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
1028
microenvironment, such as extracellular matrix (ECM) and
cell-cell interactions, influence cell fate and function.
An overview of embryonic mammary gland development
In mice, embryonic mammary gland development occurs between
embryonic day (E) 10.5 and E18.5 (Hens and Wysolmerski, 2005;
Sakakura, 1987; Veltmaat et al., 2003). It begins when the singlelayered ectoderm enlarges to form the mammary lines on E10.5.
These lines of cells extend from the anterior limb bud to the posterior
limb bud. It is thought that mammary line cells then migrate to the
location of the future mammary buds (five pairs in mice) (Hens and
Wysolmerski, 2005; Propper, 1978; Robinson, 2007). At E11.5,
lens-shaped multilayered ectodermal structures called placodes are
observed, rising slightly above the surrounding ectoderm. The
mammary placodes then become bulbs of epithelial cells that are
distinct from the surrounding epidermis. These buds are elevated
knob-like structures at E12-E13.5 but sink into the underlying
dermis at around E13.5 (Sakakura, 1987; Watson and Khaled,
2008). Mesenchymal cells around the sunken bud condense and
become the mammary mesenchyme. Androgen receptor activation
in the mesenchyme of male embryos between E13.5 and E15.5
signals for the degradation of the mammary buds (Sakakura, 1987).
A second mesenchyme, the fat pad precursor, differentiates beyond
the mammary mesenchyme at E14.5 (Sakakura, 1987). Female
mammary gland development continues at E15.5, with epithelial
cell proliferation and elongation in the bud leading to the formation
of a sprout that invades the fat pad precursor. The nipple is formed
from epidermal cells overlying the bud, and a lumen is formed in the
sprout at E16.5 (Hogg et al., 1983). The sprout then branches into
the fat pad, giving rise to the rudimentary ductal tree by E18.5
(Sakakura, 1987).
An overview of postnatal mammary gland development
From birth to puberty, the mammary epithelium originating at the
nipple remains quiescent (Fig. 1A). During puberty, and under
the control of hormones and other factors, the ductal epithelium of the
mammary anlage invades into the mammary fat pad (Fig. 1B) in a
process referred to as branching morphogenesis (Lyons, 1958; Nandi,
1958). Highly proliferative terminal end buds, which contain an outer
layer of cap epithelial cells surrounding multilayered body epithelial
cells located at the invading front of the branch, lead the way
(Silberstein and Daniel, 1982; Williams and Daniel, 1983). The
invading epithelial cells display some characteristics of mesenchymal
cells, suggesting that some degree of epithelial-to-mesenchymal
transition (EMT) occurs at the end bud (Kouros-Mehr and Werb,
2006; Nelson et al., 2006). However, unlike tumor cells, EMT genes
employed during branching morphogenesis are highly regulated.
Indeed, recent work has shown that the transcription factor Ovol2, a
master negative regulator of EMT, is required during mammary gland
morphogenesis to regulate the expression of EMT genes (Watanabe
et al., 2014). The mechanism by which the branching process stops
DEVELOPMENT
ABSTRACT
REVIEW
Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
Birth
Cell types of the mammary gland and their specification
C
E
Key
Epithelial cells
Involution
D
Lactation
Virgin
As with most glandular tissues, the adult mammary gland is
composed of multiple cell types, including epithelial, adipose,
fibroblasts, immune, lymphatic and vascular cells, that work
together to sculpt and maintain a functional organ. These different
cell types have been demonstrated to be of importance at specific
stages of mammary gland development.
Alveolar cells
Epithelial body cells
Epithelial cap cells
Luminal epithelial cells
Myoepithelial cells
Fig. 1. The mammary gland development is multistage and occurs after
birth. (A) The mammary anlage is present at birth and remains quiescent until
puberty. (B) Due to puberty hormones, the epithelial ductal cells expand into
the mammary fat pad, led by highly proliferative multilayered terminal end
buds (TEBs; inset). The TEBs have an outer layer of epithelial cap cells that
surround multilayered epithelial body cells in rodents. (C) The mammary gland
of adult virgin mice is filled with epithelial branching structures. The ducts of
this structure (inset) contain an outer layer of myoepithelial cells and an inner
layer of luminal epithelial cells. (D) Pregnancy is accompanied by different
hormonal changes that signal a large expansion of alveolar cells that mature to
milk-secreting acini/alveoli during lactation. The alveoli (inset) expand out
from the ducts filling the majority of the fat pad. (E) Upon weaning, involution
proceeds through cell death and ECM remodeling, giving rise to a state
that resembles the resting adult mammary gland. Schematic reproduced
with permission from Chuong et al. (2014) and Hennighausen and
Robinson (2005).
once the fat pad is filled involves the production and activation of
endogenous TGFβ, and is most likely regulated by mechanical and
local cues from the gland architecture (Nelson et al., 2006), although
the exact steps remain an intriguing puzzle for both cell and
developmental biologists.
The final arboreal, bilayered ductal structure is composed of
apically oriented luminal epithelial cells that are surrounded on
the basal side by contractile myoepithelial cells (Fig. 1C). In the
gland of virgin mice, the epithelium proliferates and apoptoses
during each estrus cycle (Fata et al., 2001). However, during
pregnancy, the alveolar epithelium proliferates rapidly in response
to circulating hormones, developing secretory alveoli that are
capable of producing milk (Fig. 1D). During lactation, the apically
oriented luminal epithelial cells synthesize and secrete milk
proteins into the lumen of the alveoli; the release of oxytocin
caused by the suckling infant causes the contraction of the
surrounding myoepithelial cells, thus moving the milk through the
ductal tree and to the nipple. During the process of weaning, the
stimuli for milk production are lost and the expanded epithelial
compartment apoptoses in an event referred to as ‘involution’. The
Multiple epithelial cell types can be found in the mammary gland. The
mammary bilayer found throughout the adult gland of virgin mice is
traditionally described as being composed of apically oriented
luminal epithelial cells that line the ducts and of basally oriented
myoepithelial cells in contact with the basement membrane (BM)
(Fig. 1C, inset). Luminal cells express keratins 8 and 18, whereas
myoepithelial cells express keratins 5 and 14 as well as smooth muscle
actin that mediates their contractile function. In addition to
myoepithelial cells, cell-sorting experiments have identified several
putative stem and progenitor cell types in the basal cell population
which we will discuss in later sections (Shackleton et al., 2006; Stingl
et al., 2006; Visvader and Stingl, 2014).
Throughout puberty and pregnancy, there appear to be unique
cell types undoubtedly relating to the mammary functions at these
developmental phases. During puberty, for example, cap cells and
body cells, which are both specialized epithelial cells, arise in the
end bud. Cap cells, so named because they line the end bud forming
the cap of the structure (Fig. 1B, inset), contact the surrounding
stroma through a thin basal lamina and appear to differentiate into
myoepithelial cells that generate a thicker basal lamina (Daniel and
Silberstein, 2000; Williams and Daniel, 1983). The body cells, by
contrast, fill the interior of the end bud. The central body cells then
apoptose to form the lumen, and the remaining body cells
differentiate into luminal epithelial cells, giving rise to the ductal
epithelium of the adult breast (Fig. 1C, inset) (Hennighausen and
Robinson, 2005). During pregnancy, luminal epithelial cells rapidly
expand, forming alveoli that are lined with cells primed to secrete
milk at parturition (Fig. 1D, inset).
