Developmental Biology 428 (2017) 273–282

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Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

Review article

Wnt/β-catenin signaling in adult mammalian epithelial stem cells MARK

a,b a,b,c,⁎
Kai Kretzschmar , Hans Clevers
a
Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Centre (UMC) Utrecht, 3584 CT Utrecht, The
Netherlands
b
Cancer Genomics Netherlands, UMC Utrecht, 3584 CG Utrecht, The Netherlands
c
Princess Máxima Centre for Pediatric Oncology, 3584 CT Utrecht, The Netherlands

A BS T RAC T

Adult stem cells self-renew and replenish differentiated cells in various organs and tissues throughout a
mammal's life. Over the last 25 years an ever-growing body of knowledge has unraveled the essential regulation
of adult mammalian epithelia by the canonical Wnt signaling with its key intracellular effector β-catenin. In this
review, we discuss the principles of the signaling pathway and its role in adult epithelial stem cells of the
intestine and skin during homeostasis and tumorigenesis. We further highlight the research that led to the
identification of new stem cell markers and methods to study adult stem cells ex vivo.

1. Introduction (Wg), a gene critical in segment polarity during embryonic develop-

Stem cells are the cell factories of the mammalian body. From the
zygotes to the stem cells of the fully-grown animal, all stem cells share
the capacity to self-renew as well as to produce differentiating daughter
cells. Yet, as mammalian development progresses, stem cells with
decreasing potential to differentiate are produced. In later stages of
embryonic development, specialized stem cells are born that reside
within tissues and organs and fuel their growth and regeneration
throughout adult life. These adult stem cells have the capacity to
produce one or more types of differentiated cells. During adult home-
ostasis, stem cell fate decisions are tightly regulated by extrinsic cues of
their microenvironment, the so-called stem cell niche, as well as by
intracellular signaling cascades (Watt and Hogan, 2000). In this
review, we discuss one such cascade that is heavily studied as critical
regulator of adult stem cells during homeostasis: the Wnt/β-catenin
signaling pathway. We further highlight how research on this signaling
cascade in mammalian intestine and skin expanded our understanding
of the underlying mechanisms of epithelial tumor formation and
helped to uncover new markers of stem cells and a novel culture
method for adult stem cells.
2. The Wnt/β-catenin signaling pathway
In 1982, Nusse and Varmus described the cloning of a new murine
proto-oncogene that they called integration-1 (Int-1) (Nusse and
Varmus, 1982). It later emerged that its fruit fly homolog was wingless
ment (Nüsslein-Volhard and Wieschaus, 1980; Rijsewijk et al., 1987).
Further research established that Int-1 belongs to a family of genes
highly conserved across the animal kingdom, which is now known as
the wingless-type mouse mammary tumor virus (MMTV) integration
site (Wnt) gene family. To date, 19 Wnt genes in the genomes of mouse
and human have been described (Clevers and Nusse, 2012). Wnt genes
express signaling molecules that are components of a group of signal
transduction cascades, known as Wnt signaling pathways. Wnt ligands
initiate the pathways through the interaction with cell surface receptors
of the Frizzled (FZD) family. To activate canonical Wnt signaling with
its key intracellular effector β-catenin, a co-receptor of the low-density
lipoprotein (LDL) receptor family, LDL-related protein (LRP), is also
required (Pinson et al., 2000; Tamai et al., 2000). The other Wnt
signaling pathways that do not involve β-catenin such as the non-
canonical pathways of Wnt/planar cell polarity (PCP) signaling and
Wnt/Ca2+ signaling as well as the β-catenin-independent Wnt/LRP6
pathways of Wnt/stabilization of proteins (STOP) signaling and Wnt/
target of rapamycin (TOR) have been reviewed elsewhere (Acebron and
Niehrs, 2016; van Amerongen, 2012).
In the absence of Wnt ligands, Frizzled and LRP receptors are
inactive and a destruction complex containing several components
including adenomatous polyposis coli (APC) and Axin recruit newly
synthesized cytoplasmic β-catenin (Fig. 1). Trapped β-catenin is then
phosphorylated by two kinases of the destruction complex, casein
kinase-1 (CK1) and glycogen synthase kinase-3 (GSK-3). Subsequently,
phosphorylated β-catenin is recognized by a E3 ligase harboring a β-

⁎
Corresponding author at: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Centre (UMC) Utrecht, 3584 CT Utrecht, The
Netherlands.
E-mail address: h.clevers@hubrecht.eu (H. Clevers).

