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Organizing cell renewal in the
intestine: stem cells, signals and
combinatorial control
Cécile Crosnier, Despina Stamataki and Julian Lewis
Abstract | The lining of the intestine is renewed at an extraordinary rate, outpacing all
other tissues in the vertebrate body. The renewal process is neatly organized in space, so
that the whole production line, from the ever-youthful stem cells to their dying, terminally
differentiated progeny, is laid out to view in histological sections. A flurry of recent papers
has clarified the key regulatory signals and brought us to the point where we can begin to
give a coherent account, for at least one tissue, of how these signals collaborate to
organize the architecture and behaviour of a stem-cell system.
Niche
The specific microenvironment
that stem cells inhabit.
Clonal analysis
Analysis of the composition
and distribution of the clones
of cells that are descended
from individual, heritably
marked cells, in order to
discover the fate of these
progenitors.
Vertebrate Development
Laboratory, Cancer Research
UK London Research Institute,
44 Lincoln’s Inn Fields,
London WC2A 3PX, UK.
Correspondence to J.L.
e-mail:
julian.lewis@cancer.org.uk
doi:10.1038/nrg1840
Each self-renewing tissue organizes the turnover of cells
in its own way; but wherever the process depends on
stem cells, the same fundamental questions arise. What
is special about the niche in which stem cells live, and
what mechanisms regulate its extent and location? What
are the signals that control stem-cell proliferation and
dictate whether a daughter of a stem cell shall remain a
stem cell or become committed to differentiation? How
are some stem-cell progeny directed towards one pathway of differentiation, and others to another? How are
the various cell types guided to their proper positions?
In the intestine, it is becoming possible to give a specific
and coherent answer to all of these general questions.
Mutations in the genes that govern the renewal process
can be identified and interpreted through the disruptions
they cause in normal tissue architecture, and analysis
of these mutants has clarified many of the underlying
mechanisms.
In this review, we try to describe both the new
answers that have been revealed by studies of the intestinal stem-cell system, and the problems that continue to
present a challenge. We begin with an outline of the pattern of cell renewal in the intestine. We then discuss how
feedback loops involving the hedgehog, bone morphogenetic protein (BMP), Wnt and Eph/ephrin pathways
define and localize the stem-cell niche. We shall see how
Wnt and Notch signals function together to maintain
stem cells, control proliferation and govern cell fate decisions. Finally, we consider how insights from the intestine illuminate the behaviour of other stem-cell systems,
and in particular their dependence on combinatorial
control by Wnt and Notch.
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Intestinal stem cells are confined to crypts
In the lining of the mammalian intestine, dividing cells
are confined to the crypts of Lieberkühn — finger-like
invaginations of the epithelium into the underlying connective tissue. In the small intestine, the progeny of these
dividing cells migrate upwards from the depths of the
crypts onto the surfaces of the villi — relatively large
finger-like protrusions into the gut lumen — where no
further division occurs and all the cells seem to be fully
differentiated. Here the cells are exposed to the gut contents and finally sloughed from the villus tips (FIG. 1a).
The renewal process operates continually (although with
a strong diurnal rhythm1), with cells taking two to seven
days to make the journey from the site of their final division cycle in the crypt to the point of their exfoliation
from the villus tip2. The differentiated cells generated in
this process are of four distinct types: absorptive, goblet,
enteroendocrine and Paneth cells. Absorptive cells, also
known as columnar cells or enterocytes, are the majority cell type; the other three classes are all secretory
(FIG. 1b).
This pattern of cell migration and epithelial renewal
was first demonstrated by experiments in which a
pulse of tritiated thymidine was used to label a cohort
of dividing cells and follow their fate: this method
provided evidence that all cells of the gut epithelium
originate from stem cells in the crypts 3. The same
work indicated that these stem cells are pluripotent;
each can give rise to all the differentiated cell types.
This result has been confirmed by clonal analysis4–6,
which also revealed that several such stem cells exist in
each crypt7.
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a
Villus
~3,500 cells
b
Stem cells
Paneth cells
Transit-amplifying
cells
Absorptive cells
Goblet cells
Enteroendocrine
cells
Goblet cell
Absorptive cell
Enteroendocrine cell
Paneth cell
Secretory cells
Mouth
of crypt
Crypt
~250 cells
Transit-amplifying cells
Stem cells and Paneth
cells
Intestinal stem cell
Figure 1 | The distribution of epithelial cell types in the mammalian small intestine. a | A villus with one of the
crypts that contribute to renewal of its epithelium. Arrows indicate the upwards flow of cells out of the crypts. Stem
cells lie near the crypt base; it is uncertain whether they are mixed with, or just above, the Paneth cells. Above the
stem cells are transit-amplifying cells (dividing progenitors, some of them already partially differentiated); and above
these, in the neck of the crypt and on the villus, lie post-mitotic differentiated cells (absorptive cells, goblet cells and
enteroendocrine cells; see panel b). In the colon there are no villi, but the organization is otherwise similar; cells are
discarded into the gut lumen after they emerge onto the exposed flat surfaces around the mouths of the crypts.
b | There are four classes of terminally differentiated cells. Absorptive cells have a brush border (a dense array of
microvilli) on their apical surface. The other three classes are all secretory: goblet cells secrete mucus, and their apical
cytoplasm is generally distended with mucus-filled secretory granules; enteroendocrine cells (of which there are
many subtypes) are smaller and secrete various gut hormones (peptides and catecholamines); and Paneth cells secrete
antibacterial proteins (lysozyme and cryptdins or defensins). Paneth cells differ from the other differentiated cell
types in that they lie at the bottoms of the crypts. They seem to be absent in some vertebrate species, including
zebrafish46,90, and in the mammalian large intestine. Panel a modified with permission from REF. 8 © (1998) Royal
Society of London. Panel b modified with permission from REF. 3 © (1974) Wiley-Liss.