Adipocytes
Fat-filled adipocytes comprise a large proportion of the stromal fat
pad in the adult and non-lactating gland. The dense fat pad
precursor, which is observable at E14, develops throughout
embryogenesis, yet conversion to typical white fat tissue is not
observed until two to three days after birth (Sakakura, 1987).
During pregnancy and lactation, adipocytes with reduced lipid
content are observed, suggesting that this reservoir of fat is
necessary for the metabolically demanding process of milk
production (Gregor et al., 2013; Hovey and Aimo, 2010).
Adipocytes also serve an endocrine function in the gland: they
are thought to regulate epithelial growth and mammary epithelium
function, as well as to communicate with other cell types in the
mammary gland (Bartley et al., 1981; Hovey and Aimo, 2010). As
an example, adipocytes secrete vascular endothelial growth factor
(VEGF) and probably regulate angiogenesis in the mammary
gland (Hovey et al., 2001).
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DEVELOPMENT
B
gland is remodeled by a number of proteases, of which
metalloproteinase 3 (Mmp3) is the most prominent (Talhouk
et al., 1991), to return to the ‘resting’ state of the pre-pregnant
gland (Fig. 1E). Thus, the epithelial component and the
surrounding tissue architecture go through a significant amount
of remodeling during each pregnancy.
Puberty
A
Fibroblasts
Stromal fibroblasts are embedded within the fat pad and are often
found in close proximity to the basal side of the epithelial branching
tree (Muschler and Streuli, 2010; Sakakura, 1987). Fibroblasts
serve many functions, one of which is bi-directional communication
with the epithelium during branching morphogenesis, providing
instruction in the form of growth factors, proteases and other elements
(Howard and Lu, 2014). In vivo and in vitro studies suggest that
fibroblasts play an important role in supporting both epithelial cell
survival and morphogenesis in the fat pad (Liu et al., 2012; Makarem
et al., 2013; Wang and Kaplan, 2012). Furthermore, fibroblasts are
believed to be the chief stewards of the mammary ECM: they
synthesize a number of ECM components, such as collagens,
proteoglycans and fibronectin. Additionally, fibroblasts synthesize
many enzymes, such as matrix metalloproteinases, that are capable of
not only degrading the ECM but also of releasing the growth factors
and cytokines harbored or embedded within the ECM that influence
cellular and tissue function (Simian et al., 2001; Wiseman and Werb,
2002). As such, these cells can regulate epithelial cell features and the
epithelial cancer phenotype by altering ECM composition or density
(Lühr et al., 2012). It is important to note, however, that myoepithelial
cells produce copious amounts of laminin-111, which influences
many 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 cells
The mammary gland is intercalated with extensive vascular and
lymphatic networks present throughout the fat pad. During
pubertal mammary gland morphogenesis, the lymphatic network
develops in close association with the mammary epithelial tree and
blood vasculature (Betterman et al., 2012). Lymphangiogenesis in
the mammary gland is probably driven by myoepithelial-derived
VEGF-C (Vegfc – Mouse Genome Informatics Database) and/or
VEGF-D (Vegfd – Mouse Genome Informatics Database)
(Betterman et al., 2012). Immune cells, such as macrophages and
eosinophils, are also required for branching morphogenesis, and
they are recruited to the branching tips of the epithelium to mediate
invasion into the fat pad (Gouon-Evans et al., 2000). Macrophages
are also required for epithelial cell death and adipocyte
repopulation during involution (O’Brien et al., 2012). Through
activation of their serine proteases and degranulation, mast cells are
involved in normal mammary branching during puberty, and they
accumulate, and possibly activate, plasma kallikrein, thus
activating 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 ultimate
function in a dynamic and reciprocal fashion, the vast majority
of research has focused on the epithelium alone. This perhaps can
be explained because it is the epithelial cells that show dramatic
alterations in function and structure in pregnancy and lactation,
and because mammary tumors predominantly arise in the
epithelial compartment. In an effort to better understand
epithelial behavior, recent research efforts have focused on
elucidating the hierarchy of epithelial cell differentiation, as well
as identifying factors that can influence mammary gland function.
Mammary gland stem and progenitor cells: dramatic
regenerative potential
The mammary epithelium displays dramatic regenerative potential
and the ability to undergo many cycles of growth and involution,
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Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
suggesting that the mammary gland epithelial compartment
contains mammary epithelial stem cells, that is, single epithelial
cells capable of generating the entire epithelial architecture. The
remarkable regenerative capacity of mammary epithelium was
first demonstrated in the late 1950s and early 1960s by
transplanting small numbers of epithelial cells into mammary
fat pads that had been cleared of their epithelial rudiments (Daniel,
1975; Faulkin and Deome, 1960). These cells were able to
generate branching epithelia that filled the whole fat pad (i.e.
reconstitute the glandular epithelium), and cell populations from
these epithelia were able to reconstitute the gland again in
subsequent cleared fat pads, demonstrating strong stem cell-like
behavior. More recently, it was shown that an entire functional
mammary 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 epithelium
contains a stem cell population.
Since these demonstrations, multiple studies have focused on
identifying and isolating mammary stem cell (MaSC)
populations and defining the differentiation potential of
different 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 fluorescenceactivated cell sorting (FACS), to select different epithelial cell
populations, which are then tested for reconstitution efficiency
by injecting them into cleared mammary fat pads at limiting
dilutions (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 different
epithelial cell populations with stem cell properties have been
identified, some of which might act as stem cells during the nonphysiological process of cell transplantation but not during
normal development and differentiation. To ensure clarity, we
will hereafter refer to cells with the ability to reconstitute the
glandular epithelium on transplant as mammary glandreconstituting units (MRUs). These are distinct from cells that,
during normal development, give rise to both luminal and
myoepithelial cells, which we will refer to as bipotent MaSCs,
and cells that give rise to just a single lineage during normal
development, which we will refer to as unipotent mammary
epithelial progenitors.
Markers and features of mammary gland-reconstituting cells
Using cell surface marker sorting (see Table 1) and the gland
reconstitution assay, several different markers for MRUs have
been identified. The murine mammary epithelium is typically first
isolated 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 expression
of moderate-to-high levels of CD24 (heat-stable antigen), high
levels 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 markers
appears to vary between studies, making quantitative comparisons
of the prevalence of different populations difficult (Fig. 2).
Nonetheless, recent work suggests that the cells isolated via the
sorting approach described above are all myoepithelial:
myoepithelial cells expressing smooth muscle actin were the
only cells able to repopulate the gland in one recent study (Prater
et al., 2014).
