The Rockefeller University Press $30.00
J. Cell Biol. Vol. 192 No. 5 767–780
F. Gerbe and J.H. van Es contributed equally to this paper.
Correspondence to Philippe Jay: email@example.com
Abbreviations used in this paper: Atoh1, atonal homologue 1; CBC, crypt base
columnar; ChgA, chromogranin A; COX, cyclooxygenase; DCLK1, doublecortin-
like kinase 1; Gfi1, growth factor-independent 1; Hes1, hairy/enhancer of split
1; HPGDS, hematopoietic prostaglandin-D synthase; Lgr5, leucine-rich repeat
containing G protein–coupled receptor 5; Neurog3, Neurogenin3; PCNA, pro-
liferating cell nuclear antigen; Sox9, SRY-box containing gene 9; Spdef, SAM
pointed domain containing ets transcription factor; Trpm5, transient receptor
potential cation channel, subfamily M, member 5; Wnt, Wingless-related MMTV
The intestinal epithelium is a highly dynamic tissue with con-
tinuous proliferation, migration, differentiation, and apoptosis,
resulting in complete renewal every 2–7 d, in a spatially and
temporally organized manner. This process is coordinated by a
small number of highly conserved signaling pathways (Sancho
et al., 2004). While migrating toward the villi, progenitor cells
differentiate into distinct cell types that can be identified using
morphological criteria and through expression of specific genes.
Differentiated epithelial cells belong to two classes: absorptive
enterocytes and secretory cells. Secretory cells can be further
subdivided into three cell types: mucus-producing goblet
cells, hormone-secreting enteroendocrine cells, and bactericidal
The composition of the villus epithelium mainly results
from the interaction of signaling pathways that are active in
crypt stem and progenitor cells. The most studied examples are
the Wingless-related MMTV integration site (Wnt) and Notch
pathways. Inhibition of the Wnt signaling pathway induces a
complete loss of crypt epithelial progenitors (Korinek et al.,
1998; Pinto et al., 2003). Genetic and pharmacologic inhibition
of the Notch pathway drives the cells toward a secretory fate,
of these cells with the other cell types of the intestinal epi-
thelium and describe the first marker signature allowing
their unambiguous identification. We demonstrate that al-
though mature tuft cells express DCLK1, a putative marker
of quiescent stem cells, they are post-mitotic, short lived,
derive from Lgr5-expressing epithelial stem cells, and are
found in mouse and human tumors. We show that whereas
he unique morphology of tuft cells was first revealed
by electron microscopy analyses in several endoderm-
derived epithelia. Here, we explore the relationship
the ATOH1/MATH1 transcription factor is essential for
their differentiation, Neurog3, SOX9, GFI1, and SPDEF
are dispensable, which distinguishes these cells from
enteroendocrine, Paneth, and goblet cells, and raises
from three to four the number of secretory cell types in the
intestinal epithelium. Moreover, we show that tuft cells are
the main source of endogenous intestinal opioids and
are the only epithelial cells that express cyclooxygenase
enzymes, suggesting important roles for these cells in the
intestinal epithelium physiopathology.