From the pattern of cell migration revealed by labelling
experiments with tritiated thymidine or BrdU (bromodeoxyuridine), it seems that the stem cells must lie near
the crypt base. Higher up in the crypt, cells continue to
divide but are destined, with all their progeny, to move
up out of the crypt and eventually to be discarded. The
divisions of these cells, in transit from the stem-cell
region to the villi, amplify the number of progeny that
results from each division of a stem cell. Such cells are
therefore called transit-amplifying cells, and in terms
of prospective fate they are clearly not stem cells: all
their progeny will differentiate and die. Prospective
fate, however, is not the same as intrinsic potency. If
the transit-amplifying cells were put back at the crypt
base, could they function as stem cells? There is some
indirect evidence that the answer is yes8. In terms of
350 | MAY 2006 | VOLUME 7
potency, therefore, it is possible that some or all of the
transit-amplifying cells should be classified as stem cells
on a par with the stem cells at the crypt base.
Despite uncertainties about the identifying features
of the stem cells (BOX 1), we shall see that a great deal is
known about the mechanisms governing their location
and behaviour.
How are crypts and villi made?
Stem cells are dangerous cells: they provide for renewal
and repair, but disaster can ensue if they proliferate
excessively or in the wrong place. How is the intestinal
stem-cell niche defined, how is it created, and what keeps
the stem cells in it? The most complete information
comes from studies of the mouse, and we shall take this
animal as our mammalian model here.
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Box 1 | Defining intestinal stem cells
It is still not clear at what point the progeny of intestinal stem cells lose stem-cell potency
and become irreversibly committed to differentiation. There are, indeed, many genes
that are expressed only by undifferentiated cells near the crypt base and are therefore
candidates to be molecular markers of the stem-cell state24,41,55,73–76. For none of these
genes, however, is there proof that expression is restricted to stem cells only. There is
similar uncertainty about another, more curious, type of proposed stem-cell marker. After
a pulse of tritiated thymidine or BrdU (bromodeoxyuridine) is delivered to the juvenile
gut to label cells that are in S phase, a small proportion of cells in the crypt bases are
found to retain the label for long periods, up to more than 9 weeks72. It has been
suggested that these label-retaining cells are the true stem cells. If so, two
interpretations are possible. The stem cells might divide only rarely, relying on
proliferation in the transit-amplifying stage to boost the number of their progeny.
Alternatively, they might divide at a more normal rate, but segregate their DNA strands
asymmetrically, in such a way as to retain in every division, and for each chromosome, the
specific strand that was initially labelled. The latter ‘immortal strand’ hypothesis has been
proposed as a way in which stem cells might minimize the accumulation of mutations
that are associated with DNA replication71. Wild as the idea might seem, it has received
some experimental support in the gut72, in cultured fibroblasts77, in cultured neural stem
cells78, and in the mammary gland79; but it remains contentious.
Villi begin to form at embryonic day 15; crypts
form substantially later, around postnatal day 7 (FIG. 2).
What, then, is the mechanism that organizes the formation of villi and crypts (or of intervillus pockets,
the equivalent of crypts in some non-mammalian
species)? It has long been known that the modelling
of the intestinal epithelium depends on epithelial–
mesenchymal interactions9, and recent data have identified the hedgehog, platelet-derived growth factor
(PDGF), and BMP signalling pathways as key mediators
of these two-way communications (FIG. 3): mutations in
these pathways derange the construction of crypts and
villi. Moreover, within the epithelium, cells signal to one
another through the Wnt, Notch and Eph/ephrin pathways (FIG. 3): mutations that affect these pathways cause
marked changes in the distribution of cell types along the
crypt–villus axis. The challenge is to understand not just
the action of each type of signal individually, but how the
whole set of signals operates as a system to organize
the crypt–villus architecture and to control the patterning
and renewal of the gut epithelium.
Pseudostratified
Describes an epithelium that,
because of the uneven
positions of the cell nuclei,
seems to contain several layers
of cells (stratified) but is in fact
composed of a single one, in
which all cells make contact
with the basal surface.
Hedgehog demarcates villus from crypt
In the small intestine of the developing mouse, expression of the two ligands Sonic hedgehog (Shh) and Indian
hedgehog (Ihh) is restricted to the epithelium and
becomes progressively concentrated in the intervillus
regions of the epithelium as villus morphogenesis proceeds. Meanwhile, expression of the receptors patched 1
(Ptch1) and patched 2 (Ptch2), and the effectors of
hedgehog signalling, Gli1, Gli2 and Gli3, is restricted
to the underlying mesenchyme10. Hedgehog signalling
from the gut epithelium to the mesenchyme is crucial
for development of the connective-tissue coat around
the gut tube11,12, but it is no less important for the behaviour of the epithelial cells themselves, which are powerfully affected by feedback from the mesenchymal cells.