DEVELOPMENT
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Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
Table 1. Mammary gland reconstitution studies and MRU markers
Methods
Cell markers
of MRU population
MRU frequency
or frequency of gland
reconstitution
Gland
reconstitution in
other cells studied
Markers which did
not show gland
reconstitution
Notes about MRU
populations
Smith, 2005
DNA label retention
Labeled in 3-weekold mice with
estradiol treatment
from week 3-8
DNA label retention
suggests that the
epithelium has
stem cells
Stingl et al.,
2006
FACS and gland
reconstitution from
8-14-week-old virgin
mice (in 2% FBS)
Cd31−CD45−Ter119−
CD140a−CD24med
CD49fhi
1/62 CD24med
CD49fhi (FVB mice)
1/91 CD24medCD49fhi
(C57BL/6 mice)
<1/230
CD24hiCD49flow
1/3400
CD24low
CD49flow
1/1400
unsorted cells
Sca1hi- induced
by culture, no
MaSC in Sca1hi
population
Hoechst 3342or Rhodamine
123-effluxing
cells 10% of
MaSC
Shackleton
et al.,
2006
FACS and gland
reconstitution from
8-week-old mice
(injected in 50%
FBS)
CD45−CD31−TER119−
CD24+CD29hi
1/64 Lin−CD29hi
CD24+
1/47,000 in
CD29lowCD24−
<1/7200 in
CD29low
CD24+
1/2900 in
CD29hiCD24−
0/22 in Lin+
1/30,000
Sca1hi (no
CD29loCD24+
Sca1hi cells
observed)
Hoechst 33342
exclusion – no
enrichment
over Lin−
CD29hiCD24+
Badders
et al.,
2009
FACS and gland
reconstitution from
virgin 12-14-weekold mice (injected in
50% matrigel)
CD45−CD31−
Lrp5+Lrp6+
1/485 Lrp5+
1/142 CD24+
CD49fhi
1/7760 in unsorted
population
1/100,000 Lrp5−
All Lrp5+ and Lrp6+
are K5+ or SMA+
basal cells but
not all are CD24+
CD49fhi. 40% of
Lrp5+ are Vwf+
Zeng and
Nusse,
2010
Transgenic Axin2
reporter, FACS and
gland
reconstitution from
8-12-week-old mice
(in 50% matrigel or
Wnt3a)
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 are
Axin2+
Spike et al.,
2012
FACS and gland
reconstitution from
fetal (E18.5) or
adult (age
unspecified) mice
(±50% matrigel)
CD31−CD45−Ter119−
CD24medCD49fhi
1/14 fetal CD24med
CD49fhi +matrigel
1/50 adult CD24med
CD49fhi +matrigel
1/400 fetal
CD24med
CD49f–matrigel
1/800 adult
CD24med
CD49fhi–
matrigel
dos Santos
et al.,
2013
Dox-induced DNA
label retention
+FACS and gland
reconstitution in 610-week-old virgin
mice (injected in 50%
matrigel)
CD31−CD45−Ter119−
(MACS depleted)
CD24+
CD29hiCD49fhiCD1d+
1/8 Lin−CD24+CD29hi
CD1d+
1/33 DNA-retaining
cells
1/44 Lin−CD24+
CD29hiCD49fhi
1/149 unlabeled
DNA
Machado
et al.,
2013
FACS and gland
reconstitution from
8-12-week-old mice
(in 50% matrigel)
CD3e−CD11b−CD45R−
Ly6G−Ly6C−TER119−
CD31−CD24+CD29hi
large (<10 μm
diameter)
1/66 Lin−CD24+
CD29hi large
1/132 Lin−
CD24+CD29hi
1/237 Lin− large
MRU location:
terminal end
buds
CD1d+ cells make
up 1% of basal
cells
Continued
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DEVELOPMENT
MaSC
marker
study
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Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
Table 1. Continued
MaSC
marker
study
Cell markers
of MRU population
Methods
MRU frequency
or frequency of gland
reconstitution
Gland
reconstitution in
other cells studied
Plaks et al.,
2013
FACS and gland
reconstitution on
cells from 7-9-weekold mice (in 50%
matrigel+FGF2)
CD31−CD45−Ter119−
CD24+CD49fhi
Ck14+Lgr5+
5/16 glands Lin−
CD24+CD49fhi
Ck14+Lgr5+
(10 cells)
Prater et al.,
2014
FACS, in vitro culture
and gland
reconstitution on
cells from 10-14week-old mice (in
25% matrigel)
CD31−CD45−Ter119−
CD49fhiEpCAMhi or
SMA+
1/57 EpCAMhi
1/79 cultured CD49fhi
cells
15/18 cultured
EpCAMhi colonies
1/93 SMA+
1/300 EpCAMlow
1/205 non-cultured
CD49fhi
11/18 cultured
EpCAMlow
colonies
>1/300 SMA−
Wang et al.,
2015
FACS and gland
reconstitution on
cells from 8-12week-old mice (in
50% 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+
Markers which did
not show gland
reconstitution
0/16 glands Lin−
CD24+CD49fhi
Ck14+Lgr5− (10
cells)
Notes about MRU
populations
Lgr5+ cells are 6%
of K14+ basal
cells
0/16 glands
CD24+ CD29hi
ProcR–Lgr5−
Abbreviations: MRU, mammary reconstituting unit; FACS, fluorescence-activated cell sorting; MACS, magnetic-activated cell sorting; Vwf, Von Willebrand factor;
SMA, smooth muscle actin; Ck, cytokeratin; FBS, fetal bovine serum; CD31−CD45−Ter119− (Lin−), lineage-marker cocktail negative.
B
CD24
A
CD29
Fig. 2. Flow cytometry-based studies of MRUs: discordance between
studies. Cell-surface marker expression levels in luminal cells (circled in blue)
and myoepithelial cells (circled in red) from adult (8-12-week old) murine
mammary glands show discordance between studies using similar
methodologies. (A) In one study (Shackleton et al., 2006), similar levels of
CD24 were reported in luminal and myoepithelial cells, with CD29 serving to
distinguish 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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Refining MRU surface markers by isolating DNA label-retaining cells
Several subsequent studies have since attempted to refine the surface
markers of MRUs and have identified several disparate populations
with gland-reconstituting capacity. The adult mammary gland
contains cells that retain their parental DNA strand, suggesting that
these 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 have
fivefold higher gland-reconstituting capacity compared with nonlabeled and non-label-retaining epithelial cells in a recent study (dos
Santos et al., 2013). Furthermore, the isolation and subsequent
profiling of DNA-retaining CD29highCD49fhighCD24+ cells has
been used to identify improved MRU markers. Through this work, an
additional cell surface marker, CD1d, a glycoprotein typically found
on antigen-presenting cells, was identified in the DNA labelretaining population (dos Santos et al., 2013). Adding this marker to
the current sorting technique led to a fivefold enrichment of MRUs
in the Lin−CD29highCD49fhighCD24+CD1d+ population to 1/8.
However, CD1d+ cells might not represent the entire stem cell
population, given the rarity of the CD1d+ population (1% of
CD29highCD49fhighCD24+) compared with the frequency of MRUs
observed in this study (1/44 Lin−CD24+CD29hi versus 1/8
Lin−CD24+CD29hiCD1d+), suggesting that CD1d+ cells are a
subset of MRUs. This study also did not determine whether the
Lin−CD24+CD29hiCD1d− population was depleted of stem cell
activity.