Distinct ATOH1 and Neurog3 requirements define
tuft cells as a new secretory cell type in the
François Gerbe,1 Johan H. van Es,2 Leila Makrini,1 Bénédicte Brulin,1 Georg Mellitzer,3 Sylvie Robine,4
Béatrice Romagnolo,5 Noah F. Shroyer,6 Jean-François Bourgaux,7 Christine Pignodel,8 Hans Clevers,2 and Philippe Jay1
1CNRS UMR5203, Montpellier F-34094; INSERM U661, Montpellier F-34094; Montpellier I University, Montpellier F-34094; Montpellier II University,
Montpellier F-34094; Institute of Functional Genomics; 34094 Montpellier Cedex 5, France
2Hubrecht Institute for Developmental Biology and Stem Cell Research & University Medical Centre Utrecht, 3584CT Utrecht, Netherlands
3Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67412 Illkirch, France
4Morphogenesis and Intracellular Signaling, Institut Curie-CNRS, 75248 Paris Cedex 5, France
5Institut Cochin, Université Paris Descartes; CNRS (UMR 8104); INSERM, U1016; 75014 Paris, France
6Gastroenterology, Hepatology, and Nutrition, Cincinnati Children’s Hospital, Cincinnati, OH 45229
7Service d’Hépato-Gastroentérologie and 8Service d’Anatomie-pathologie, CHU, 30029 Nîmes, France
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as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 192 • NUMBER 5 • 2011 768
cell population in the intestinal epithelium is still a matter of
debate. For instance, we have recently demonstrated that the
solitary cells expressing the doublecortin-like kinase 1 protein
(DCLK1, also called DCAMKL1), which have been considered
as putative quiescent stem cells (Giannakis et al., 2006; May
et al., 2008, 2009; Dekaney et al., 2009; Jin et al., 2009; Sureban
et al., 2009), are bona fide tuft cells (Gerbe et al., 2009). Since
their first identification in the rat trachea (Rhodin and Dalhamn,
1956) and mouse gastrointestinal tract (Jarvi and Keyrilainen,
1956), tuft cells (also known as brush cells) have been found in
several endoderm-derived epithelia. These cells are character-
ized by long and blunt microvilli with prominent rootlets, and
by a well-developed tubulovesicular system in the supranuclear
cytoplasm (Sato, 2007). Several markers have been proposed
for tuft cells, including villin, fimbrin (Höfer and Drenckhahn,
1992), neuronal nitric oxyde synthase (Kugler et al., 1994),
-gustducin (Höfer et al., 1996), Ulex europaeus lectin 1
(Gebhard and Gebert, 1999; Gebert et al., 2000), Cytokeratin 18
(Höfer and Drenckhahn, 1996), and the transient receptor poten-
tial cation channel, subfamily M, member 5 (TRPM5; Bezençon
et al., 2007). However, due to their ubiquitous expression in the
intestinal epithelium, villin and fimbrin are not very suitable
markers of intestinal tuft cells (Höfer and Drenckhahn, 1996).
Similarly, -gustducin, Trpm5, and Ulex europaeus lectin 1 ex-
pression have also been reported in subtypes of enteroendocrine
cells (Jang et al., 2007; Sutherland et al., 2007; Bezençon et al.,
2008; Kokrashvili et al., 2009). Finally, the high neuronal nitric
oxyde synthase expression reported for stomachal and pancre-
atic tuft cells (Kugler et al., 1994) is not a property of intestinal
tuft cells (Sutherland et al., 2007), and the validity of Cytokera-
tin 18 as a marker for mouse intestinal tuft cells is controversial
(Gebert et al., 2000). Thus, none of the above markers is strictly
tuft cell specific and, more than 50 years after their initial dis-
covery, functional studies of tuft cells are still nonexistent. Here,
we report a marker signature that allows unambiguous identifi-
cation of mouse and human tuft cells, both in the small and
large intestines. We extend our previous study to demonstrate
that DCLK1-expressing tuft cells are short lived, post-mitotic
cells that are permanently generated from Lgr5-expressing stem
cells. Furthermore, unlike what is commonly thought, we show
that tuft cells do not belong to the enteroendocrine lineage, but
rather constitute a distinct entity with transcription factor re-
quirements for differentiation that differ from those of entero-
cytes, enteroendocrine, Paneth, and goblet cells.