http://dx.doi.org/10.1016/j.ydbio.2017.05.015
Received 1 February 2017; Received in revised form 15 May 2017; Accepted 16 May 2017
Available online 17 May 2017
0012-1606/ © 2017 Elsevier Inc. All rights reserved.
K. Kretzschmar, H. Clevers
extracellular
Frizzled
LRP
Dvl
Axin
CK1
GSK-3
-cat
APC
P P
P
-TrCP
-cat
P P U
P U
U
Groucho
TCF
nucleus
Fig. 1. Schematic of the canonical Wnt signaling, which is inactive in the absence of Wnt ligands (left panel) and active upon binding of Wnt ligands (right panel).
transduction repeat-containing protein (β-TrCP) that ubiquitinates β-
catenin for proteasomal degradation.
Canonical Wnt signaling remains inactive until Wnt ligands bind
with both Frizzled and LRP receptors causing the phosphorylation of
the latter receptor (Janda et al., 2012) (Fig. 1). Dishevelled (Dvl), as
part of the destruction complex, acts as adaptor for phosphorylated
LRP to recruit the entire intact complex to the plasma membrane (Li
et al., 2012). While the destruction complex is still able to bind β-
catenin and phosphorylate it, ubiquitination by β-TrCP is inhibited.
Newly synthesized β-catenin accumulates in the cytoplasm eventually
translocating into the nucleus. Nuclear β-catenin then replaces a
repressor of T-cell factor (TCF)/lymphoid enhancer factor (LEF)
transcription factors named Groucho (Roose et al., 1998; van de
Wetering et al., 1991; Verbeek et al., 1995). Interaction of β-catenin
and TCF/LEF recruits transcriptional co-activators and histone modi-
fiers such as the ATP-dependent helicase Brahma-related gene 1
(BRG1, also known as SMARCA4), cyclic adenosine monophosphate-
response element (CREB)-binding protein (CBP), p300, B-cell lympho-
ma 9 (BCL9) and pygopus (Korinek et al., 1997; Molenaar et al., 1996;
Morin et al., 1997; Schuijers et al., 2014; van de Wetering et al., 1997).
This complex is transcriptionally active allowing expression of β-
catenin target genes, which mediate an array of developmental and
homeostatic processes, some to be discussed in this review. Wnt/β-
catenin signal strength and the resulting transcriptional activity are
highly context dependent. It has been suggested that even low levels of
nuclear β-catenin accumulation may be sufficient to induce the
transcriptional program independent of the absolute levels of β-catenin
(Goentoro and Kirschner, 2009). Furthermore, cell type-specific Wnt/
β-catenin target gene expression may also be determined by splice
variants of TCF/LEF1 transcription factors present in the transcrip-
tional complex (Hovanes et al., 2001; Van de Wetering et al., 1996;
Wallmen et al., 2012).
A variety of extracellular and membrane-associated modulators
acting either as inhibitors or agonists tightly regulate Wnt/β-catenin
signaling. Several extracellular secreted molecules are Wnt pathway
inhibitors that directly bind Wnt ligands such as secreted Frizzled-
related proteins (SFRPs) (Leyns et al., 1997; Wang et al., 1997) or Wnt
inhibitory factor (WIF) (Hsieh et al., 1999). Other inhibitors, for
instance, members of the Dickkopf family (DKKs) (Glinka et al.,
1998; Mao et al., 2001), sclerostin (SOST) (Semenov et al., 2005)
and sclerostin domain containing 1 (SOSTDC1) (Ahn et al., 2010;
Developmental Biology 428 (2017) 273–282
Wnt OFF extracellular Wnt ON
Wnt
Frizzled
cytoplasmic LRP
P
Dvl P
cytoplasmic Axin
CK1
GSK-3
-cat APC
P
P
P
-cat
-TrCP
-cat
Proteasome
-cat
-cat
TCF
nucleus
Itasaki et al., 2003), block Wnt signaling through interaction with
membrane-associated LRPs. Both Wnt ligands and LRPs are simulta-
neously bound by adenomatosis polyposis down-regulated 1 (APCDD1)
to inhibit Wnt/β-catenin activation (Shimomura et al., 2010). Apart
from its role as cytoplasmic effector of canonical Wnt signaling, β-
catenin interacts with the cytoplasmic domain of E-cadherin and is also
an intracellular component of adhesion junctions (Ozawa et al., 1989).
As such binding of β-catenin by E-cadherin at the plasma membrane
may therefore reduce the cytoplasmic pool of β-catenin available for
canonical Wnt signaling (Huelsken et al., 1994).
Wnt agonists, in contrast, potentate signal strength of canonical
Wnt signaling. The secreted protein Norrin, for instance, binds
Frizzled-4−LRP5 in the absence of Wnt ligands and, subsequently,
activates β-catenin target gene expression (Junge et al., 2009; Xu et al.,
2004). Secreted proteins of the roof plate-specific spondin (R-spondin
or RSPO) family were initially described to be co-expressed with Wnt
ligands and agonists of canonical Wnt signaling (Kazanskaya et al.,
2004). Later, research conducted independently by three different
groups revealed that R-spondins are the long unknown ligands of the
leucine rich repeat containing G protein-coupled receptor (LGR) family
(de Lau et al., 2011; Glinka et al., 2011; Ruffner et al., 2012).
Subsequently, it was discovered that the active LGR−RSPO complex
binds and inactivates the transmembrane E3 ubiquitin ligases zinc and
ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43), which are
both β-catenin target genes and Wnt antagonists (Hao et al., 2012; Koo
et al., 2012). The above described mechanism for the inactivation of
ZNRF3 and RNF43 through LGR−RSPO is prerequisite for the
prolonged stabilization of β-catenin, subsequent expression of β-
catenin target gene and stem cell self-renewal (Yan et al., 2017), which
plays an essential role for adult homeostasis of epithelial tissues. Work
in the 1990s established that Wnt/β-catenin signaling is not only
required for early embryonic development in vertebrates (Korinek
et al., 1998b), but is also critical for regulating adult homeostasis in
mammals. In 1998, a study linked canonical Wnt signaling with the
maintenance of the adult stem cell compartment first using the mouse
intestine as a model stimulating a new direction of research (Korinek
et al., 1998a).
3. Wnt/β-catenin signaling in intestinal stem cells
The intestinal tract can be separated into two major parts, the small
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K. Kretzschmar, H. Clevers Developmental Biology 428 (2017) 273–282