Refining MRU surface markers by interrogating the Wnt signaling
pathway
Another fruitful approach for isolating MRUs has been to identify
elements of the Wnt signaling pathway that are active in mammary
development, given the importance of Wnt signaling in stem cell
behavior (Reya and Clevers, 2005) and the high activation of the
Wnt/β-catenin pathway in DNA label-retaining cells (dos Santos
et al., 2013). Indeed, a downstream target of Wnt, the G-protein-
DEVELOPMENT
The reported likelihood of a single cell from this MRU-enriched
population in a limiting dilution giving rise to a full glandular
epithelium 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 be
compared with 1/1400 to 1/7600 in the unsorted Lin− epithelial
population (Badders et al., 2009; Shackleton et al., 2006) or less
than 1/3000 in the Lin−CD29− or Lin−CD24− MRU-depleted
epithelial populations (Shackleton et al., 2006). However,
Lin−CD24med-hiCD29highCD49fhigh populations are not pure
MRU populations; rather, they are MRU-enriched populations,
and the complement populations are mostly, but not completely,
MRU-depleted (Shackleton et al., 2006).
coupled receptor Lgr5, appears to act as a marker of stem cells in
several other organ systems (Barker et al., 2010, 2007; Jaks et al.,
2008). In the mammary gland, Lgr5+ cells are a subset of the keratin
14+ (K14) basal Lin−CD24+CD49fhigh MRU-enriched population
and are superior to their parent population in regenerating functional
mammary glands (de Visser et al., 2012; Plaks et al., 2013).
Supporting a role for Lgr5 in not just gland reconstitution but normal
development, loss-of-function and deletion experiments show that
Lgr5 and its principal ligand, R-spondin, are necessary for normal
postnatal mammary gland organogenesis (Chadi et al., 2009; de
Visser et al., 2012; Plaks et al., 2013). The Lin−CD24+CD49fhigh
Lgr5−-depleted population showed no gland-reconstituting ability in
one study (Plaks et al., 2013), whereas Lgr5− cells showed rare
repopulating activity in another (Rios et al., 2014). However, a third
study found the opposite, such that Lgr5− cells that also expressed
protein C receptor (ProcR) showed stronger MRU behavior than
Lgr5+ cells (Wang et al., 2015).
Other Wnt pathway elements, including the canonical Wnt
pathway receptors Lrp5 and Lrp6 (Goel et al., 2012), also appear to
mark MaSC-enriched populations (Badders et al., 2009; Lindvall
et al., 2009) compared with epithelial cells as a whole. Accordingly,
selection for Lrp5, an LDL receptor-related protein, increases gland
reconstitution efficiency compared with unsorted epithelial cells
(Badders et al., 2009), and Lrp6 is necessary for normal mammary
branching invasion in vivo (Lindvall et al., 2009). A very recent
study reported that ProcR, which is a Wnt3A target, is also a marker
of MRUs: CD24+CD29hiProcR+ cells showed much higher gland
reconstitution potential than their parent or ProcR-depleted
populations (Wang et al., 2015). Furthermore, anthrax toxin
receptor 1 (Antxr1), which also acts in the Wnt pathway, might
act as a stem cell marker based on in vitro colony-forming assays
(Chen et al., 2013). It will be of interest to investigate whether the
cell populations characterized by these various Wnt signaling
components overlap with the CD1d+ MaSC-enriched populations.
Issues with MRU studies
Although flow cytometry-based studies have been useful for
identifying various populations of MRUs and the markers that
they express, variations in FACS results and the frequency of
MRU detection are observed from study to study (Fig. 2). These
might partially be explained by methodological variations,
including those in donor age and transplant conditions. As mice
undergo puberty and massive changes in epithelial architecture
around week 6-8, followed by estrous cycles every 4 days, 6-weekold murine mammary glands might show a very different cellular
composition to that from 12-week-old mice. The estrus cycle can
also dramatically alter gland reconstitution efficiency, resulting in
tenfold higher MRU numbers during progesterone-high luteal
phases (Joshi et al., 2010). Likewise, we recently demonstrated that
very subtle differences in cell isolation can result in discordance in
FACS data (Hines et al., 2014). Furthermore, transplant conditions
can dramatically affect reconstitution efficiency, leading to a tenfold
increase in gland reconstitution in one study (Spike et al., 2012).
For example, the use of matrigel to prevent anoikis, and the
injection of epithelia in their native configuration with attached
stroma, both strongly increased gland reconstitution efficiency
(Spike et al., 2012). Likewise, the culture of myoepithelial cells for
one week in vitro was sufficient to massively increase MRU activity
(Prater et al., 2014). Given these many potential variables, a
consensus technical paper, comparing leading separation methods
and providing guidance for future studies, has been published
(Smalley et al., 2012).
Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
It should also be noted that, although gland reconstitution
studies have demonstrated the remarkable regenerative capacity
of MRUs, it is unclear whether normal mammary development
relies on the same cellular populations. Gland reconstitution
likewise involves injection site wounding, which might induce
different fate decisions than those occurring during normal
gland development (Plaks et al., 2013; Shackleton et al., 2006;
Van Keymeulen et al., 2011).
Mammary gland stem and progenitor cells: insights from
lineage tracing
Given some of the issues with FACS-based approaches, lineage
tracing of cell populations in transgenic animals is increasingly being
employed to define and better understand the stem and progenitor cell
populations in the mammary gland. However, such studies have also
yielded some conflicting results on the nature and potency of
mammary stem and progenitor cells during pubertal development,
pregnancy and involution (summarized in Figs 3 and 4).
Pubertal development might rely on bipotent MaSCs
Two single-color lineage-tracing studies concluded that
mammary development during puberty predominantly occurs
through unipotent progenitor cells, such that all basal cells arise
from basal progenitor cells alone, and all luminal cells from
luminal progenitors (Fig. 4C) (Van Keymeulen et al., 2011; van
Amerongen et al., 2012). By contrast, recent, extensive lineagetracing studies demonstrated that the pubertal gland contains
several different stem and progenitor cell populations, including
bipotent stem cells that give rise to both the luminal and
myoepithelial cells of the duct (Rios et al., 2014) (Figs 3 and 4).
This work used multicolor lineage tracing to mark single cells
and their clonal progeny, and confocal microscopy on thick
sections to analyze large regions of the ductal tree, thereby
allowing for more sensitive detection of rare cell types.
In this study, keratin 5 (K5)-expressing cells labeled at puberty
were all found in the basal compartment at the time of labeling, but
gave rise to contiguous patches of both luminal and basal cells,
suggesting bipotency (Rios et al., 2014). At the initial labeling time
point, all K5-labeled cells were in the basal compartment, as
detected both by FACS analysis (91% CD29hi, CD24+ and less
than 1% luminal CD29lo, CD24+) and by histopathology. At
1 week after K5 induction, no single-colored luminal patches were
observed, and 65% of clonal patches were solely myoepithelial.
After an 8-week chase, 61% of contiguous, single-color patches
had both luminal and myoepithelial cells, a result unlikely to be
due to adjacent, commonly colored stem or progenitor cells giving
rise to single-color patches (Fig. 4A′). Indeed, cells labeled for
either K5, K14 or Lgr5 in the prepubertal gland all appeared to give
rise to clonal patches of cells from both lineages when assessed
either by confocal microscopy or by FACS (Rios et al., 2014). A
small subset of dividing K5+ cells at puberty were also observed to
express markers of multiple lineages, including the basal marker
K14, the luminal marker Elf5 and the putative stem cell marker
Lgr5, suggesting that this cell population represents a progenitor
population.