A set of molecular markers allows
unambiguous identification of tuft cells in
the mouse intestinal epithelium
Trpm5-expressing cells, hypothesized to be tuft cells, were pre-
viously shown to express the cyclooxygenase 1 and 2 (COX1 and
COX2) enzymes (Bezençon et al., 2008), and we recently found
that expression of the DCLK1 protein is a specific marker of tuft
cells (Gerbe et al., 2009). We have now extended these observa-
tions with multiple costaining experiments of the mouse small
intestinal epithelium, based on previously published micro-array
even though the Wnt cascade remains active (Fre et al., 2005;
van Es et al., 2005b) and, accordingly, deletion of the Notch
effector hairy/enhancer of split 1 (Hes1) results in the generation
of excessive numbers of goblet, enteroendocrine, and Paneth
cells (Jensen et al., 2000; Suzuki et al., 2005). Conversely,
the basic-helix-loop-helix transcription factor encoded by the
Atonal homologue 1 gene (Atoh1, also called Math1), which is
repressed by the HES1 transcription factor, is required for a pro-
genitor cell to adopt a secretory fate (Yang et al., 2001; Shroyer
et al., 2007; van Es et al., 2010). It is often thought that a single
Atoh1-dependent secretory progenitor exists for all three secre-
tory cell types. However, some data instead point toward the ex-
istence of several bi-potential progenitors that can each produce
either an enterocyte or a secretory cell belonging to the goblet,
enteroendocrine, or Paneth cell type, a fate choice that likely re-
lies on Notch signaling (Bjerknes and Cheng, 1999). In addition
to ATOH1, a set of transcription factors determines the cell fate
choice and differentiation toward goblet, Paneth, or enteroendo-
crine cell types. Neurogenin 3 (Neurog3, also called Ngn3) is
essential for all intestinal enteroendocrine cells (Jenny et al.,
2002; Mellitzer et al., 2010) and has been reported to be re-
pressed by the growth factor-independent 1 (GFI1) transcrip-
tion factor, which is normally expressed in both Paneth and
goblet cells (Bjerknes and Cheng, 2010). Deletion of the Gfi1
gene, in turn, results in an increased enteroendocrine cell popu-
lation at the expense of Paneth and goblet cells (Shroyer et al.,
2005), likely due to cellular reprogramming of Paneth and gob-
let cells toward a Neurog3+ enteroendocrine cell phenotype
(Bjerknes and Cheng, 2010). The Kruppel-like factor 4 (Klf4)
and SAM pointed domain containing Ets transcription factor
(Spdef) genes are required for terminal differentiation of goblet
cells (Katz et al., 2002). Spdef is necessary for Paneth cell matu-
ration (Gregorieff et al., 2009), and differentiation is shifted
toward the goblet cell type, at the expense of the absorptive as
well as Paneth and enteroendocrine cell types in the intestinal
epithelium of transgenic animals overexpressing Spdef (Noah
et al., 2010). Finally, the SRY-box containing gene 9 (Sox9) is
essential for differentiation of Paneth cells (Bastide et al., 2007;
Mori-Akiyama et al., 2007), and Wnt signaling through the
Frizzled-5 receptor is required for their terminal maturation
(van Es et al., 2005a).
The permanent turnover of the intestinal epithelium relies
on the self-renewing capacity of stem cells. The Wnt target gene
Leucine-rich repeat containing G protein–coupled receptor 5
(Lgr5) has been identified as a marker of crypt base columnar
(CBC) cells (Barker et al., 2007). Genetic lineage-tracing ex-
periments revealed that CBC cells are multipotent and self-
renewing, thus representing genuine intestinal stem cells (Bjerknes
and Cheng, 1999; Barker et al., 2007). In addition, cells located
above the Paneth cell compartment (also known as the +4 posi-
tion) and expressing the Bmi1 polycomb ring finger oncogene
(Bmi1) have been reported to have features of stem cells (Sangiorgi
and Capecchi, 2008). However, the presence of these cells is
limited to the duodenum, and recent studies showed that Bmi1
expression is, at least partially, overlapping with Lgr5 expres-
sion (van der Flier et al., 2009). Both Lgr5+ and Bmi1+ cells are
actively cycling, and the presence of a long-lived quiescent stem
769Tuft cells: a new intestinal epithelial lineage • Gerbe et al.
