The role of canonical Wnt signaling in
mammary stem cells
Eline van Kappel
September 2012
The mammary gland undergoes extensive growth and involution during postnatal
mammary gland development. Tissue expansion and remodeling of the mammary
gland suggests the presence of adult stem cells. Transplantation studies validated the
existence of mammary stem cells that are able to reconstitute a functional mammary
gland. Mammary stem cells are located in the basal cell lineage of the epithelial ducts,
but the exact population is not identified yet. However, the Lin- CD29hi CD24+ cell
population is reported as stem cell-enriched cell population. Canonical Wnt signaling
is shown to be important in adult stem cell populations of different organs. Recent
studies also suggest a role for canonical Wnt signaling in mammary stem cells. The
first indication came from the MMTV-Wnt1 mouse model, where Wnt signaling
increases stem/progenitor cell activity. Another study showed that depletion of Lrp5,
the essential co-receptor of Wnt signaling, affects proliferation in the basal cell
lineage in vivo. Using Axin2 as marker for Wnt-responsive cells, it was possible to
further enrich the stem cell population within the Lin- CD29hi CD24+ cell population and
increased Wnt-responsive cells have a competitive advantage in transplantation
assays. Together these data indicate an important role for Wnt signaling in mammary
stem cells. Recently, the hierarchy of stem cells and progenitor cells was studied
using a genetic lineage tracing approach. These experiments showed the existence of
two distinct progenitor cell populations for basal and luminal cell lineages instead of a
stem cell population.
Both progenitor cell populations are Wnt-responsive.
Interestingly, under regenerative circumstance a single basal progenitor cell was able
to reconstitute a functional mammary gland consisting of both luminal and basal cell
lineages. Different studies showed the importance of Wnt signaling in mammary stem
or progenitor cells, but further studies are required to assign the exact role of
canonical Wnt signaling in the mammary stem cells.
1
Introduction
The primary function of the mammary gland is the production of milk to feed the young.
Production of milk starts during pregnancy and stops after nursing. In order to produce
enough milk the mammary gland undergoes extensive growth of the epithelial ductal network
during pregnancy. After the lactation phase the mammary gland returns to its virgin state, a
process called involution (1). The mammary gland is composed of an epithelial ductal tree
embedded in a fat pad surrounded by stromal cells. This organ is known to develop through
three different stages throughout embryonic and pubertal development and during the
reproductive life (Figure 1) (1). During embryonic development the position of the mammary
gland is specified by formation of the fat pad. At birth only a small rudimentary epithelial tree
around the nipple area is present in the fat pad and growth is arrested until the onset of
puberty (2). During puberty the epithelial ductal tree further develops into a highly branched
tubular structure invading the whole fat pad. The ductal branches are bi-layered structures
consisting of luminal cells surrounding the lumen of the ducts and an outer layer of
basal/myoepithelial cells. During pregnancy the ductal network further expands and forms
extra branches and milk-producing alveoli. The luminal cells covering the inside of the alveoli
are the milk-producing cells. The basal/myoepithelial cell layer contracts to help excrete the
milk from the gland. Alveoli regress at the end of lactation by apoptosis and remodeling and
the mammary gland returns to its original morphological state (1, 2).
The development of the mammary gland is relatively unique, since the majority of organ
development takes place during puberty. Together with the massive tissue remodeling
around pregnancy and lactation, mammary gland homeostasis is a complex process. Adult
stem cells are reported to maintain tissue homeostasis in epithelial tissues, including
intestine and skin (3, 4). In these organs, the canonical Wnt signaling pathway is important
for the function of adult stem cell populations (3). Within the mammary gland, canonical Wnt
signaling might also play a role in maintaining a mammary stem cell population. To study
canonical Wnt signaling in mammary stem cells, the population of mammary stem cells first
needs to be identified.
Recent studies reported stem cell-enriched cell populations in the mammary gland using
different combinations of cell surface markers (5, 6). The exact stem cell population is still not
identified due to the lack of specific markers. Here we discuss the main techniques used to
identify stem cell properties, including transplantation assays and lineage tracing
experiments. We also discuss stem cell-enriched populations and the possible role of Wnt
signaling in mammary stem cells.