Blocking of the hedgehog signal by strong overexpression
of an inhibitor, hedgehog-interacting protein (HHIP),
NATURE REVIEWS | GENETICS
leads to a complete absence of villi and the persistence
of a highly proliferative, and sometimes pseudostratified,
intestinal epithelium, with increased activation of the
Wnt pathway and a deficit of properly differentiated
cells10. Partial inhibition of hedgehog signalling leads to
a milder effect: abnormally branched villi are formed on
which there is ectopic epithelial proliferation and ectopic
activation of the Wnt pathway 10. Blocking of the pathway with a pan-hedgehog antibody produces a similar
phenotype13. Although unconditional knockouts of Shh
and Ihh individually have complex effects on embryonic
development of the gut14–16, the general conclusion is that
hedgehog signalling from epithelium to mesenchyme is
required for the formation of villi and the concomitant
restriction of proliferation to the intervillus regions
within the intestinal epithelium. This process must
depend on a feedback loop in which mesenchymal cells
respond directly to hedgehog from the epithelium, and
deliver a signal back to the epithelium by some other
signalling pathway.
In the small intestine, both SHH and IHH are produced only in those sites at which proliferation persists
— the intervillus pockets, and subsequently the bases
of the crypts (although the pattern is different in the
colon17). The suggestion, therefore, is that hedgehog
proteins diffuse outwards from these sites and exert
their effect at a distance, promoting villus formation and
inhibiting crypt-like behaviour in the neighbourhood of
each established crypt, so as to maintain a proper spacing
between one crypt and the next, as discussed below.
Hedgehog is not the only signal passing from epithelium to mesenchyme. PDGFA, like SHH and IHH, is
made by the epithelial cells, and its receptor, PDGFRA,
is expressed in the mesenchyme. PDGF signalling helps
to control the behaviour of the mesenchyme and the
shaping of villi, but does not, apparently, evoke signals
that act back on the epithelium to regulate its proliferation
or differentiation18.
BMP inhibits crypt formation
If hedgehog and PDGF signals are both delivered from
the epithelium to the mesenchyme, what molecules convey signals from the mesenchyme back to the epithelium
to control its regional differentiation? One pathway, at
least, has been identified: BMP2 and BMP4 are both
expressed in the mesenchyme, where they are positively regulated by hedgehog signalling10; their receptor,
BMPR1A, is expressed in the epithelium. The BMP
antagonist noggin is expressed in the neighbourhood of
the crypts, whereas activation of the BMP pathway, as
indicated by the presence of phosphoSMAD1, 5 and 8,
is seen most strongly in the epithelium of the villi18–20.
When the receptor is knocked out, or the antagonist
noggin is overexpressed, excessive quantities of cryptlike structures develop. In the noggin overexpression
mutant, these occur on the sides of the villi19; in the
mouse Bmpr1a knockout, there is also a marked increase
in the number of crypts in the region within which crypts
normally lie20. Similar abnormalities are seen in humans
with juvenile polyposis syndrome, which can be traced
to mutations in BMPR1A or SMAD4 (a key downstream
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Mesenchyme
Epithelium
Villus
Crypt
Early embryo
(E14)
Neonate
Adult
(from P7)
Figure 2 | Morphogenesis of the small intestine in the mouse. The digestive tract originates from the folding of an
endodermal sheet, which undergoes remodelling to form a tube that is lined with stratified epithelium91. Until around
embryonic day 14 (E14), all of the intestinal epithelial cells, which are still undifferentiated, proliferate actively. Villus
morphogenesis starts at E15 and involves a reshaping of the mesenchyme (yellow) that underlies the intestinal epithelium;
the mesenchyme seems to drive the formation of protrusions into the gut lumen. This process is accompanied by marked
effects on the epithelial cells (red and blue cells): the epithelium becomes monolayered, and epithelial proliferation (red)
becomes restricted to the intervillus pockets, and ultimately to crypts. The crypts themselves develop relatively late,
beginning around postnatal day 7 (P7), by invagination of the intervillus epithelium. In some vertebrate species they never
develop. In zebrafish, for example, crypts are absent, but stem cells are maintained in the intervillus pockets 46,92.
effector of BMP signalling)21,22. All this evidence strongly
suggests that (in the adult at least19) BMP signalling is a
key factor, if not the key factor, that mediates the action
of hedgehog, blocking the formation of ectopic crypts,
and that the expression of noggin in the neighbourhood
of each crypt base protects the epithelium in this region
from the action of BMPs, thereby enabling proliferation
to continue.
Crypts and villi: a self-organizing system?
Through hedgehog and BMP, each crypt or intervillus
pocket delivers a long-range signal that inhibits crypt
formation in its neighbourhood. But what then sustains
the crypt itself, and how does the pattern of crypts and
villi arise? Crypts form by invagination of the proliferative intervillus pockets that lie between the villi in
the fetal intestine. The mechanism of this invagination
is unclear, but the origin of the initial spacing pattern
of villi and intervillus pockets can be explained quite
simply (FIG. 3c,d). As Gierer and Meinhardt have shown
mathematically 23, a tissue can pattern itself autonomously by means of a pair of diffusible signals — a shortrange (weakly diffusible) activator and a long-range
(more freely diffusible) inhibitor — both of which are
secreted at the same time by the same cells and regulate
their own production. The positive feedback that is due
to the activator can give rise to self-sustaining foci of
signal production, with a regular spacing between them.
If the hedgehog–BMP relay provides the long-range
inhibition in the intestine, what could provide the shortrange activation? Evidence that we discuss below points
to an obvious candidate: the Wnt signalling pathway.