intestine (subdivided into duodenum, jejunum and ileum) and the

large intestine (consisting of cecum, colon and rectum). To increase the
surface of the absorptive epithelium, the small intestine comprises villi
that harbor differentiated cell types such as enterocytes, enteroendo-
crine cells and goblet cells, while the large intestine is devoid of villi.
Cellular input for the constant loss of differentiated cells is governed by
a compartment of proliferative cells residing in specialized stem cell
niches, called crypts of Lieberkühn, which are embedded in the
mesenchymal tissue of both small and large intestine (Lieberkühn,
1745).
To investigate the role of canonical Wnt signaling for adult home-
ostasis of the gut, Korinek et al. (1998a) inactivated β-catenin target
gene expression through depletion of Tcf7l2, the gene encoding TCF4,
which is specifically expressed in intestinal crypts. Mice lacking TCF4
developed a severe phenotype upon the transition from embryonic
development to postnatal homeostasis of the gut. Tcf7l2-/- mice died
shortly after birth and while the villus compartment of the small
intestine remained unchanged, the entire crypt epithelium with
harboring the adult stem cells was absent (Korinek et al., 1998a).
Overexpression of the Wnt inhibitor DKK1 using transgenic mice or Fig. 3. Crypt bottom of the intestinal epithelium with different cell types described in
adenoviral treatment of wildtype mice completely disrupts proliferation this compartment. *Lrig1 expression specifically in +4 position cells remains contro-
in the adult intestinal crypts (Kuhnert et al., 2004; Pinto et al., 2003) versial.
(Fig. 2A). Later, two different studies inactivated Wnt/β-catenin
signaling postnatally in the intestinal epithelium in vivo (Fevr et al., 1887) and were named crypt base columnar (CBC) cells by Cheng and
2007; van Es et al., 2012a). This resulted in increased intestinal Leblond more than forty years ago (Cheng and Leblond, 1974);
differentiation and complete loss of stem cell proliferation in the crypt however, their existence was neglected until the discovery of Lgr5
demonstrating the critical role of the pathway for adult intestinal brought them back under attention (Fig. 3). To further study these
homeostasis (Fevr et al., 2007; van Es et al., 2012a) (Fig. 2B). To Lgr5+ CBC cells, a genetic mouse models was generated that allowed
further study adult stem cells in the intestine and their specific role of purification of Lgr5+ cells by flow cytometry as well as in vivo lineage-
homeostasis, markers were required. tracing experiments using the inducible Cre/loxP system (Barker et al.,
A screen of previously identified β-catenin target genes provided 2007). When Barker and coworkers injected tamoxifen into double-
several candidate marker genes enriched in putative adult stem cells mutant mice carrying both Lgr5 lineage tracer and LacZ reporter, only
(van de Wetering et al., 2002). The gene Lgr5 was one of them and single Lgr5+ cells in the bottom of the crypts were β-gal+ the following
showed a unique expression pattern marking proliferating cells inter- day. Subsequently, histological analysis performed at later time points,
spersed between Paneth cells in the bottom of intestinal crypts (Barker revealed that the number of β-gal+ intestinal cells dramatically
et al., 2007). These actively cycling cells were described by Josef Paneth increased within five days and labelled clones had expanded out of
140 years ago as “schmale Zellen” (German for slender cells) (Paneth, the crypt bottom reaching high into the villus compartment of the small