2
Figure 1: The mammary gland develops in 3 different stages. At birth the fat pad and a small
rudimentary epithelial tree are present. During puberty the epithelium expands and the ductal network
invades the whole fat pad. During pregnancy the epithelial ductal tree forms alveoli to produce milk
for lactation. After weaning of the pups the milk-producing cells undergo apoptosis, a process termed
involution and the gland returns to its virgin state. Figure adapted from: (1)
Studying stem cell characteristics
The hallmark characteristics of stem cells are the ability to differentiate into different
specialized cells and the ability to self-renew (5, 7). Proliferation of stem cells will give rise to
daughter stem cells to maintain the population (self-renewal) and daughter cells able to
differentiate into other cell types (multipotency). These stem cell properties are essential for
tissue maintenance and regeneration of a particular organ. Tissue-specific adult stem cells
give rise to all the different cell types within the organ of origin.
Since the architecture and development of the human mammary gland is highly similar to the
mouse mammary gland, mice are frequently used as model organism (7). Recent studies
have isolated stem cell-enriched cell populations from the mouse mammary epithelium. To
isolate stem cell populations mammary glands are first harvested and processed into single
cells. Thereafter, stem cell-enriched populations are isolated by fluorescence activated cell
sorting (FACS) based on combinations of cell surface markers. To isolate mammary stem
3
cells, hematopoietic and endothelial cells should be removed by applying a FACS-strategy
that excludes CD45+, TER119+ and CD31+ cells. This method selects for the lineage (Lin)negative cell population (5). Other cell surface markers are used to further enrich for
mammary stem cells and will be discussed later (5, 6).
FACS isolated cell populations are tested for stem cell properties using different techniques,
including transplantation assays. The ability to reconstitute a fully functional organ from a
single cell is believed to be the gold standard for identifying stem cells (7). This assay proofs
the ability of a single cell to differentiate in all different cell types of the organ, a property
termed ‘multipotency’. In the mammary gland, single cells are transplanted in recipient mice
with fat pads cleared of their endogenous epithelium. Reconstitution ability is proven by
outgrowth of a differentiated and functional epithelial ductal tree (5, 8). Self-renewal
properties of a stem cells population are investigated by serial transplantations. In this
experimental set-up the outgrowth of the first transplantation assay is used to transplant
single cells in another recipient mouse with cleared fat pads. Secondary and tertiary
outgrowths proof the self-renewal capacity of a stem cell (5, 8).
In transplantation assays the original mammary epithelium is removed, mimicking a
regenerative state. Under these conditions stem cells might contribute to cell lineages to
which they do not contribute under physiological circumstances. Using genetic lineagetracing experiments the contribution of a single cell to mammary gland development and
homeostasis can be traced under physiological conditions (9-11). Genetic lineage tracing
experiments are based on inducible markers in (stem) cells. For these experiments genetic
modified mouse models are generated. These mouse models often carry a tamoxifeninducible Cre recombinase into an endogenous gene, which is cell population specific.
Activation of Cre upon tamoxifen administration is cell type specific and leads to
recombination of floxed reporter loci elsewhere in the genome. This activates a permanent
genetic mark in the genome, for example LacZ (9). During proliferation the marker will be
passed on to all daughter cells and therefore the cellular hierarchy of an organ can be
studied. In the mammary gland this method has long remained unexplored but recently van
Keymeulen et al. used genetic lineage-tracing experiments to study the cellular hierarchy
(10, 11).
4
Using the above-mentioned techniques, different research groups attempted to identify the
stem cell population in the mammary gland. Currently, the existence of a stem cell population
in the mammary gland is well accepted (12), although the exact phenotype of the stem cell
population is unknown. Different studies attempt to isolate stem cells in the mammary gland
using cell surface markers.
Mammary stem cells
It has been shown that different parts of the epithelial tree of the mammary gland are able to
reconstitute a fully functionally mammary gland after transplantation in a recipient mouse (8).