The epithelial cells in the crypts produce both the signal
molecules — Wnt proteins — and the receptors for the
so-called canonical branch of the Wnt pathway, and they
respond to activation of these receptors by increasing
352 | MAY 2006 | VOLUME 7
their production of at least one of the Wnt molecules
(WNT6 (REF. 24)). The noggin protein produced in the
neighbourhood of the Wnt-active region (and produced
perhaps in response to Wnt) would help to make the
system robust by protecting the crypt or intervillus cells
from inhibition.
This explanation of the origin of the pattern of crypts
and villi in terms of a Gierer–Meinhardt mechanism is
speculative. But the importance of Wnt signalling in the
intestine is firmly established, as we now explain.
Wnt signalling maintains proliferation
The Wnt signalling pathway 25 was the first to be implicated in the control of the gut stem-cell system, and a
large body of evidence shows that activation of the Wnt
pathway is the key factor that maintains the crypt cell
population in a proliferative state. When the pathway
is overactivated, crypts enlarge; when the pathway is
blocked, they disappear.
A subset of members of the Wnt family are
thought to be specifically responsible for canonical
Wnt signalling, and these proteins (WNT3, WNT6
and WNT9B) are expressed only in the epithelium,
where they are restricted to the crypts (at least in the
small intestine 24; FIG. 3 ). The crypt epithelial cells
also express members of the corresponding receptor families (frizzled 5, 6 and 7, and LRP5 and 6)
and show activation of the canonical pathway, which is
manifest in high levels of intranuclear β-catenin. Other
Wnt family members are expressed in the villus mesenchyme, and other frizzled molecules are expressed in the
villus epithelium, but these are thought to mediate only
non-canonical Wnt signalling: the villus epithelial cells
do not contain intranuclear β-catenin.
Deletion of transcription factor 4 (Tcf4), the main
downstream effector of the pathway in the small intestine,
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a
c
Villus
Villus
Villus
BMP
BMP
BMP
Epithelium
Mesenchyme
BMP pathway:
BMPR1A;
phosphoSMAD1, 5, 8
Eph/ephrin pathway:
ephrin B1
Hedgehog
pathway:
PTCH1, 2;
GLI1, 2, 3
BMP pathway:
BMP2, 4
Hedgehog pathway:
SHH, IHH
PDGF pathway:
PDGFA
Canonical Wnt pathway:
WNT3, 6, 9B;
frizzled 5, 6, 7;
LRP5, 6
Eph/ephrin pathway:
EPHB2, EPHB3
Notch pathway:
NOTCH1, 2;
DLL1, 4;
JAG1;
HES1, 6, 7
PDGF pathway:
PDGFRA
BMP pathway:
noggin
b
HH
Wnt
HH
Wnt
Noggin
Noggin
Crypt
Crypt
d
Cell A
Cell B
Wnt
Frizzled
β-Catenin
Mib
APC
HH
Nucleus
β-Catenin*
LEF/TCF
?
Wnt
RBPSUH
Delta
Notch
BMP
NICD
Figure 3 | Signalling pathways in the small intestine. a | Components of the hedgehog (HH), platelet-derived growth
factor (PDGF), bone morphogenetic protein (BMP), Wnt, Eph/ephrin and Notch pathways are expressed in different
regions along the crypt–villus axis — some in the epithelium and some in the mesenchyme. The brackets list, for each
indicated region, the pathway components that are expressed there. Only those molecules mentioned in the text are
included. Because all these signalling pathways have essential functions in many other tissues, experiments to study their
role in the gut have rested heavily on conditional mutagenesis and conditional misexpression, using gut-specific
promoters. Two such promoters have been especially valuable: the villin promoter 93–95, and the Cyp1A1 (cytochrome P450,
family 1, subfamily A, polypeptide 1) promoter 96. Both of these drive expression in all cells of the gut epithelium, including
the stem cells. b | A simplified diagram of the Wnt and Notch pathways, showing the functional relationships among
components mentioned in the text. c | A model of how the HH, BMP and Wnt signalling pathways combine to organize the
pattern of villi and crypts or intervillus pockets. Epithelial cells in each crypt or intervillus pocket form a signalling centre,
which functions as a source of long-range inhibition through the HH–BMP relay, and of short-range auto-activation
through Wnt signalling. HH signalling activates the expression of BMP in the mesenchyme. BMP feeds back on the
intestinal epithelium to repress Wnt signalling. The expression of the BMP inhibitor noggin in the neighbourhood of the
crypts counteracts the effect of BMP so that Wnt activity is maintained in the crypt epithelium. d | The logic of the gene
control network corresponding to the spatial patterning mechanism that is suggested in panel c. NICD, intracellular
domain of Notch.
leads to an absence of proliferating cells in the intestinal
epithelium26. Villi are reduced in number, and the animal
dies soon after birth. Similarly, forced expression of the
diffusible Wnt inhibitor, dickkopf homologue 1 (DKK1),
leads to a loss of crypts and a decrease of villus size and
numbers in adult mice27,28.
Converse effects are seen when the Wnt pathway is
overactivated by mutations in adenomatous polyposis
coli (Apc), β-catenin, or other Wnt pathway components,
leading to the constitutive activation of downstream
targets of the canonical Wnt pathway 29. Therefore,
when Apc is knocked out in the adult gut epithelium,
crypts become enormously enlarged within four to five
days, whereas the villi become smaller, as if the normal
NATURE REVIEWS | GENETICS
progression from a crypt-like proliferative state to a postmitotic differentiated villus state has been blocked30,31.
The outward migration of cells along the villi is also
reduced, implying that the mutant cells remain cryptlike not only in their proliferative behaviour but also in
their affinity for the stem-cell niche30,31.