Fig. 2. Overexpression of Wnt inhibitor DKK1 (A) or inactivation of the transcription factor TCF4 (B) in the intestinal epithelium causes a loss of the stem cell compartment (positive for
the proliferation marker Ki67 in the left panels and absent in right panels) in the crypt bottom. Images adapted from Pinto et al. (2003) in (A) and from van Es et al. (2012a) in (B).

275
K. Kretzschmar, H. Clevers
intestine (Barker et al., 2007). Further characterization using cell
morphology and co-marker expression confirmed that all differentiated
cell types present in the epithelium of the small intestine including the
absorptive enterocytes as well as the secretory enteroendocrine cells,
goblet cells and Paneth cells were indeed β-gal+ progeny of Lgr5+ CBC
cells. Long-term lineage tracing showed persistence of the β-gal+
progeny in the intestinal epithelium for the life time of a mouse,
demonstrating that Lgr5+ cells are bona fide adult stem cells in vivo
(Barker et al., 2007). Several follow-up studies utilized multicolor
lineage tracing as well as intravital imaging experiments to define that
Lgr5+ stem cells divide symmetrically and stochastically compete for
space in the crypt bottom stem cell niche, a process called neutral drift
(Lopez-Garcia et al., 2010; Ritsma et al., 2014; Snippert et al., 2010b).
Additional β-catenin target genes were later found to be specific or at
least enriched in these CBC cells, such as achaete scute-like 2 (Ascl2)
(Schuijers et al., 2015; van der Flier et al., 2009), prominin-1/cluster of
differentiation 133 (CD133) (Snippert et al., 2009; Zhu et al., 2009)
and Musashi-1 (Potten et al., 2003).
The discovery of rapidly cycling, but long-lived Lgr5+ stem cells was
in stark contrast to the previous dogma that quiescent, DNA label-
retaining intestinal stem cells reside in the so-called +4 position just
above the Paneth cells (Potten et al., 1978) (Fig. 3). However, the
putative stemness character of the +4 cells had not been assessed
experimentally before, because of missing specific markers.
Subsequently, several markers such as B lumphoma Mo-MLV insertion
region 1 homolog (Bmi1), mouse telomerase reverse transcriptase
(mTert), homeodomain-only protein (Hopx) and leucine-rich repeats
and immunoglobulin-like domains protein 1 (Lrig1) were proposed to
specifically label quiescent stem cells at the +4 position (Fig. 3). Several
studies demonstrated that cells expressing these markers are compe-
tent to regenerate the intestinal epithelium including Lgr5+ stem cells
during homeostasis and upon injury or are, in contrast to Lgr5+ stem
cells, radiation-resistant (Li et al., 2014; Montgomery et al., 2011;
Powell et al., 2012; Sangiorgi and Capecchi, 2008; Takeda et al., 2011;
Tian et al., 2011; Yan et al., 2012). However, the interpretation of these
results remains controversial. For instance, the exact expression
pattern of Lrig1 is disputed, as Jensen and colleagues described at
the same time as Powell et al. that Lrig1 is broadly expressed in rapidly
cycling cells in the lower intestinal crypt with the highest levels found
in Lgr5+ stem cells (Wong et al., 2012). Furthermore, crypt expression
of the other +4 cell markers Bmi1, mTert and Hopx is broader than
previously anticipated with high levels of these markers indeed being
found in Lgr5+ stem cells (Grün et al., 2015; Munoz et al., 2012; Wang
et al., 2013). On the other hand, robust evidence suggests that the
intestinal epithelium is unexpectedly plastic, which makes drawing
conclusions on a possible stem cell hierarchy difficult (Tetteh et al.,
2015). Our group showed that secretory precursor cells expressing the
Notch ligand Delta-like 1 (Dll1) are competent to replenish the stem
cell niche upon loss of Lgr5+ stem cells (van Es et al., 2012b). A study
by Winton and colleagues revealed that a pool of Bmi1+ intestinal
label-retaining cells (LRCs) acting as secretory precursors of the
lineages of Paneth cells and enteroendocrine cells during normal
homeostasis, can replenish the entire intestinal epithelium upon
damage (Buczacki et al., 2013). Recent work demonstrated that also
the precursors of the most abundant intestinal epithelial cell type, the
absorptive enterocytes, have the capacity to replace lost Lgr5+ stem
cells (Tetteh et al., 2016a). It therefore remains unclear if Lgr5+ CBC
cells and +4 cells are distinct stem cell compartments or indeed
interdependent stem cell pools during intestinal homeostasis and
regeneration in vivo (Takeda et al., 2011). This unresolved question
requires further research to uncover the exact nature of the relation-
ship of these intestinal stem cell populations.
The remarkable capacity of Lgr5+ CBC cells to renew the intestinal
epithelium long-term can not only be observed in vivo. Utilizing the
EGFP tag it was possible to purify single Lgr5−EGFP+ stem cells by
flow cytometry and embed them into extracellular matrix (ECM)-rich
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Developmental Biology 428 (2017) 273–282
Matrigel (Sato et al., 2009). Providing the embedded single cells with
culture medium rich in stem cell niche factors such as epidermal
growth factor (EGF), the bone morphogenetic protein (BMP) inhibitor
Noggin and the above discussed Wnt agonist R-spondin, spontaneously
generated three-dimensional self-organizing cell clusters that re-
sembled the anatomy of the intestinal epithelium (Sato et al., 2009).
These in-vitro grown structures, so-called organoids, not only con-
tained Lgr5+ stem cells, but all differentiated cell types of the intestinal
epithelium (Grün et al., 2015; Sato et al., 2009). Using minced pieces of
murine intestinal tissue Kuo and colleagues succeeded in generating
organoid cultures, which, however, required mesenchymal cells as
support (Ootani et al., 2009). These organoids contained stem cells
expressing Lgr5 as well as Bmi1 that increased in number upon R-
spondin treatment. Organoid cultures can be maintained in culture
long-term and can be used to selectively induce particular types of
differentiated intestinal epithelial cells by manipulating intracellular
signaling pathways including the Wnt/β-catenin pathway (Basak et al.,