In addition to these bipotent stem cell populations, Elf5+ luminal
cells labeled before puberty gave rise to luminal and alveolar cells
alone, suggesting that Elf5+ cells contain a luminal progenitor pool
(Fig. 4D) (Rios et al., 2014). This result is mirrored by the earlier
finding that keratin 8+ (K8) and keratin 18+ (K18) luminal cells in
the prepubertal gland give rise to luminal and alveolar cells alone
(Van Keymeulen et al., 2011).
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Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
Fig. 3. An overview of lineage tracing in
vivo. Different labeling-based
experiments, in which the fate of Elf5-,
K5-, K8-, K14-, K18-, Lgr5- or Axin2expressing cells were followed, are
represented by arrows, starting at the
developmental time of labeling and
ending at the time of harvest and analysis.
The location of labeled cells at the onset
of the experiment is listed (when
available) and the cell types derived from
the labeled cells in each case are listed at
the endpoint. Gray boxes indicate single
studies [from top to bottom: Rios et al.
(2014); van Amerongen et al. (2012); Van
Keymeulen et al. (2011)]. It is instantly
apparent that K5, Lgr5 and K14 labeling is
not consistent across studies.
When using lineage tracing in a postpubertal mammary gland of
virgin adult mice, contiguous patches of labeled cells were observed
in multiple studies (Rios et al., 2014; Van Keymeulen et al., 2011),
suggesting that, even in the quiescent gland, extensive cell turnover
and remodeling occurs. Using lineage tracing of Elf5+ luminal cells
to assess adult animals, large patches of luminal cells derived from
the same progenitor were observed at 8 weeks, but fewer clonal
patches were observed at 20 weeks, consistent with the expansion
and slow depletion of luminal progenitors over time (Rios et al.,
2014). When using lineage tracing of K5+ basal cells in the adult
gland, patches of both luminal and myoepithelial cells were
observed, including some physically coupled cell pairs,
suggesting recent asymmetric division (Rios et al., 2014). Over
long periods of time, fewer, larger patches, containing both luminal
and basal labeled cells were observed, suggesting massive clonal
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expansion of these K5+ bipotent progenitors during glandular
remodeling (Rios et al., 2014). Additionally, over long chases, a
shift from mixed or myoepithelial-only clonal patches to
predominantly luminal clonal patches was observed with this
labeling scheme, suggesting that bipotent progenitors give rise to a
population of luminal progenitors that then expands for luminal
ductal maintenance (Fig. 4B) (Rios et al., 2014).
Conflicting evidence for stem/progenitor cells in pregnancy and
involution
During pregnancy, the glands of virgin adult mice develop an
extensive network of secretory alveoli lined by specialized luminal
cells. These alveolar luminal cells are believed to be derived from
the ductal luminal cells seen in the adult virgin mice, based on
lineage-tracing experiments using luminal lineage markers, such as
Elf5, K8 or K18 (Rios et al., 2014; Van Keymeulen et al., 2011). In
DEVELOPMENT
Remodeling in the adult mammary gland of virgin mice
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Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
Fig. 4. Mammary gland stem cells in normal development: models of stem cell behavior. (A) During puberty, MaSCs can be found, among other places, in
the 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) give
rise 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 both
luminal 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 separate
study, 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 often
labeled 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). Images
shown in A′,B′,C′,D′ are reproduced with permission from Nature Publishing Group (Rios et al., 2014; Van Keymeulen et al., 2011).
that progeny of secretory alveolar cells, marked according to whey
acidic protein promoter (Wap) expression, persist through multiple
cycles of pregnancy and involution. These parity-induced epithelial
cells are found in the lumens between pregnancy and in the
secretory alveoli during pregnancy; they express markers of luminal
cells, including Elf5, and appear to not express hormone receptors
(Chang et al., 2014).
Potential causes of discrepancies in lineage-tracing studies
The discrepancies between these various lineage-tracing studies can
be explained partially by differences in gene-labeling techniques,
labeling times and induction agents (Rios et al., 2014). Some studies
choose to label fewer total cells by inducing with lower drug
concentrations (Van Keymeulen et al., 2011), which aids in the
identification of clonal patches, whereas others use stronger
induction techniques to label all potential cells of a given lineage
(Rios et al., 2014), which compromises some ability to identify
clones. In addition, tamoxifen, which is commonly used for cre-lox
recombination, is in fact an estrogen blocker, and the doses of this
drug used in floxing appear to alter mammary epithelial cell fate
decisions (Rios et al., 2014; Visvader and Stingl, 2014).
When a lineage is traced with a single-color label, it is very
difficult to determine whether adjacent, labeled cells are derived
from a single precursor or from two different ones (Van Keymeulen
et al., 2011). The more recent use of multi-color labeling schemes
makes this less likely, as the probability of two cells being labeled
with the same color is related to the probability of labeling squared
times the probability of the two having the same color (1/16 for fourcolor labeling), but it is still possible that some single-color patches
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multicolor labeling approaches, most alveoli were shown to express
one to four distinct colors, suggesting that, on average, two luminal
progenitors give rise to each alveolus (Fig. 4D) (Rios et al., 2014).
When tracing the bipotent K5+, K14+ or Lgr5+ populations, both
labeled luminal alveolar and myoepithelial cells are observed.
However, multicolor experiments suggest that contiguous alveolar
and myoepithelial cells do not arise from the same progenitor. This
suggests that, although bipotent progenitors in the adult gland can
give rise to alveolar cells, these pass through a luminal progenitor
state first (Rios et al., 2014). However, a separate study using
lineage tracing of Axin2, which is expressed exclusively in basal
cells in the gland of virgin mice, showed that the induction of
pregnancy resulted in both basal cells and luminal alveolar cells
within the same alveoli being labeled, suggesting that alveolar cells
arise from a bipotent stem cell instead of a luminal progenitor (van
Amerongen et al., 2012). Additionally, a separate, rare, population
of Notch2-expressing luminal cells appears necessary for tertiary
branching and formation of alveoli during pregnancy, although the
lineage 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 showed
that a new pool of Elf5-labeled luminal progenitors gave rise to
alveolar luminal cells in pregnancy, but that this labeled population
then died off during involution, to be replaced with a new pool of
progenitors for subsequent rounds of pregnancy (Rios et al., 2014).
However, a different study showed that descendants of K8- or K18labeled luminal progenitor cells persist through multiple cycles of
pregnancy (Van Keymeulen et al., 2011). A different study found
were not clonal. Although lineage tracing studies appear to be
superior for identifying the normal developmental potential of
various mammary gland stem and progenitor cell populations,
further studies are needed to reconcile the relationship between the
K5+, K8+, K14+, Lgr5+, Axin2+, CD1d+ and parity-induced
mammary epithelial cell populations (Chang et al., 2014; dos
Santos et al., 2013; Plaks et al., 2013; Šale et al., 2013; Van
Keymeulen et al., 2011; van Amerongen et al., 2012; Wagner et al.,
2002).