In a similar way, polyps consisting of masses of giant
crypts develop in the human intestine at sites where
both copies of APC have been lost or inactivated32. In
fact, loss of APC through somatic mutations seems to
be the initiating event in most human colorectal cancers. A mouse model shows similar phenomena: the
Min (Apc Min/+) mouse, carrying a germline mutation in
Apc, forms intestinal adenomas at sites where a somatic
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mutation has eliminated the remaining functional
allele of Apc33. Apc mutant zebrafish similarly develop
intestinal tumours34.
Wnt maintains stem cells cooperatively
Do the effects of Wnt on proliferation reflect an action on
the stem cells themselves, or only on transit-amplifying
cells? Because loss of Apc gives rise to polyps that can
apparently grow without limit, it seems that a high level
of Wnt signalling can keep cells proliferating indefinitely,
or at least for a large number of cycles. If the ability to
divide indefinitely is the feature that distinguishes stem
cells from transit-amplifying cells, it follows that Wnt
signalling can keep cells in a stem-cell state.
A puzzling observation is that the diffusible Wnt
antagonist, SFRP5 (secreted frizzled-related sequence
protein 5), is also produced by some cells in the stemcell region24. We do not know how the effects of Wnt and
its antagonist balance out as a function of distance from
their source; for example, if SFRP5 is the more diffusible factor, the net effect could be autostimulation within
the stem-cell region but inhibition outside that region,
limiting the size of the stem-cell population. In any case,
other evidence — from mutants and the distribution of
nuclear β-catenin — indicates that the Wnt pathway is
strongly activated in the stem-cell region and essential
for maintaining the stem-cell character.
Therefore, it seems that the cells in the crypt stimulate
one another by both making and responding to Wnt.
Furthermore, if the cells respond to Wnt by making more
Wnt24, crypt-like behaviour would be expected to be a
cooperative phenomenon, and cells in which the Wnt
pathway is hyperactive should tend to make it hyperactive
in their neighbours too. Bjerknes and Cheng have examined the adenomas in Min mice35. In these Apc +/– mutants,
the bulk of the intestinal epithelium is phenotypically
normal, with adenomas forming only where a somatic
mutation has given rise to Apc –/– tissue. However, in the
close neighbourhood of the Apc –/– tissue, the Apc +/– cells
proliferate abnormally, forming colossal crypts that are
up to ten times the normal length. This is just what would
be expected if the Apc –/– cells secrete excessive quantities
of Wnt protein. Positive feedback, such that Wnt signal
reception stimulates production of Wnt signal, will tend
to amplify and propagate the effect. The same mechanism
could help to explain the development of polyclonal adenomas, which are observed in both mouse Apc +/– (REF. 36)
and human APC +/– (REF. 37) mutants.
Eph/ephrin signals control cell segregation
Through selective cell migration, the different categories of cells in the intestinal epithelium are segregated
into separate regions: the cluster of Wnt-activated cells
avoids becoming diluted with differentiated cells that
lack Wnt activity (Paneth cells being a special case; see
below), whereas the population of differentiated cells
on the villi avoids contamination with dangerously
proliferative stem cells. This segregation is controlled
by the level of Wnt pathway activation, through the
effects of Wnt signalling on ephrin B1 and its receptors
EPHB2 and EPHB3.
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Eph receptors and their ligands (ephrins) are
membrane-associated proteins that mediate communication between adjacent cells. Repulsion between Eph- and
ephrin-expressing cells seems to be a general mechanism
for creating tissue boundaries38 and for restricting and
defining paths of cell migration39. In the gut, Wnt signalling switches on expression of EphB2 and EphB3 and
inhibits expression of ephrin B1 (REFS 40,41). Therefore,
in the neonatal mouse, ephrin B1 is expressed by
the epithelial cells of the villi, and EphB2 and EphB3
by cells of the intervillus pockets41. As the intestinal
epithelium matures, ephrin B1 expression becomes
confined to the crypt–villus junction and EphB3 to the
Paneth cells, whereas EphB2 is mainly expressed by cells
located near the base of the crypts, with the exception
of Paneth cells.
Deletion of the EphB2 and EphB3 genes leads to
marked changes in the arrangement of the different
cell populations within the crypts: Paneth cells and
proliferating cells are no longer restricted to the bottom
of the crypts but are abnormally scattered along the
crypt–villus axis, and cells expressing ephrin B1 become
distributed throughout the crypts41. Therefore it seems
that control of EphB2 and EphB3 expression by Wnt
ensures the proper segregation of proliferating (and
Paneth) cells from post-mitotic cells. The importance
of this mechanism is indicated by the finding that, in
colorectal cancer, loss of expression of EPHB receptors
is correlated with the onset of invasive behaviour 42.
Wnt makes cells competent for secretory fates
Wnt signalling is crucial in controlling cell proliferation
in the gut epithelium. But there is more to the character
of a gut epithelial cell than the simple dichotomy between
proliferative and non-proliferative behaviour. What part
does Wnt signalling have in guiding the choices the cells
must make between different modes of terminal differentiation? Only one of the terminally differentiated
cell types in the gut — the Paneth cell — shows signs of
sustained Wnt pathway activation. Paneth cells reside
at the base of crypts, where Wnt protein is plentiful,
and Wnt signalling drives their differentiation30,43. All
the other terminally differentiated intestinal cell types
— absorptive, goblet and enteroendocrine — maintain
their differentiated characters in areas where canonical
Wnt signalling is not active, but have those characters
assigned to them while under the influence of Wnt
signalling or shortly after.