2016; Sato et al., 2009; Yin et al., 2014) further highlighting that Wnt-
active intestinal crypt cells such as those expressing Lgr5+ are indeed
stem cells in vivo and ex vivo.
Since intestinal stem cell homeostasis is dependent on the activity
of the Wnt/β-catenin signaling cascade, it does not come as a surprise
that components of the pathway are frequently mutated in colorectal
carcinomas (CRCs). The main molecular mechanism behind tumor
initiation in the intestine are loss-of-function mutations in the APC
gene cause cytoplasmic stabilization of β-catenin allowing for its
subsequent nuclear translocation and activation of target gene expres-
sion (Korinek et al., 1997; Rubinfeld et al., 1996; Su et al., 1993). In
rare instances activating mutations in CTNNB1, the gene encoding β-
catenin, or inactivating mutations in the AXIN2 gene have been found
to be tumor-initiating (Lammi et al., 2004; Liu et al., 2000; Morin
et al., 1997). Crypt stem cells expressing markers such as Lgr5, Bmi1
or CD133/Prominin-1 that acquire APC mutations are likely cells-of-
origin of intestinal cancers (Barker et al., 2009; Sangiorgi and
Capecchi, 2008; Schepers et al., 2012; Zhu et al., 2009). APC loss-of-
function in progenitor cells is insufficient to drive intestinal tumor-
igenesis (Barker et al., 2009; Tetteh et al., 2016a). However, when APC
is inactivated in the presence of mutations in KRAS, TP53 and SMAD4
that are associated with tumor propagation (Drost et al., 2015; Fearon
and Vogelstein, 1990; Fumagalli et al., 2017), CRCs can also arise from
differentiated intestinal cells in an experimental setting (Tetteh et al.,
2016b).
4. Wnt/β-catenin signaling in epidermal stem cells
Canonical Wnt signaling is also heavily studied in another epithelial
tissue: mammalian epidermis, which comprises a stratified epithelium
called interfollicular epidermis and several specialized appendages,
hair follicles, sebaceous glands and sweat glands. The stem cell
compartment of the epidermis is the so-called basal layer that is
embedded in an extracellular matrix (ECM)-protein-rich basement
membrane deposited by the dermis, a mesenchymal niche containing
different cell types such as fibroblasts. Committed cells leave the basal
layer, move into the suprabasal layers and terminally differentiate into
specialized epidermal lineages contributing the protective barrier of the
skin. For instance, sebocytes produce a moist and oily sebum contain-
ing antimicrobial products, follicular keratinocytes generate hair shafts
that collectively built the heat-protective fur coat and interfollicular
keratinocytes express crosslinking structural proteins (keratinization)
to eventually building an impenetrable physical barrier of highly
keratinized dead cells, the cornified envelope. While interfollicular
epidermis and sebaceous glands undergo constant cellular turnover,
hair follicles self-renew in a cyclic fashion with phases of hair growth
(anagen), regression (catagen) and rest (telogen).
In 2001, an elegant study provided first evidence for the critical role
of Wnt/β-catenin signaling during hair follicle formation and regen-
K. Kretzschmar, H. Clevers
eration in murine skin epidermis (Huelsken et al., 2001). In their
study, Birchmeier and colleagues specifically depleted of β-catenin in
the epidermal basal layer. This resulted in perturbed hair follicle
morphogenesis during skin development, while maintenance of the
hair follicle stem cell compartment was disrupted postnatally triggering
hair follicle loss in young adult mice (Huelsken et al., 2001). However,
formation of the stratified epithelium in between hair follicles, called
interfollicular epidermis, was not effected in neonatal mice. Depletion
of the genes encoding LEF1, TCF3 and TCF4 resulted in similar
phenotypes as those described for epidermal specific β-catenin deletion
(Nguyen et al., 2009; van Genderen et al., 1994). While transgenic mice
overexpressing LEF1, the main transcription factor interacting with β-
catenin to trigger expression of hair differentiation-specific Wnt target
genes, under the control of the keratin 14 (K14) promoter (specifically
expressed in the epidermal basal layer) showed aberrant formation of
hair placodes in epidermal compartments such as the interfollicular
epidermis (Zhou et al., 1995). These early studies suggested that Wnt/
β-catenin signaling may be essential in epidermal stem cell fate
decisions selecting the differentiation towards either the follicular or
the epidermal lineage.
In agreement with this work, several studies by the laboratories of
Fuchs and Watt demonstrated that Wnt/β-catenin signaling in post-
natal epidermis acts as a critical regulator of the lineage selection
program of epidermal stem cells. Different mouse models with genetic
stabilization of β-catenin in the epidermal basal layer showed de novo
formation of hair follicles in adult skin (Baker et al., 2010; Gat et al.,
1998; Lo Celso et al., 2004; Silva-Vargas et al., 2005). Transient
stabilization of β-catenin in specific regions of the epidermal basal layer
of adult mouse skin induced formation of so-called ectopic hair follicles
from epidermal compartments such as the interfollicular epidermis
and the sebaceous glands, normally avoid of hair follicle differentiation
(Baker et al., 2010; Silva-Vargas et al., 2005) (Fig. 4). To demonstrate
the opposite, the two groups independently generated transgenic mice
expressing a N-terminally truncated version of LEF1 (ΔNLEF1) under
the control of the K14 promoter that allowed for inhibition of the
transcriptional activity of canonical Wnt signaling, even in the presence
of β-catenin in the nucleus (Merrill et al., 2001; Niemann et al., 2002).
Gradually, this resulted in a loss of hair differentiation and a conver-
sion of the hair follicles into cysts, expressing markers of both
interfollicular epidermis and sebaceous gland (Merrill et al., 2001;
Niemann et al., 2002). Critical component of the hair follicle stem cell
niche are mesenchymal cells that aggregate together to form the dermal
papilla (Driskell et al., 2009; Greco et al., 2009; Jahoda et al., 1984;
Rompolas et al., 2012). Modulation of Wnt/β-catenin signaling within