The differentiation of mammary gland cell types
Lineage tracing-based studies have thus revealed that, during
mammary gland development and homeostasis, bipotent stem cells
in the prepubertal gland give rise to both luminal and myoepithelial
cells, possibly by passing through a unipotent progenitor state. The
mechanisms governing the differentiation of mammary progenitors
and/or their progeny into luminal, alveolar or myoepithelial cells are
not well understood, but recent transcriptional profiling studies
coupled with mouse knockout models are very useful in providing
new insights into these cellular differentiation pathways. Here, we
summarize the factors known to act on epithelial cell differentiation
in the murine mammary gland.
Luminal progenitor cell differentiation
During pregnancy, luminal cells or luminal progenitors give rise to
alveolar epithelial cells, and this differentiation is driven by a
number of factors. The transcription factor Gata-3, for example, is
known to be a crucial regulator necessary for luminal epithelial
differentiation from luminal progenitors (Asselin-Labat et al., 2007;
Kouros-Mehr et al., 2006). It has been demonstrated that
β3-integrin/CD61 is a marker of luminal progenitor cells (AsselinLabat et al., 2007, 2011); using transgenic models it was shown that
Gata-3 expression is necessary for the differentiation of the CD61+
population into luminal cells and for alveolar development in
pregnancy (Asselin-Labat et al., 2007). Furthermore, induction of
Gata-3 in a MaSC-enriched population drove differentiation to an
alveolar luminal phenotype, defined by Wap and β-casein (Csn2 –
Mouse Genome Informatics Database) expression (Asselin-Labat
et al., 2007). These studies indicate that Gata-3 is a crucial regulator
of commitment and maturation of the luminal epithelial lineage.
The massive hormonal changes in puberty, menstruation and
pregnancy profoundly alter breast structure, yet luminal progenitor
cells themselves are believed to be hormone insensitive (AsselinLabat et al., 2010; Joshi et al., 2010). The effects of hormones on
MaSCs and progenitors are thus believed to occur via paracrine
signaling from adjacent cells. Indeed, recent studies have shown
that, in pregnancy, progesterone induces mature luminal cells to
signal to MaSCs via RANK Ligand (Tnfsf11) in a Stat5a-dependent
manner (Obr et al., 2013). In line with this, using a floxed Stat5a/b
allele and mammary-specific Cre expression, Yamaji et al. (2009)
showed that Stat5a/b was required for alveolar differentiation but not
for ductal branching. It was shown also that RANK Ligand induces
expression of the transcription factor Elf5 in CD61+ luminal
progenitors (Lee et al., 2013). When Elf5 expression is blocked, the
population of K8+K14+p63+ MaSCs expands and overaccumulates
in the mammary gland of pregnant mice, and the mammary glands
of these animals fail to lactate (Chakrabarti et al., 2012; Lee et al.,
2013; Oakes et al., 2008). Accordingly, Elf5 conditional knockouts
exhibit 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
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(Vafaizadeh et al., 2010; Forster et al., 2014). Nrg1 is expressed
in myoepithelial cells in a p63-dependent manner and is detected by
luminal cells via the Erbb4 receptor. This signaling induces the
expression of the Stat5a targets Elf5 and cyclin D1, necessary for
luminal and luminal progenitor cell function (Forster et al., 2014).
The expansion of progenitor cells also requires Myc, suggesting that
this proto-oncogene has a physiological role in mammary
development (Moumen et al., 2012). Finally, Lrg5 appears to act
as a receptor for an alveologenic signal in pregnancy (de Visser
et al., 2012; Plaks et al., 2013; Rios et al., 2014), and when either
Lgr5 or its ligand, R-spondin (de Lau et al., 2011), are absent,
secretory alveoli fail to develop (Chadi et al., 2009; Chakrabarti
et al., 2012).
Functional mammary differentiation is also mediated by contact
of mammary epithelial cells with the ECM via laminin-111-ligation
of integrins. Luminal cells express β-casein in response to laminin111 signaling, independent of cell-cell contact (Streuli et al., 1991,
1995). It was further demonstrated that there is a transcriptional
enhancer (BCE1) in the bovine β-casein promoter that is responsive
to prolactin and ECM signaling (Schmidhauser et al., 1992), and
laminin-111 was shown to be the specific protein mediating the
ECM signaling (Streuli et al., 1995). Molecular characterization of
the BCE1 enhancer revealed two essential regions that bind C/EBPβ (Cebpb – Mouse Genome Informatics Database) and Stat5 (Myers
et al., 1998). Importantly, the enhancer had to be integrated into the
genome in order to be activated by laminin-111 and prolactin,
suggesting that chromatin structure probably plays an essential role
in activation of this element (Myers et al., 1998). In line with this
finding, later studies determined that sustained activation of Stat5
mediated by laminin-111 is essential for chromatin remodeling and
β-casein transcription (Xu et al., 2009).
Myoepithelial cell differentiation
Mammary myoepithelial cells are identified by the expression of
specific proteins, including keratin isoforms and contractile
proteins [reviewed by Moumen et al. (2011)]. However, the
transcription factors that mediate the differentiation of a basal
progenitor into a myoepithelial cell in vivo are not well understood.
In the human breast, p63 has been identified as a potential mediator
of the basal phenotype (Yalcin-Ozuysal et al., 2010). The
differentiation of myoepithelial cells is dependent on serum
response factor (Srf ), as demonstrated by studies of the knockout
mouse model of the Srf coactivator myocardin-related
transcription factor A (MRTF-A; Mkl1 – Mouse Genome
Informatics Database), which reveal that MRTF-A is required for
myoepithelial differentiation upon lactation (Li et al., 2006; Sun
et al., 2006). Additional transcription factors, such as Slug and
Smad3, as well as Notch signaling, have been implicated in the
basal phenotype [reviewed by Moumen et al. (2011)]; however, it
is clear that still more studies are required to understand the
mechanisms underlying myoepithelial differentiation.
The microenvironment as a regulator of mammary gland
development and homeostasis
The maintenance and differentiation of the various mammary
gland cell types is also dependent on the features and properties of
the local tissue microenvironment, in particular those of the
surrounding ECM. The importance of the ECM and stroma in
mammary gland development and function were proposed several
decades ago [(Williams and Daniel, 1983), reviewed by Varner
and Nelson (2014)]. Chimeric recombination models [reviewed by
Nelson and Bissell (2006)] demonstrated the power of the stroma
DEVELOPMENT
REVIEW
to influence the developing epithelium. When mammary
epithelium was recombined with mammary mesenchyme, not
surprisingly, the outcome was that of a typical mammary ductal
tree. However, mammary epithelium recombined with salivary
gland mesenchyme resulted in structures that resembled salivary
gland epithelium (Sakakura et al., 1976). On the other hand,
outgrowths of salivary epithelium in contact with mammary
mesenchyme resembled a mammary gland ductal tree and could
even become competent for lactation and respond to hormonal
stimuli (Cunha et al., 1995). These and other studies revealed that
the epithelial component is highly malleable and that cell fate and
tissue function are strongly influenced by the stromal component
of the gland.