When Wnt signalling is defective, as a result of loss
of Tcf4, the gut epithelial cells differentiate as practically normal absorptive cells, but enteroendocrine
cells fail to develop26. When Wnt signalling is blocked
by overexpression of DKK1, a more extreme effect is
seen: absorptive cells still differentiate normally, but
all classes of secretory cells seem to be lost 27. In the
opposite circumstance, where the Wnt pathway is
overactivated by the loss of Apc, there is a more general
failure to differentiate, leading to reduced expression
of markers of absorptive, goblet and enteroendocrine cells, but with an overproduction of Paneth cell
precursors30,31.
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From all this evidence one can tentatively conclude
that Wnt signalling has three basic effects: it keeps the
cells dividing; it stops them from differentiating (except
as Paneth cells); and it confers the potential — but not
the obligation — to differentiate as a secretory cell type
once the cell escapes from the influence of Wnt. Some
other mechanism must guide the final choice between
the various secretory and absorptive fates.
Secretory precursors exert lateral inhibition
Notch and its ligands of the Delta and Serrate/Jagged
subfamilies are transmembrane proteins that mediate communication between cells that are in contact
with one another. These and other components of the
Notch pathway are expressed in the epithelium of
the adult intestinal crypts and for the most part not
in the villus epithelium44, which suggests that Notch
signals are mainly exchanged between cells in the crypts
(FIG. 3a,b).
When the Notch pathway is partially inactivated
by deletion of Hes1 (hairy and enhancer of split 1)
in mouse45 or by a nonsense mutation in deltaD in
zebrafish 46, excessive numbers of goblet and enteroendocrine cells are produced. Stronger inhibition of
Notch signalling by deletion of Rbpsuh (recombining
binding protein suppressor of hairless)47 (which mediates control of gene expression by Notch), or by use of
a γ-secretase inhibitor that prevents the release of NICD
(REFS 47,48) (‘activated Notch’ — the active intracellular
domain of Notch), results in a more extreme effect: the
intestinal epithelium of the mouse becomes almost
exclusively composed of goblet cells. Similarly in the
zebrafish mutant mind bomb (mib), in which Notch
is not activated49, almost all the epithelial cells adopt
a secretory character 46. Increased activity of Notch
signalling has the opposite effect on intestinal cell differentiation: mice that express NICD constitutively in
the gut epithelium show a severe reduction of all three
secretory cell types50.
It therefore seems that in the normal tissue, the cells
that become secretory are those that escape Notch activation. These cells are also the ones that express Delta
proteins46, enabling them to activate Notch in their
neighbours. Notch signalling in the gut epithelium
therefore seems to mediate lateral inhibition in the
standard fashion: like nascent neurons in the CNS51,
cells in the gut that become committed to a secretory fate
express Notch ligands and inhibit their neighbours from
differentiating in the same way.
All three secretory cell types derive from a precursor
expressing Math1 (mouse atonal homologue 1), which
encodes a bHLH (basic helix-loop-helix) transcription
factor that is also expressed in some classes of neuron52
and in sensory hair cells in the ear 53. Math1 mutant
mice lack all three secretory gut cell types54 but still
generate absorptive cells. The Notch-controlled choice
between an absorptive fate (MATH1-negative, receiving lateral inhibition) and a secretory fate (MATH1positive, delivering lateral inhibition) might therefore
be the first of the decisions made by daughters of stem
cells as they become committed to differentiation.
NATURE REVIEWS | GENETICS
Crypt cells that are homozygous for a LacZ knockin
mutation in Math1 form a dense cluster of LacZexpressing cells at the base of each crypt 54. This pattern
suggests that Notch-mediated lateral inhibition has
failed, and that functional MATH1 protein is required
not only for cells to progress along the secretory
pathway of differentiation, but also to deliver lateral
inhibition to their neighbours as they do so. A possible
interpretation of this result is that MATH1 is needed
to drive the expression of Delta.
Cells keep on dividing after fate commitment
Cells in the gut epithelium that express Math1 often
seem to be still capable of dividing (as indicated by
expression of Ki67, a marker of proliferative cells)54.
Therefore, commitment to a secretory fate probably
precedes withdrawal from the cell cycle. This tallies with
the findings of clonal analysis: single genetically marked
cells in the mouse intestine can give rise to large clones
that consist of nothing but secretory cells — as many
as 19 (REF. 6). Clones that consist entirely of absorptive
cells also occur and are on average five times as big.
Therefore different numbers of transit-amplifying divisions in the two committed sublineages could explain
why absorptive cells are so strongly in the majority
on the villi.
Wnt and Notch jointly maintain stem cells
It might be tempting to suggest that there is a simple
division of function between the Wnt and Notch pathways, with Wnt signalling in the crypt base maintaining
the stem-cell proliferative state, and Notch signalling
in the transit-amplifying compartment controlling the
choice between differentiating as an absorptive cell and
differentiating as a secretory cell.
The truth, however, seems to be more interesting.
Notch, Delta and Hes proteins are in fact chiefly expressed
in the neighbourhood of the crypt base, in the stem-cell
region44,55. When Notch signalling is blocked, secretory
cells are overproduced. However, this does not only occur
at the expense of differentiated absorptive cells: it seems
that the whole cell population of the adult intestinal crypt
is converted to a secretory character and stops proliferating 47. Overactivation of the Wnt signalling pathway, at
least as seen in adenomas, is not sufficient to overcome
this proliferation failure: when Min mice are treated
with a γ-secretase inhibitor that abolishes Notch signalling, proliferation is blocked within the tumour tissue47.