dermal papilla cells has been shown to control hair follicle growth
(Kishimoto et al., 2000; Kitagawa et al., 2009). However, there is
growing evidence that β-catenin activation within hair follicle stem
cells is sufficient to drive hair growth (Deschene et al., 2014;
Kretzschmar et al., 2015) (Fig. 4). Collectively, these studies exemplify
the critical role of Wnt/β-catenin signaling for postnatal skin home-
ostasis and epidermal stem cell biology.
Epidermal stem cells were initially believed to be solely localized to
a region of the hair follicle enriched for label-retaining cells (LRCs), the
so-called bulge (Braun et al., 2003; Cotsarelis et al., 1990; Tumbar
et al., 2004). However, in the past ten years more and more stem cell
populations have been described in the epidermis (Kretzschmar and
Watt, 2014). Several components of canonical Wnt pathway have been
shown to be specifically expressed in epidermal stem cells. A study in
2008, for instance, found that Lgr5 marks rapidly cycling, Wnt-active
stem cells in the hair follicle (Jaks et al., 2008) (Fig. 5). Lgr5 expression
did overlap with known bulge stem cell markers such as CD34 and K15,
while being absent from LRCs. Yet, Lgr5+ hair follicle stem cells exhibit
the highest colony-forming capacity of all tested epidermal stem cell
populations tested in vitro. Furthermore, lineage tracing experiments
revealed robust contribution of Lgr5+ stem cells to the cellular input in
vivo to the hair follicle lineage and in skin reconstitution assays to all
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Developmental Biology 428 (2017) 273–282
epidermal lineages (Jaks et al., 2008). While Lgr5 expression in skin
development was not detected until late in embryogenesis at embryonic
day (E)18.5, its close relative Lgr6 is expressed as early as E14.5 in hair
placodes forming in the skin (Snippert et al., 2010a). Postnatally, Lgr6
marks actively cycling stem cells in the isthmus, a region of the hair
follicle just above the bulge, the sebaceous gland and the interfollicular
epidermis (Füllgrabe et al., 2015; Kretzschmar et al., 2016; Page et al.,
2013; Snippert et al., 2010a) (Fig. 5). These epidermal compartments
are also regenerated long-term by Lgr6+ stem cells, which further
contribute to wound repair and new hair follicles arising in adult mouse
skin (Füllgrabe et al., 2015; Kretzschmar et al., 2016; Snippert et al.,
2010a). Interestingly, Lgr5 is co-expressed with Lgr4 in the lower hair
follicle, while Lgr6 is not co-expressed with either in the epidermis
(Snippert et al., 2010a). The reason for this distinct compartmentaliza-
tion of the Lgr family members within the epidermis is not known and
requires further investigations.
Since Lgr6 is, in contrast to Lgr5, no direct Wnt/β-catenin target
gene, and considering earlier studies showing no obvious effect of
inactivating β-catenin in the interfollicular epidermis and sebaceous
glands (Huelsken et al., 2001), it remained unclear whether Wnt-
responsive cells existed outside of the lower hair follicle (i.e. hair bulge
and germ). A study by Nusse and colleagues addressed this issue and
discovered that stem cells in the basal layer of interfollicular epidermis
expressed the Wnt/β-catenin target gene Axin2 (Lim et al., 2013)
(Fig. 5). These stem cells not only robustly contributed to epidermal
wound repair, but also required activation of canonical Wnt signaling
in an autocrine manner to proliferate and maintain adult homeostasis
of the epidermis. Interestingly, a subsequent study showed that Axin2
is also expressed by a population of hair follicle stem cells that are
maintained by a similar autocrine mechanism, yet, these cells located
in the outer bulge appear to be quiescent (Lim et al., 2016) (Fig. 5).
Collectively, these results demonstrate that Wnt/β-catenin signaling
plays an essential role for the homeostasis of adult skin epidermis.
Some of its components and target genes are further markers of
distinct epidermal stem cell populations that govern cellular input into
the autonomously maintained lineages of epidermal differentiation
(Kretzschmar et al., 2016; Page et al., 2013).
In the epidermis, sustained Wnt/β-catenin signaling is critical for
the cell differentiation into the hair follicle lineage. In line with this
observation, activating mutations of pathway components, in particular
of β-catenin itself, have been associated with the initiation of hair
follicle tumors such as pilomatricomas (Chan et al., 1999; Gat et al.,
1998; Lo Celso et al., 2004). The most common skin cancer, the basal
cell carcinoma (BCC), has been shown to arise from activating
mutations of both Wnt/β-catenin and Hedgehog signaling (Palmer
et al., 2008; Salto-Tellez et al., 2006; Youssef et al., 2012). The cellular
origin of BCCs remained controversial, since this tumor type resembles
undifferentiated follicular and interfollicular basal keratinocytes.
Several studies by the Blanpain and co-workers suggested that BCCs
are initiated by interfollicular epidermal cells that adopt a hair follicle-
like phenotype (Youssef et al., 2012; Youssef et al., 2010), while other
reports defined hair follicle stem cells as tumor-initiating cells
(Peterson et al., 2015; Wang et al., 2011). On the other hand,
Grachtchouk and colleagues showed in mice that different BCC
subtypes arise from different epidermal subcompartments with the
interfollicular epidermis being the origin of superficial BCCs and
nodular BCCs developing from the hair follicle (Grachtchouk et al.,
2011). Another type of β-catenin-dependent epidermal tumors are
trichofolliculomas, which are benign and contain mostly differentiated
cells (Gat et al., 1998), possibly because of activation of vitamin D
receptor signaling (Palmer et al., 2008). While most β-catenin-depen-
dent epidermal tumors adopt a HF-like phenotype, inactivating muta-
tions in LEF1 trigger formation of sebaceous gland tumors (Niemann
et al., 2002; Niemann et al., 2003; Takeda et al., 2006). Huelsken and
co-workers showed that in squamous cell carcinomas (SCCs) generated
in the epidermis of mice using the classical two-step chemical
K. Kretzschmar, H. Clevers Developmental Biology 428 (2017) 273–282