More recent studies (Bruno and Smith, 2012; Bussard and Smith,
2012) demonstrated, amazingly, that non-mammary cells and even
human cancer cells can be reprogrammed and incorporated into
mammary outgrowths that are capable of self-renewal upon further
transplantation into a murine cleared mammary fat pad (Boulanger
et al., 2013, 2012, 2007). In these studies, co-injection with
mammary epithelial cells was required, indicating that a ‘stem cell
niche’, which includes the microenvironment and ECM
components, is also necessary for signaling from bona fide
mammary epithelial cells. How does the tissue microenvironment
support growth, cellular differentiation and development of the
mammary gland? As we discuss below, an intricate signaling
network exists between the epithelium and its microenvironment,
and includes signaling in response to mechanical forces and cell-cell
contact, signaling from the ECM molecules, stromal-derived growth
factors and cytokines, and the activities of proteolytic enzymes in
the microenvironment.
The role of cell contacts and mechanical forces
Mammary cells are tightly connected both to each other and to their
surrounding environment, and they require this connection for
normal function. Myoepithelial cells, for example, are anchored to
other myoepithelial cells and luminal epithelial cells via
desmosomes, and to the BM via hemidesmosomes (Adriance
et al., 2005; Pandey et al., 2010). Similar properties, mediated by the
integrin α6β4 complex, are exhibited by mammary luminal cells
when cultured in laminin-rich, three-dimensional (3D) ECM gels
(Weaver et al., 2002). In vivo, the two layers of the duct express
different cell-cell contact mediators (Chanson et al., 2011; Huebner
et al., 2014; Mroue et al., 2014) and cell-ECM connections (Brizzi
et al., 2012); as a result, the layered structure of the duct selforganizes when luminal and myoepithelial cells are mixed in 3D
cultures (Chanson et al., 2011; Gudjonsson et al., 2002; Huebner
et al., 2014; Runswick et al., 2001). Furthermore, these connections
are necessary for normal cell function: mammary cells in the
lactating gland require E-cadherin to survive (Boussadia et al.,
2002). Together, these tight connections between mammary cells
allow the transmission of mechanical forces and biochemical
signals throughout the growing epithelium and allow collective cell
motion (Gjorevski and Nelson, 2010), in which mechanically active
cells push or pull more passive cells along with them (Rørth, 2012).
ECM-mediated control of mammary gland development
The ECM is a major regulator of epithelial architecture and function.
Within the mammary gland, myoepithelial cells reside on a
specialized layer of ECM, termed the basement membrane (BM).
Myoepithelial and stromal cells synthesize ECM components, such
as laminins, collagens, fibronectin and proteoglycans, which are
incorporated into the ECM as well as the BM. These matrices provide
Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
not only physical support for correct tissue architecture but are also
essential biochemical signaling networks that guide cellular fate and
function of the gland. The BM also anchors the myoepithelial cells
by ligating cell surface molecules into position and by organizing the
basal side of the myoepithelium, through myoepithelial-secreted
laminin-111 and hemidesmosomes (Inman et al., 2011).
Studies using microenvironmental protein microarrays have
started to uncover the ECM molecules that influence cell fate
decisions in mammary progenitor cells (LaBarge et al., 2009; Lin
et al., 2012). These studies revealed that laminin-111 regulates
mammary progenitor self-renewal, whereas other combinations of
ECM proteins result in growth, differentiation and apoptosis
(LaBarge et al., 2009). As discussed above, myoepithelial cells
are a major source of laminin-111 of the BM, and it is this laminin
that is essentially responsible for establishing luminal epithelial cell
apical-basal polarity (Gudjonsson et al., 2002; Weir et al., 2006).
Furthermore, because of their position and function within the
mammary tissue, myoepithelial cells are thought to act as tumor
suppressors in the adult gland (Adriance et al., 2005; Bissell and
LaBarge, 2005; Pandey et al., 2010; Sternlicht and Barsky, 1997;
Sternlicht et al., 1997).
Other stromal ECM proteins are important for instructing the
development of the mammary arboreal structure. For example, recent
studies show that collagen I fiber orientation in the mammary fat pad
is a patterning cue for mammary branch orientation during
development (Brownfield et al., 2013). These collagen I fibers
appear to be oriented prior to mammary branching morphogenesis,
indicating that epithelial architecture might be pre-patterned in the
stroma of the pubertal mammary gland. Such structural components,
which support tissue architecture, are often overlooked as regulators
of morphogenesis, but we now understand that correct tissue
architecture and stiffness of the ECM are essential components of
normal development, differentiation and function within the
mammary gland (Bissell et al., 1982; Maller et al., 2013; Schedin
and Keely, 2011). Future studies of the stroma in mammary gland
development would undoubtedly provide additional clues into the
role of stroma in breast cancer initiation and progression.
Proteases are essential regulators of mammary gland
morphogenesis and differentiation
Proteolytic action remodels the ECM and stroma, and releases
sequestered growth factors and cytokines. Not surprisingly,
proteases are essential for mammary gland development and
function. For the epithelial rudiment to fill the fat pad upon
hormonal signals during puberty it is essential for the cells to take on
an invasive phenotype and for proteinases, in this case matrix
metalloproteinases (MMPs), to pave the way by remodeling the
ECM (Talhouk et al., 1991). To understand the role of proteinases in
the mammary gland, a number of transgenic mice in which a
particular proteinase (or proteinase inhibitor) is silenced or
overexpressed have been developed. Although compensation by
different MMPs makes it difficult to exclusively define the function
of a single proteinase in vivo, the transgenic mouse models have
provided insight into the importance of proteinases during
mammary gland development and function. Additionally, 3D
physiologically relevant organotypic culture models have shed
light on the importance of both the catalytic activity and the newly
discovered functions of the non-catalytic domains of proteinases in
mammary gland development (Correia et al., 2013; Kessenbrock
et al., 2013; Mori et al., 2013).
Several different classes of proteases are necessary for proper
mammary gland development and function. Proteinases provide
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local environmental signals to promote EMT and invasion. For
example, we showed more than two decades ago that Mmp3 has a
crucial role in remodeling the mammary gland in involution
(Talhouk et al., 1991), and that its aberrant activity in 3D collagen
gels leads to EMT and a premalignant phenotype (Lochter et al.,
1997). Others have shown that cathepsins play important roles
during involution and apoptosis of the mammary gland (Sloane,
2012; Watson and Kreuzaler, 2009). Serine proteases are
important as well: mice deficient for the serine protease
plasminogen have difficulties supporting lactation, due to a
disruption in factors that control involution (Green et al., 2006).
Other studies identified the serine protease plasminogen activator
kallikrein as being important for adipocyte differentiation in the
mammary gland. Kallikrein is thought to play a role in the
plasminogen cascade of remodeling the fibrin-rich pre-adipocyte
stromal ECM (Selvarajan et al., 2001).