Each part of the epithelial ductal tree was able to regenerate an entire mammary epithelial
tree, suggesting that mammary stem cells are present in all branches of the ductal network
(8). According to Shackleton et al. the cell population Lin- CD29hi CD24+ is highly enriched for
adult mammary stem cells (5). These cell surface markers are based on previous stem cell
studies in different organs. Selecting for the Lin negative cell population excludes the
endothelial and hematopoietic cells, CD29 is known as a stem cell marker in the skin and
CD24 has been used to enrich neural stem cells. Shackleton et al. showed that a single cell
of the Lin- CD29hi CD24+ population is able to reconstitute an entire functional mammary tree
in recipient mice (5). These outgrowths exhibit complete differentiation of the epithelial ductal
tree and are able to produce milk during pregnancy. The self-renewing capacity of the
isolated stem cells was proven by serial transplantation of the clonal outgrowths (5). The
mammary stem cells are suggested to reside in the basal layer of the mammary ducts (5, 6).
Although the isolated stem cell-enriched cell population showed stem cell properties, it
consisted of more than pure stem cells (5). Therefore, more specific markers are necessary
to purify the stem cell population in the mammary gland. In other epithelial organs the
canonical Wnt signaling pathway is associated with adult stem cell populations (3) and
therefore Wnt-responsiveness might also be a marker for stem cells in the mammary gland.
Canonical Wnt signaling
In different epithelial tissues Wnt-responsiveness is associated with adult stem cell properties
(13, 14). Survival of tissue-specific stem cell populations is shown to be dependent on
canonical Wnt signaling (3). In the mammary gland multiple Wnt genes are expressed and
Wnt signaling is found to be essential for mammary gland development during
embryogenesis (15, 16). Moreover, the canonical Wnt signaling pathway is often affected in
cancer, including breast cancer (17). Together these findings provided strong clues for
further studies searching for a role of canonical Wnt signaling in the mammary gland and the
mammary stem cell population.
5
Wnt signaling was first discovered in the model organism Drosophila Melanogaster and is a
highly conserved signaling pathway. In humans the Wnt family contains 19 genes, which are
widely expressed during embryonic development. Aberrant expression of Wnt genes during
embryonic development leads to birth defects and can even be embryonic lethal (18). Wnt
ligands are known to activate three different signaling cascades in cells from which the
canonical Wnt signaling pathway is studied most extensively (19).
The canonical Wnt signaling pathway describes the cell signaling pathway in response to
engagement of Wnt ligand with the Frizzled receptor on the cell membrane. In the absence
of Wnt the cytosolic pool of β-catenin is bound to a destruction complex consisting of
regulatory proteins (Figure 2A) (19). β-catenin will be phosphorylated and ubiquitinated,
resulting in degradation by the proteasome. Constant degradation of β-catenin maintains a
low cytosolic concentration of β-catenin (19). In the presence of Wnt ligand, Frizzled and the
co-receptor low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), activate the Wnt
signaling cascade (Figure 2B) (19). This leads to inactivation of the β-catenin destruction
complex in the cytosol. The cytosolic pool of β-catenin will be stabilized and accumulates in
the cytoplasm. β-catenin will translocate to the cell nucleus, where it functions as cotranscription factor for transcription factor, T-cell factor (TCF). The TCF/β-catenin complex
transcribes Wnt-responsive genes, including c-Myc, Cyclin D, Tcf-1 and Axin2 (19, 20).
Wnt signaling is required to maintain self-renewal properties in stem cell populations (3). In
vitro stem cell populations differentiate and lose their self-renewal properties, probably by the
lack of specific factors (21) It was recently shown that in the presence of Wnt ligands
mammary stem cell populations could be maintained in vitro for several cycles and that their
stem-cell properties were retained (21). This indicates a function of Wnt signaling in the
maintenance of mammary stem cells.