The opposite combination of signals — overactivation of
the Notch pathway along the villus epithelium, where the
canonical Wnt pathway is inactive56 — is equally unable
to drive proliferation. Forced expression of NICD in the
newborn mouse does indeed increase the population of
proliferating cells, but mainly in the intervillus regions,
where Wnt signalling is active50.
The ultimate stem cells might respond to Wnt and
Notch signalling according to different rules. As they
are a small minority of the crypt population, different
proliferative behaviour on their part could go unnoticed.
But the simplest interpretation of the observations is that
all the proliferating cells, including the stem cells, depend
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a Intestine
Absorptive cells
b Central nervous system
Secretory cells
Glial cells
c Haemopoietic system
Neuron
Lymphoid and myeloid blood cells
Terminal
differentiation
Amplifying
divisions
Transitamplifying
cells
Secretory
precursor
Lateral
inhibition
Nascent
neuron
Lateral
inhibition
Committed
precursor
(short-term
stem cell)
Stem cells
Stem-cell
divisions
Wnt pathway
activated
Notch pathway
activated
Figure 4 | Wnt and Notch signalling cooperate to maintain stem cells. The diagrams show, in simplified form, the
lineage history and sequence of cell fate choices made by the cells in three stem-cell systems: the intestine (a), the CNS (b),
and the haemopoietic system (c). The differentiated cell types and the programmes of transit-amplifying divisions are
different for the three tissues, but the rules of stem-cell maintenance by Wnt and Notch signalling seem to be the same.
Note, however, that for haemopoietic stem cells the Notch-activating stimulus is thought to be provided by the stromal
bone marrow cells that form the stem-cell niche (probably JAG1 (jagged 1)-expressing osteoblasts), rather than by
adjacent haemopoietic stem cells. The transit-amplifying stages for all these systems are certainly more complex than we
have indicated, and the numbers of amplifying divisions and their timing in relation to commitment to a specific
differentiated fate are uncertain. Although we show Notch signalling only in the stem-cell region, it might also be involved
in later cell fate choices.
on Notch and Wnt signals in combination to keep them
in a proliferating state: neither Wnt pathway activation
nor Notch pathway activation is sufficient by itself.
Wnt signalling evokes Notch signalling
A Wnt pathway mutation, such as loss of Apc, is sufficient to give rise to a tumour that grows without limit.
This suggests that the mutation, in ectopically activating the Wnt pathway, has also ectopically activated the
Notch pathway, as otherwise proliferation would not be
expected to continue. In fact, active Wnt signalling seems
able to switch on Notch activity, if we can judge from the
evidence of the Min mouse: in the adenomatous tissue,
expression of the Notch target gene Hes1 is increased47.
The converse does not seem to apply: there is no obvious
effect on β-catenin nuclear localization when the Notch
pathway is constitutively activated50 or blocked47.
These observations suggest the following model
(FIG. 4a). Wnt signalling drives expression of Notch pathway components. Notch pathway components mediate
lateral inhibition within the Wnt-activated (Wnt+) population, so that some cells express Delta and escape Notch
activation (Notch–) while others fail to express Delta and
have Notch activation (Notch+) imposed on them. The
(Wnt+, Notch–) cells become committed to a secretory
fate and eventually stop dividing. The (Wnt+, Notch+)
cells continue to divide without differentiating, generating
daughters like themselves that again interact through
Box 2 | Notch signalling and epidermal stem cells
In the interfollicular regions of mammalian epidermis, it is the stem cells that express Notch ligands and activate Notch in
their neighbours. Notch activation in these cells, far from helping to maintain the stem-cell state, actually drives cells
towards terminal differentation80, and loss of Notch leads to uncontrolled proliferation81. Therefore, in respect to Notch
signalling, the epidermis behaves in an opposite way to the vertebrate gut. Wnt signalling in the epidermis has complex
functions and, in contrast to what occurs in the vertebrate intestine, hedgehog signalling in the epidermis seems to be a
key positive regulator of cell proliferation82,83.
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Box 3 | Insect gut stem cells obey different rules
A recent surprise has been the finding that the intestine (or at least the midgut) of Drosophila melanogaster also contains
stem cells, providing for continual renewal of the gut lining in the adult fly 84,85. These stem cells give rise to absorptive cells
(with a brush border) and to enteroendocrine cells. Both these differentiated cell types are non-dividing and have a
lifetime of about a week before they are replaced. Given these parallels with the vertebrate system, are the regulatory
mechanisms also similar?
The role of Wnt signalling in the adult D. melanogaster gut is not yet defined, but Notch signalling is crucial. However,
upsetting all expectations, it seems that Notch has a role opposite to the one we have described for the vertebrate gut:
activation of Notch, instead of favouring proliferation, is required to drive cells into a post-mitotic state; and when
Notch activation is artificially blocked, cells that would normally stop dividing as part of the programme of terminal
differentiation instead continue to proliferate. Indirect arguments suggest that the Notch-activating signal is delivered
by the stem cell to its neighbours — the exact opposite to the situation in the vertebrate gut.
Notch and diversify. The size of the group of Wntactivated cells is limited, however, as a result of the shortand long-range spatial signals we discussed in the first
half of this review. Some cells therefore have to move
out, losing Wnt activation. These cells differentiate as
absorptive cells if Notch was still activated in them at the
time of their exit, and as secretory cells if not.