(Giannakis et al., 2006; May et al., 2009), that DCLK1+ cells
are never observed in a proliferative state. We further confirm
this using the proliferating cell nuclear antigen (PCNA; Fig. 2 A),
and Ki67 or phospho-histone H3 (Fig. S1 D) as proliferation
markers (n > 200 cells). Furthermore, DCLK1-expressing cells
express typical tuft cell markers (Gerbe et al., 2009). Yet, the
possibility existed that we had overlooked a second, nontuft
fraction of DCLK1+ cells, which might represent quiescent stem
cells. To clarify this important point we first compared the level
of DCLK1 expression with that of other tuft cell markers and
found that 98.1% of DCLK1+ cells were COX1+ (n = 253), the
rare DCLK1+ COX1 cells being mainly found in the lower half
of the crypts. To further characterize these DCLK1+ COX1
cells present in crypt bottoms, we exploited one of the unique
morphological features of tuft cells that is not found in other
epithelial cells: the axial bundles of actin filaments supporting
the microvilli (Höfer and Drenckhahn, 1998), which can be vi-
sualized with phalloidin. An intense, apical staining of F-actin
with phalloidin was found in 100% of DCLK1+ tuft cells pres-
ent in crypt bottoms (n = 74). In addition, we found that SOX9
expression is higher in tuft cells present in crypt bottoms than in
Paneth or CBC stem cells. This demonstrates that although
COX1 is barely detectable in differentiating tuft cells, strong
SOX9 expression or the pattern of actin filaments unambigu-
ously identify all DCLK1-expressing cells as tuft cells.
DCLK1+ tuft cells are postmitotic and
undergo permanent turnover fueled by
Lgr5-expressing CBC stem cells
If all DCLK1-expressing cells are indeed differentiated cells,
we would expect them to have a turnover rate similar to that of
the other cell types of the intestinal epithelium, and, like other
intestinal epithelial cell types, they should originate from Lgr5+
CBC stem cells. To measure their turnover rate, we birth-dated
the DCLK1+ tuft cell population with BrdU. Wild-type C57BL/6
mice were treated with BrdU in drinking water for 1 or 2 wk.
After 1 wk of BrdU treatment, we found that 96% DCLK1+
crypt tuft cells were BrdU+. This proportion reached 100% after
2 wk of BrdU treatment (Fig. 2 B). After 2 wk of BrdU treat-
ment, 93% and 100% of tuft cells were BrdU after 1 and 2 wk
of chase with normal drinking water, respectively (Fig. 2 B).
Therefore, DCLK1+ tuft cells are not quiescent stem cells but
instead are postmitotic, short-lived differentiated cells, and their
turnover time is close to 7 d.
We then performed a lineage tracing experiment using the
Lgr5-EGFP-IRES-CreERT2;Rosa26-LacZ compound knock-in mouse
line, in which the expression of the LacZ reporter gene is pre-
vented by a stop cassette flanked by LoxP sequences (Barker et al.,
2007). Upon tamoxifen injection, CRE activity is induced in Lgr5+
stem cells and the stop cassette is excised, leading to permanent
LacZ expression in the progeny of recombined Lgr5-expressing
CBC stem cells. Of note, the intrinsically mosaic Cre expres-
sion in intestinal crypts of Lgr5-EGFP-IRES-CreERT2;Rosa26-LacZ
mice results in a mosaic pattern of -galactosidase in the corre-
sponding villi (Fig. 3 A). If tuft cells originate from Lgr5-
expressing stem cells, we would expect that the proportion
of total -galacosidase+ epithelial cells is identical to that of
data (Bezençon et al., 2008). This allows us to identify a unique
marker signature of tuft cells, which includes coexpression of
SOX9, COX1, COX2, hematopoietic prostaglandin-D synthase
(HPGDS), and DCLK1 (Fig. 1, A–C and F). Compared with
other epithelial cells, cells displaying this signature show a
stronger immunoreactivity toward villin, -tubulin and F-actin,
which is a typical feature of tuft cells (Höfer and Drenckhahn,
1996; Fig. 1, D, E, and G). The SOX9–COX1 or the HPGDS–
COX1 limited signature allowed unambiguous identification of
tuft cells in the mouse and human colon, as well as in other epi-
thelia such as in the mouse gall bladder (Fig. S1, A–C).