6
Figure 2: Canonical Wnt signaling pathway. In the absence of Wnt ligand the β-catenin destruction
complex is intact and phosphorylates cytosolic β-catenin. Phosphorylated β-catenin is recognized by
β-Trcp leading to ubiquitination and degradation of β-catenin (A). When Wnt ligand is bound to the
Frizzled receptor and the co-receptor Lrp5 or 6, Dishevelled and Axin1 are recruited to the cell
membrane. The β-catenin destruction complex is inactivated and β-catenin accumulates in the
cytosol. Accumulation of β-catenin results in translocation to the cell nucleus and activation of the
transcription factor TCF leading to transcription of Wnt-responsive genes (B). Figure from: (20)
Wnt signaling in mammary stem cells
Wnt signaling plays an important role during embryonic mammary gland development and
different Wnt proteins are expressed in the mammary gland (15, 16). Inhibition of Wnt
signaling during embryogenesis even prevents mammary gland formation (15). Both the fat
pad and rudimentary epithelial tree are not formed in the absence of Wnt signaling. Proper
Wnt signaling is important during mammary gland development and is hijacked in breast
cancer (17). To study the effect of Wnt signaling on mammary progenitor cells Liu et al. used
mouse models expressing the oncogenes Wnt1 or ΔNβcat under control of mouse mammary
tumor virus (MMTV) (22). The truncated form of β-catenin (ΔNβcat) escapes degradation
leading to cytosolic accumulation independent of Wnt signaling and activation of the
canonical Wnt signaling pathway. Breast tumors from both mouse models consist of both
luminal and basal cell lineages. Mammary stem cell activity is increased in mammary glands
of ΔNβcat mice, indicating accumulation of mammary progenitor cells upon Wnt signaling
activation (22). These data indicate a function for Wnt signaling in the stem cell population of
the mammary gland.
7
In order to understand the function of Wnt signaling in the mammary gland Badders et al.
studied loss-of-function of canonical Wnt signaling in the mammary gland (23). Both the Wnt
ligands and the Frizzled receptor have many homologues and therefore Badders et al.
choose to study canonical Wnt signaling by loss-of-function of the required co-receptors,
Lrp5 or 6. Ablation of Lrp5 or Lrp6 does result in different phenotypes in mice. Lrp6
expression is essential for embryonic development, including mammary gland development.
Both the fat pad and epithelial rudimentary tree are affected by the loss of Lrp6 (24). Lrp5
knockout mouse models are viable and in wild type the mRNA of Lrp5 is widely expressed in
adult tissues (23). Interestingly, mammary glands of Lrp5 knockout mice are resistant to
Wnt1-induced tumor development, indicating a reduced Wnt-responsiveness although Lrp6
is still functional.
Further study of Lrp5 and 6 expression patterns revealed that most basal cells of the
mammary epithelium express both proteins (23). Badders et al. showed that the Lrp5 high
population co-localizes with the Cd24+CD49f+ stem cell-enriched population reported before
(6, 23). Depletion of Lrp5 results in senescence of the basal cell layer and a reduction in the
number of cells from the basal cell lineage (23). Together these data suggest a role for Wnt
signaling in proliferative activity in the mammary gland. Lrp5 depletion studies do not provide
sufficient data to discover the function of Wnt signaling in adult mammary stem cells. The
use of other specific markers for canonical Wnt signaling can help to uncover the function of
Wnt in the mammary stem cell.
Axin2 as mammary stem cell marker
To identify the Wnt-responsive cell population in the mammary gland Zeng et al. used an
Axin2-LacZ reporter mouse model (21). Axin2 is a specific target gene of the canonical Wnt
signaling pathway and LacZ will therefore mark the Wnt-responsive cells. Histological
analysis of the mammary gland showed the presence of Wnt-responsive cells in the basal
layer of the mammary epithelial ducts. This localization of the presumed mammary stem cell
corresponds with previous publications (5) where Lin- CD29hi CD24+ cells were identified as a
stem cell-enriched cell population. Wnt-responsive cells were only detected in the Lin- CD29hi
CD24+ cell population and represent about 5% this cell population (21). Lin- CD29hi CD24+
Wnt-responsive cells showed increased number of mammary epithelial outgrowths compared
to the total Lin- CD29hi CD24+ cell population in transplantation assays (21). This indicates
that selecting for Wnt-responsiveness can further enrich the mammary stem cell population.
8
Insertion of LacZ into the Axin2 gene leads to inactivation of Axin2 and blocks the negative
regulatory function of Axin2 (21). As a result, canonical Wnt signaling loses a part of its
negative feedback regulation and cells become more Wnt-responsive. These increased Wntresponsive cells were able to out-compete the normal cells in in vivo repopulation assays.