Fig. 4. Activation of β-catenin in adult epidermis induces formation of ectopic hair follicle from sebaceous glands and other non-follicular epidermal compartments. Wild-type (A) or
ΔK5ΔNβ-cateninER transgenic mice (B, C) received one to three doses of 1.5 mg 4-hydroxytamoxifen (4-OHT) and tissue was collected one or two weeks later as indicated. Back skin
sections were stained with hematoxylin and eosin (H & E). Lines indicate ectopic HFs and dashed lines demarcate sebaceous glands. Scale bars: 200 µm.
Adapted from Kretzschmar et al. (2016).

carcinogenesis protocol, hair follicle stem cells with high levels of
nuclear β-catenin acted as tumor-propagating cells (Malanchi et al.,
2008). This study suggests that SCCs may also be β-catenin-dependent
epidermal tumors, as epidermal loss of β-catenin caused complete
tumor regression. The involvement of Wnt/β-catenin signaling in many
different types of epidermal tumors demonstrates its critical role for
neoplasms of the skin. Compartmentalization of epidermal stem cells
as well as cross-talk with other signaling pathways may underlie
epidermal tumor heterogeneity arising from altered Wnt/β-catenin
signaling (Kretzschmar et al., 2016; Watt and Collins, 2008).
5. Concluding remarks
In this review, we have presented an overview over the current
knowledge about the Wnt/β-catenin signaling cascade and its role for
the epithelia in adult intestine and skin. We have further highlighted
how research into this pathway has uncovered several adult stem cell
markers expressed in these tissues, some of them even functional such
as Lgr5 (de Lau et al., 2011) and Axin2 (Lim et al., 2013). Based on
these discoveries, Wnt pathway components and β-catenin target genes
were identified as adult stem cell markers in many other epithelial
tissues of the mammalian body. Stem cells in the stomach (Barker
et al., 2010), kidney (Barker et al., 2012), mammary glands (de Visser
et al., 2012; Plaks et al., 2013; Rios et al., 2014; Shackleton et al., 2006;
Van Keymeulen et al., 2011), cochlea of the ear (Bramhall et al., 2014;
Chai et al., 2012; McLean et al., 2016; Shi et al., 2012), ovary (Flesken-
Nikitin et al., 2013) and taste buds (Ren et al., 2014; Takeda et al.,
2013; Yee et al., 2013) have been shown to express Lgr5, while Lgr6+
stem cells are described for mammary glands (Blaas et al., 2016), taste
buds (Ren et al., 2014), nails (Lehoczky and Tabin, 2015) and cochlea
(Zhang et al., 2015). Axin2+ stem and progenitor cells also have been
identified in mammary gland (van Amerongen et al., 2012), kidney