All DCLK1+ cells are post mitotic tuft cells
Because DCLK1-expressing cells lack differentiation markers
typical of the other intestinal epithelial cells, and do not prolif-
erate, DCLK1 has been considered as a marker of putative qui-
escent intestinal epithelial stem cells (Giannakis et al., 2006;
May et al., 2008, 2009; Dekaney et al., 2009; Jin et al., 2009;
Sureban et al., 2009). We previously reported, like others
Figure 1. Molecular characterization of mouse intestinal tuft cells. Immuno-
fluorescent stainings for (A) SOX9 and COX1, (B) SOX9 and COX2,
(C) HPGDS and COX1, (D) villin and COX1, (E) -tubulin and COX1, and
(F) DCLK1 and COX1. Each panel contains a merged image on the left,
and gray level pictures of the indicated individual markers corresponding
to the yellow inset area on the right. (G) Whole-mount immunofluorescent
staining for DCLK1 and F-actin on a dissociated fragment of intestinal epi-
thelium. Panels on the right show higher magnification of the cropped area
of the overlay image. Yellow arrowheads point at tuft cells. Nuclei are
stained with Hoechst (blue). Bars, 10 µm.
JCB • VOLUME 192 • NUMBER 5 • 2011 770
epithelium, tuft cells derive from Lgr5-expressing intestinal
crypt base columnar stem cells.
Differentiated tuft cells appear postnatally
The first tuft cells appear around d 7 after birth and become
readily detectable a week later (Fig. 4). In adult mice, tuft cells
are scattered throughout the crypt and villus epithelium, and
represent 0.4% of all epithelial cells (Fig. S2 A). In adult ani-
mals, representation of the tuft cell population is similar
throughout the entire length of the small and large intestines
(Fig. S2 B). Except for SOX9, also expressed in Paneth cells,
-galactosidase+ tuft cells. When we quantified this, we found
53 -galacosidase+ tuft cells out of 10,650 -galactosidase+ epi-
thelial cells (0.49%), and 51 -galacosidase tuft cells out of
14,340 -galacosidase epithelial cells (0.35%). The difference
between the two values was not significant (P = 0.23), and both
values were close to the representation of tuft cells in wild-type
intestinal tissue sections (0.4%; see Fig. S2 A). In addition, tuft
cells located within a stretch of -galacosidase+ epithelial cells
were invariably -galacosidase+, as shown with SOX9 (Fig. 3 B)
and DCLK1 (Fig. 3 C) stainings. This demonstrates that, like
the four established differentiated cytotypes of the intestinal
Figure 2. DCLK1-expressing tuft cells are post-mitotic and continuously renewed. (A) Immunofluorescent staining for COX1, DCLK1, PCNA, and Hoechst.
The PCNA nucleus of a tuft cell is highlighted by a yellow dotted circle. (B) Experimental scheme of the BrdU birth dating experiment. Relative proportion
and number of crypt DCLK1-expressing cells positive for BrdU are indicated. Two representative immunofluorescent stainings for DCLK1 and BrdU are
shown for the indicated time point. Nuclei are stained with Hoechst (blue). Yellow dotted circles highlight tuft cell nuclei.
771Tuft cells: a new intestinal epithelial lineage • Gerbe et al.
tuft cells do not share expression of other markers with entero-
cytes, goblet, or enteroendocrine cells (Fig. S3), suggesting that
they constitute a distinct differentiated cell type.