This logical scheme provides a simple explanation of
why Wnt pathway mutations frequently give rise to intestinal cancers, whereas Notch pathway mutations have
not been reported to do so. A Wnt pathway mutation is
sufficient to switch on both of the two activities required
to make a gut cell proliferate indefinitely as a stem cell; a
Notch pathway mutation is not sufficient to do this.
Skin, bone marrow and brain: same principles?
The Wnt and Notch pathways are essential for regulating other stem-cell systems, including the CNS, the epidermis and the bone marrow. Is the logic of control the
same in all these tissues? For the vertebrate epidermis,
the rules are almost certainly different, and the same is
true, rather surprisingly, for the midgut of Drosophila.
In both these cases, Notch signalling has a function that
is diametrically opposite to its role in the vertebrate gut:
the stem cells deliver the Notch-activating signal, and
this drives their neighbours to differentiate and cease
division (BOXES 2,3).
For the CNS (discussed below) and the haemopoietic system (BOX 4; FIG. 4c), however, the parallels with
the gut seem remarkably close: their stem cells seem to
depend on Wnt and Notch in almost the same way as
the stem cells of the intestine.
In the CNS, dividing neuroepithelial progenitors and
stem cells give rise to neurons and glial cells, and this
process can be compared with the way in which intestinal stem cells give rise to secretory cells and absorptive
cells in the gut (FIG. 4b). The geometry is different, but
the control machinery is surprisingly similar. In the
embryonic chick retina, for example, blocking Notch
signalling causes all the dividing neuroepithelial cells
to halt division and differentiate prematurely into neurons; conversely, forced activation of the Notch pathway
inhibits adoption of a neuronal character and maintains the cells as dividing progenitors57 or drives them
towards a glial fate58. This exactly parallels the effects of
manipulating Notch pathway activity in the intestine.
Many other studies point to similar conclusions, both
in the intact neuroepithelium59–62 and for neural stem
cells in culture63–65.
Moreover, in the CNS, as in the gut, Wnt signalling
also is crucial for maintenance of the progenitor or
stem-cell population. Therefore, when canonical Wnt
signalling is artificially blocked in the neural tube of
the developing chick or mouse, the neuroepithelial cells
fail to proliferate. Conversely, when this signalling pathway is artificially overactivated (by forced expression
of stabilized β-catenin), the cells proliferate excessively,
giving rise to a giant brain or spinal cord66–68. In the
hippocampus, where neurogenesis normally continues
in adult life, the neural stem cells generate neurons at
an increased rate when Wnt signalling is overactivated,
and at a decreased rate or not at all when Wnt signalling is blocked69. These findings also parallel the effects
of corresponding treatments on stem-cell proliferation
Box 4 | Notch, Wnt and haemopoietic stem cells
The spatial organization of the haemopoietic stem-cell system is completely different from that of either gut or CNS.
However, in this case, as Duncan et al.86 have pointed out, the stem cells again seem to be regulated by Wnt and Notch
signalling according to combinatorial rules that are similar to those that operate in the intestine (FIG. 4c).
Activation of Wnt signalling by WNT3A or by constitutively activated β-catenin maintains haemopoietic stem cells in a
stem-cell state, stimulates their expression of Notch1, and promotes their proliferation, whereas blocking Wnt signalling
has the opposite effect 87. Studies in transgenic mice that contain a reporter for activation of the Notch pathway show that
Notch is activated in these stem cells86, and that this activation helps to keep them in a stem-cell state both in vitro 88,89 and
in vivo 86. Conversely, when the Notch pathway is artificially blocked by a dominant negative form of RBPSUH (recombining
binding protein suppressor of hairless), the cells differentiate precociously, even though they are exposed to a constant
source of Wnt signal (WNT3A) (REF. 86).
So, as in the intestine and the CNS, it seems that Wnt activity and Notch activity are required in combination to maintain
the stem-cell state in the haemopoietic system.
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REVIEWS
in the gut (although some parts of the CNS might follow different rules66,70). And in the neural tube as in the
intestine, it seems that Wnt signalling brings the Notch
pathway and the machinery of lateral inhibition into
action, regulating the extent of the domain within which
one finds cells expressing the Notch target genes Hes1
and Hes5 (REF. 67). Wnt pathway activation and Notch
pathway activation seem to be required in combination
to keep a cell in the neural progenitor/stem-cell state.
Concluding remarks
In this review, we have tried to highlight some basic
principles through which molecular signals organize the
intestinal stem cell system. Together, the hedgehog, Wnt,
BMP and Eph/ephrin pathways create and maintain the
crypt–villus architecture. The Wnt and Notch signalling pathways combine to control the detailed pattern
of cell fate choices as stem cells divide and give rise to
differentiated progeny. The stem cells of the vertebrate
intestine, CNS and bone marrow all seem to be governed
by Wnt and Notch according to similar combinatorial
logic. But equally it is clear that some other stem-cell
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Acknowledgements
We thank N. Wright and the anonymous referees for helpful
comments, and Cancer Research UK for financial support.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
Apc | BMP2 | BMP4 | BMPR1A | ephrin B1 | EPHB2 | EPHB3 |
Gli1 | Gli2 | Gli3 | Hes1 | HHIP | Ihh | Math1 | noggin | PDGFA |
PDGFRA | Ptch1 | Ptch2 | Rbpsuh | Shh | Tcf4 | WNT3 | WNT6 |
WNT9B
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM
juvenile polyposis syndrome
Access to this links box is available online.
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