278
K. Kretzschmar, H. Clevers
Fig. 5. Epidermal stem cell populations in adult telogen skin (hair follicles are resting)
marked by components of the Wnt/β-catenin signaling pathway.
(Rinkevich et al., 2014), liver (Wang et al., 2015) and cochlea (Jan
et al., 2013). Further, the understanding of the intestinal stem cell
niche requiring Wnt ligands and R-spondins and the ability to purify
adult intestinal stem cells led to one of the most exiting discovery of the
last ten year, the generation of organoid cultures (Sato et al., 2009).
Organoid technology has transformed the fields of developmental
biology and stem cell research (Kretzschmar and Clevers, 2016). It is
also a promising application in disease research and clinical therapeu-
tics. And yet, it all started from a simple observation in the mouse
intestine linking canonical Wnt signaling with the maintenance of the
adult stem cell compartment (Korinek et al., 1998a). We look forward
to more exiting research shedding light on unknown roles of Wnt/β-
catenin signaling cascade on adult epithelia and their stem cell niches.
Acknowledgments
We are grateful to all colleagues in the field who contributed to the
work described in this review. We apologize to those whose research
could not be discussed due to space limitations. We are thankful to Kim
Boonekamp for critical reading the manuscript. KK is supported by a
VENI grant from the Netherlands Organisation for Scientific
Research (NWO-ZonMW, 016.166.140) and is a long-term fellow of
the Human Frontier Science Program Organization (HFSPO, LT771/
2015). HC is named inventor on several patents related to Lgr5 stem-
cell-based organoid technology.
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