The epithelial ductal trees grown from increased Wnt-responsive cells have a normal bilayered structure and differentiate normally during pregnancy. Hyperplasia or tumor
formation was not observed in contrast with the MMTV-Wnt1 mouse model, where Wnt1
overexpression leads to tumor formation (25). This suggests that increased Wnt
responsiveness by the lack of functional Axin2, as negative feedback regulator, is less
excessive than over-expression of the Wnt ligand itself.
Lineage tracing assay with Axin2
Van Amerongen et al. recently published Axin2 lineage tracing experiments in the mammary
gland to show the hierarchy during mammary gland development (10). For this study the
mouse model Axin2CreERT2 was used to label the Wnt/ β-catenin-responsive cells and their
daughter cells (10). To validate the model Van Amerongen et al. marked intestinal stem cells
and monitored their offspring. Intestinal stem cells where located at the crypt base and
offspring of these cells migrated up along the crypt and villus, which was shown before by
Barker et al. (14). Labeling of Wnt/β-catenin-responsive cells in the mammary gland at
different developmental time points allows the identification of the behavior and contribution
of mammary stem cells during these developmental processes (10).
Remarkably, the contribution of Wnt-responsive cells differs between the embryonic and
postnatal mammary gland (10). Axin2+ cells in the embryonic mammary gland will finally give
rise to the luminal cell lineage in the adult mammary gland (Figure 3). After birth, in two
week-old pups (prepubescent), a switch is observed. Wnt/β-catenin-responsive cells at this
stage give rise to the basal cell lineage in the mammary gland (Figure 3). These cells retain
long-term proliferative potential and are unipotent for the basal cell lineage. However, when
these unipotent cells are transplanted in cleared recipient fat pads they were able to
regenerate a functional epithelial ductal tree consisting of both luminal and basal cells (10).
Secondary transplants confirmed the self-renewal properties of the stem cells in the newly
generated epithelial ductal trees. These data suggest that basal lineage-restricted stem cells
are able to give rise to luminal cells under regenerative circumstances and behave as
multipotent stem cells of the mammary gland (10). This experiment might explain why
transplantation studies and cell lineage tracing experiments in the mammary gland lead to
different opinions about the role of stem cells in the mammary gland.
9
Further experiments investigated the function and location of Wnt-responsive cells in the
adult mammary gland. During puberty both the luminal and basal cell lineage origin from
Wnt/β-catenin-responsive cells, but probably basal cells and luminal cells both have their
own Axin2+ precursor cell (Figure 3) (10, 11). In the adult mouse Axin2+ cells are mainly
found in the Lin- CD29hi CD24+ basal mammary epithelium cell population, corresponding
with the stem cell-enriched population marked by Shackleton et al. (5). During pregnancy the
mammary gland undergoes extreme remodeling including the growth of milk-producing
alveoli. During the first pregnancy both luminal and basal epithelial cells in the alveoli arise
from Axin2+ cells. The Axin2CreERT2 adult mammary stem cells are shown to be long-lived,
since they survive involution of the mammary gland and multiple rounds of pregnancy (10).
The clustering of labeled cells in the alveoli suggests that bipotent Wnt/β-catenin-responsive
cells give rise to both basal and luminal cells in the alveoli (Figure 3) (10).
Figure 3: Model of contribution of Wnt-responsive cells to the mammary epithelium. Axin2+ cells
in the embryonic mammary gland will contribute to the luminal cell lineage in the adult mammary
gland. Axin2+ cells in the prepubescent will contribute to the basal epithelial cell lineage. During
puberty both basal and luminal cell lineage develop from Axin2+ cells, but probably from distinct
progenitors both Axin2+. During pregnancy the basal and luminal cell lineage of the alveoli are thought
to reside from one Axin2+ progenitor. Figure from: (10)
10
Discussion
The mammary gland is an excellent organ to study adult stem cells because of its unique
postnatal development and accessibility (21). The human and mouse mammary gland share
a similar organ development, which makes mice a good model for mammary stem cell
studies (7). The primary function of the mammary gland is to produce milk to feed the young.
In order to produce enough milk the mammary gland undergoes extensive growth during
pregnancy followed by involution after weaning of the pups (2). This complex homeostatic
process does imply the existence of multipotent mammary stem cells to maintain the
structure and function of the mammary gland.