The most widely studied enzymes in the context of mammary gland
development and differentiation are metalloproteinases, which
include MMPs and the ‘a dysintegrin and metalloproteinases’
(ADAMs). MMPs consist of a family of over 20 zinc-dependent
proteinases synthesized as latent enzymes that must be activated posttranslationally (Kessenbrock et al., 2010). Their activity is modulated
by endogenous inhibitors referred to as tissue inhibitors of
metalloproteinases (TIMPs) (Murphy, 2011). Collectively, MMPs
can degrade all protein components of the ECM, and we now know of
a number of other proteins, including E-cadherin (Lochter et al.,
1997). Over the past decades, many transgenic mouse models have
been developed to investigate MMP function in development and
cancer (Gill et al., 2010; Wiseman and Werb, 2002). Interestingly, the
Mmp14 knockout is the only single MMP gene knockout model that is
lethal. Overexpression of Mmp14 (Ha et al., 2001) or Mmp3
(Sympson et al., 1994) in the mammary gland leads to excessive side
branching and advanced alveolar morphogenesis (Fata et al., 2004).
Increased levels of the active form of Mmp3 not only causes
supernumerary branching in the mammary gland but is responsible for
causing production of reactive oxygen species (ROS), changes in
splicing of Rac1, EMT and genomic instability (Radisky et al., 2005),
which precedes mammary tumor development (Sternlicht et al.,
1999). Crossing the Mmp3-overexpressing mouse model with a
mouse overexpressing Timp1 decreases the detrimental effects of
Mmp3 overexpression/activity. These data support the notion that
Mmp3 activity in the mammary gland is important for branching
morphogenesis, but indicate that unscheduled activity alters the
mammary tissue microenvironment. Furthermore, the fact that Mmp3
activity could be tempered by Timp1 emphasizes the importance of a
balance between enzymes and inhibitors for proper tissue
development and homeostasis, and might explain why TIMPs are
also 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 adipocyte
differentiation (Alexander et al., 2001). Timp1 overexpression gives
rise 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 gland
involution. In studies in which slow-release pellets of Timp1, 2, 3 or
4 were implanted into mouse mammary glands, it was shown that
Timp1, 3 and 4 inhibited ductal elongation, most likely via
inhibition of MMP activity. However, Timp2 had an elongation
promoting effect (Hojilla et al., 2007). On the surface, the Timp2
result seems contradictory, but increased levels of Timp2 most
likely raise the levels of Mmp2 activation by increasing the
formation of a tertiary complex (Mmp14-Timp2-Mmp2) that is
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Development (2015) 142, 1028-1042 doi:10.1242/dev.087643
responsible for Mmp2 activation in vivo (Ellerbroek and Stack,
1999; English et al., 2006).
To unravel the mechanisms underlying these observations
in vivo, cell culture studies have been most informative. These
studies have revealed that active Mmp3 increases ROS levels,
leading to genetic instability and EMT (Radisky et al., 2005), which
contribute 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 branching
morphogenesis (Correia et al., 2013). Proteomic analysis of Mmp3
hemopexin domain binding partners revealed, surprisingly, that the
chaperone heat shock protein, 90β (Hsp90β) interacts specifically
with the hemopexin domain of Mmp3 in the extracellular space and
that this interaction is crucial for Mmp3 function. Other work has
implicated the hemopexin domain of Mmp3 as a regulator of Wnt
signaling and MaSC activity (Kessenbrock et al., 2013). In line with
this, mice deficient for Mmp3 exhibit decreased numbers of MaSCs
and diminished mammary-reconstituting activity. Conversely, in
the same study it was shown that Mmp3 overexpression elevated
MaSC function. Together, these observations suggest that Mmp3
activity is necessary for the maintenance of MaSCs.
Three-dimensional cell culture models have also been essential
for elucidating the many functions of MMPs in normal and
malignant mammary glands (Barcellos-Hoff et al., 1989; Petersen
et al., 1992). For example, investigations into the role of Mmp14 in
mammary development were hampered due to the lethality of this
gene knockout. However, using micropatterned gels of collagen I, it
was revealed that the hemopexin domain of Mmp14 is important for
sorting mammary epithelial cells to points of branching, thus
highlighting that the non-proteolytic domains of Mmp14 also are
essential for proper branching morphogenesis (Mori et al., 2009). In
addition to the hemopexin domain, recent evidence indicates that
the short intracellular domain of Mmp14 is crucial for epithelial cell
invasion (Mori et al., 2013). Using engineered Mmp14 constructs in
which different domains were deleted, we discovered that only the
short intracellular domain of Mmp14 is needed to rescue branching
morphogenesis in Mmp14-deficient cells, despite the fact that this
sequence does not contain kinase activity. This deficiency is
compensated for by integrin β1, which interacts with the short
cytoplasmic domain of Mmp14. It is required for interaction with
the ECM, and for transducing the extracellular signals needed for
epithelial cells to invade. These recent observations provide strong
evidence that MMPs are important not only for ECM remodeling
but also for the microenvironmental signaling necessary for
morphogenic programs within the mammary gland.
Combining mouse models with physiologically relevant 3D
models of human cells will allow further investigations into the
mechanisms of action of these proteinases. This information is
important not only for understanding tissue development and
mammary function, but also for identifying new targets for cancer
therapy, as researchers focus on the new functions of the non-catalytic
domains of MMPs as regulators of tissue morphogenesis and tumor
development (Dufour and Overall, 2013; Rodríguez et al., 2010;
Strongin, 2010). The non-catalytic domains are targetable using
antibodies or blocking peptides directed specifically against those
domains (Basu et al., 2012). Such biological pharmaceuticals directed
to domains other than the catalytic domains of these proteinases surely
will provide more specificity in therapy.
Conclusions
Like all other organs, the mammary gland is composed of many
specialized cell types that carry out mammary functions, with
DEVELOPMENT
REVIEW
interconnected signaling occurring between the different cellular
compartments. With its unique developmental mode occurring
essentially after birth and its remarkable regenerative properties, the
mammary gland provides a superb model for investigating
developmental programs, stem and progenitor cell properties, and
the stability of the differentiated state.
Early serial transplant experiments and transgenic mouse models
have shed light on the identity and role of MaSCs and progenitor
cells. More recently, lineage-tracing experiments have identified
multiple different, and somewhat conflicting, populations of stem
and progenitor cells. Despite the discrepancies, these studies have
begun to fine-tune our understanding of MaSCs and how they drive
development of the gland and maintain homeostasis of the resulting
arboreal architecture. Additionally, the results of a number of recent
studies are revealing the role MaSCs play in the many cycles of
proliferation and apoptosis needed both to expand and maintain the
gland form and functions during pregnancy and to return it to a
quiescent 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 the
tissue microenvironment, especially from the ECM in general and
the BM in particular. For decades, the ECM and BM were thought
to be the inert ‘bricks and mortar’ of a tissue, simply providing
physical structure. We now know that correct tissue architecture,
including the organization and stiffness of the ECM, together with
the reservoir of growth factors, cytokines and proteinases within
which the stem cell niche nestles, are essential for mammary
glandular tissue to develop and function properly.
Competing interests
The authors declare no competing or financial interests.
Funding
C.R. was supported by a fellowship from the Congressionally Directed Medical
Research Programs of the US Department of Defense (DoD). M.J.B. was funded by
Congressionally Directed Medical Research Programs of the DoD, The Breast
Cancer Research Foundation and The National Cancer Institute. This work was also
supported by the US Department of Energy Office of Biological and Environmental
Research and Low Dose Radiation Program.
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