Canonical Wnt signaling is shown to play an important role in tissue homeostasis and stem
cell maintenance (3). In the mammary gland different studies showed the importance of Wnt
signaling for progenitor cell populations (10, 21, 23). Lrp5 is essential to maintain proliferation
in the basal cell lineage (23) and the Wnt-responsive gene Axin2 is upregulated in
stem/progenitor cells (10, 21). Increased Wnt-responsiveness by inhibiting the Axin2
negative feedback loop does increase proliferation potential (10). However, a remarkable
switch in Wnt/β-catenin signaling is observed around birth. Wnt-responsive cells present
before birth will contribute to the prospective luminal cell lineage of the mammary gland. On
the other hand, the Wnt-responsive cells in the mammary gland after birth will mainly
contribute to the basal cell lineage of the mammary epithelium (10). Together these data
suggest an important role for canonical Wnt signaling in mammary stem/progenitor cells.
Transplantation studies in the mammary gland confirm the existence of multipotent
mammary stem cells (5, 8). These cells are able to regenerate a fully functional mammary
gland consisting of all different cell types. Recently two studies are published using genetic
lineage tracing to identify the stem/progenitor cells in the mammary gland (10, 11). These
experiments are performed under physiological conditions while transplantation assays
mimic regenerative conditions. Lineage tracing experiments do not reveal a multipotent stem
cells population in the adult mammary gland and suggest the existence of two distinct
progenitor populations for basal and luminal stem cells in adulthood (10, 11). However, when
the progenitor cells of the basal cell lineage were used in a transplantation assay, it was
shown that these cells are multipotent under regenerative circumstances (10). The signals
inducing the switch from unipotent to multipotent progenitor cell are still unclear. Stem cells
are thought to be intrinsically destined to differentiate and extrinsic factors are necessary to
maintain multipotent properties (26). The niche surrounding the stem cell supplies extrinsic
factors under physiological conditions (27). It would be interesting to identify the “niche” of
the mammary stem cells under physiological conditions and generative circumstances.
11
A stem cell niche is a well-accepted idea in the intestine, where the adult stem cell population
is recently identified. Lgr5 was found as a stem cell-specific marker in the intestine (14). This
finding accelerated stem cell studies in the intestine and other organs and by now Lgr5 is
known as stem cell marker in the stomach, small intestine, colon and hair follicles (3, 14).
The inner surface of the intestine undergoes constant homeostatic renewal and is the fastest
proliferating tissue in adult animals (3). In the intestine this self-renewal potential is
generated by the Lgr5 cell population, which resided in the crypts and is surrounded by
Paneth cells (28). Within this stem cell niche, Wnt signaling is essential to maintain stem cell
properties (28). After proliferation one daughter cell retains self-renewal properties and
remains in the stem cell niche. The other daughter cell will migrate towards the villus and
differentiate into intestinal epithelial cells (14).
Lgr molecules, including Lgr5, are identified as R-spondin receptors that enhance Wnt
signaling (29). Up till now Lgr5 is found as stem cell marker in highly proliferative tissues like
the intestine and skin (3, 14). The mammary gland does also undergo massive remodeling,
but this has a periodically character. However, a rare population of Lgr5+ cells is detected in
the basal cell lineage of the mammary gland (14). All together, canonical Wnt signaling is
associated with stem and progenitor cells in the mammary gland. Lgr5 as receptor might play
a role in the Wnt-responsiveness of the mammary stem cells, but further research is
necessary to confirm this idea. The source of Wnt ligands in the mammary gland needs
further investigation and may uncover the existence of a stem cell niche in the mammary
gland.
12
References:
1.
Watson CJ, Oliver CH, & Khaled WT (2011) Cytokine signalling in mammary gland
development. J Reprod Immunol 88(2):124-129.
2.
Watson CJ & Khaled WT (2008) Mammary development in the embryo and adult: a
journey of morphogenesis and commitment. Development 135(6):995-1003.
3.
Schuijers J & Clevers H (2012) Adult mammalian stem cells: the role of Wnt, Lgr5
and R-spondins. EMBO J 31(12):2685-2696.
4.
Tumbar T (2012) Ontogeny and Homeostasis of Adult Epithelial Skin Stem Cells.
Stem Cell Rev.
5.
Shackleton M, et al. (2006) Generation of a functional mammary gland from a single
stem cell. Nature 439(7072):84-88.
6.
Stingl J, et al. (2006) Purification and unique properties of mammary epithelial stem
cells. Nature 439(7079):993-997.
7.
Tiede B & Kang Y (2011) From milk to malignancy: the role of mammary stem cells in
development, pregnancy and breast cancer. Cell Res 21(2):245-257.
8.
Kordon EC & Smith GH (1998) An entire functional mammary gland may comprise
the progeny from a single cell. Development 125(10):1921-1930.
9.
Barker N & Clevers H (2010) Lineage tracing in the intestinal epithelium. Curr Protoc
Stem Cell Biol Chapter 5:Unit5A 4.
10.
van Amerongen R, Bowman AN, & Nusse R (2012) Developmental Stage and Time
Dictate the Fate of Wnt/beta-Catenin-Responsive Stem Cells in the Mammary Gland.
Cell Stem Cell 11(3):387-400.
11.
Van Keymeulen A, et al. (2011) Distinct stem cells contribute to mammary gland
development and maintenance. Nature 479(7372):189-193.
12.
Visvader JE (2009) Keeping abreast of the mammary epithelial hierarchy and breast
tumorigenesis. Genes Dev 23(22):2563-2577.
13.
Barker N, et al. (2010) Lgr5(+ve) stem cells drive self-renewal in the stomach and
build long-lived gastric units in vitro. Cell Stem Cell 6(1):25-36.
14.
Barker N, et al. (2007) Identification of stem cells in small intestine and colon by
marker gene Lgr5. Nature 449(7165):1003-1007.
15.
Andl T, Reddy ST, Gaddapara T, & Millar SE (2002) WNT signals are required for the
initiation of hair follicle development. Dev Cell 2(5):643-653.
16.
Chu EY, et al. (2004) Canonical WNT signaling promotes mammary placode
development and is essential for initiation of mammary gland morphogenesis.
Development 131(19):4819-4829.
13
17.
Wang YZ, et al. (2012) Differential gene expression of Wnt signaling pathway in
benign, premalignant, and malignant human breast epithelial cells. Tumour Biol.
18.
Logan CY & Nusse R (2004) The Wnt signaling pathway in development and
disease. Annu Rev Cell Dev Biol 20:781-810.
19.
Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell
127(3):469-480.
20.
MacDonald BT, Tamai K, & He X (2009) Wnt/beta-catenin signaling: components,
mechanisms, and diseases. Dev Cell 17(1):9-26.
21.
Zeng YA & Nusse R (2010) Wnt proteins are self-renewal factors for mammary stem
cells and promote their long-term expansion in culture. Cell Stem Cell 6(6):568-577.
22.
Liu BY, McDermott SP, Khwaja SS, & Alexander CM (2004) The transforming activity
of Wnt effectors correlates with their ability to induce the accumulation of mammary
progenitor cells. Proc Natl Acad Sci U S A 101(12):4158-4163.
23.
Badders NM, et al. (2009) The Wnt receptor, Lrp5, is expressed by mouse mammary
stem cells and is required to maintain the basal lineage. PLoS One 4(8):e6594.
24.
Lindvall C, et al. (2009) The Wnt co-receptor Lrp6 is required for normal mouse
mammary gland development. PLoS One 4(6):e5813.
25.
Nusse R & Varmus HE (1982) Many tumors induced by the mouse mammary tumor
virus contain a provirus integrated in the same region of the host genome. Cell
31(1):99-109.
26.
Clevers H & Nusse R (2012) Wnt/beta-catenin signaling and disease. Cell
149(6):1192-1205.
27.
Morrison SJ & Spradling AC (2008) Stem cells and niches: mechanisms that promote
stem cell maintenance throughout life. Cell 132(4):598-611.
28.
Sato T, et al. (2011) Paneth cells constitute the niche for Lgr5 stem cells in intestinal
crypts. Nature 469(7330):415-418.
29.
de Lau W, et al. (2011) Lgr5 homologues associate with Wnt receptors and mediate
R-spondin signalling. Nature 476(7360):293-297.
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