Presence of cells expressing tuft cell
differentiation markers in intestinal tumors
Because tuft cells are the only epithelial cells expressing the
COX1 and COX2 enzymes in the healthy intestinal epithelium,
and these enzymes are strongly linked to intestinal tumori-
genesis (Wang and Dubois, 2010), we analyzed the status of tuft
cells in mouse small intestinal and colon tumors originating
from two different oncogenic initiating events. Surprisingly, tuft
cells could still be identified in the transformed lesions of mice
carrying a k-RasV12G activating mutation (Janssen et al., 2002;
Fig. 5, A and B) or a mutation in the Adenomatosis polyposis
coli (Apc) tumor suppressor gene (Colnot et al., 2004; Fig. 5, C–F).
In both cases, tuft cells invariably stained negative for expres-
sion of proliferation markers such as PCNA and did not incor-
porate BrdU (Fig. 5 F). In human lesions, tuft cells were also
found in adenomas (Fig. 5 G), but rarely in adenocarcinoma
(Fig. 5 H). This indicates that the tuft cell differentiation path-
way is remarkably conserved in early tumor tissue, but not in
their more malignant counterpart, raising the question of the
potential role played by tuft cells during tumorigenesis.
The ATOH1 transcription factor is required
for tuft cell differentiation, which identifies
them as secretory cells
Having shown that tuft cells are differentiated and constantly
renewed, we then asked to what extent they share the differ-
entiation pathways previously reported for enterocytes, goblet,
Paneth, and enteroendocrine cells and, therefore, to what extent
tuft cells are related to these four cell types. It is generally as-
sumed that commitment toward a specific differentiated cyto-
type involves the choice between one of the secretory fates
(goblet, Paneth, or enteroendocrine cells) and the absorptive
fate (enterocyte). This early cell fate decision is controlled by
ATOH1 because differentiation of all secretory cell types is im-
paired in Atoh1 knock-out mice, whereas differentiation of the
absorptive enterocytes is unaffected (Shroyer et al., 2007).
Compared with Atoh1LoxP/LoxP or Villin-CreERT2 control mice (n = 5),
in which tuft cell representation is unchanged, the intestinal
epithelium of tamoxifen-injected Atoh1LoxP/LoxP; Villin-CreERT2
mice (n = 5) is completely devoid of tuft cells (Fig. 6).
Figure 3. Tuft cells derive from Lgr5+ CBC stem cells. (A) Scheme ex-
plaining how chimeric Cre expression in crypts results in heterogeneous
-galactosidase staining in the adjacent villi (several crypts contribute to
the generation of the cells constituting each villus). Wild-type (gray) and
-galactosidase (blue) cells coming from un-recombined (gray) and recombined
(blue) crypts can migrate and colonize the same villus. The resulting cross sec-
tion is shown. (B) Immunofluorescent staining for SOX9, -galactosidase,
-catenin, and Hoechst in the Lgr5-EGFP-IRES-creERT2; Rosa26-LacZ mouse. Arrow-
heads point at SOX9+ tuft cells. The inset shows higher magnification of a
SOX9+ tuft cells nucleus within a stretch of -galactosidase+ cells. (C) Immuno-
fluorescent staining for DCLK1, -galactosidase, -catenin, and Hoechst
in intestinal sections from the Lgr5-EGFP-IRES-creERT2; Rosa26-LacZ mouse line.
Arrowheads point at DCLK1+ tuft cells. The inset shows higher magnifica-
tion of two DCLK1+ tuft cells within -galactosidase+ crypts. -galactosidase-
crypts are shown by white dotted lines. Bars, 10 µm.
JCB • VOLUME 192 • NUMBER 5 • 2011 772
required for tuft cells. We analyzed the tuft cell population in
the intestinal epithelium of Neurog3LoxP/LoxP; Villin-Cre mice
(n = 4), in which the Neurog3 gene is constitutively deleted at
mid-gestation in all intestinal epithelial cells, and in control
Neurog3LoxP/LoxP mice (n = 4). DCLK1 and chromogranin A (ChgA)
immunohistochemical staining of sections from Neurog3-deficient
mice and littermate controls showed that tuft cells are still pres-
ent in the absence of Neurog3, whereas no enteroendocrine cells
are found (Fig. 7, A and B). This indicates that tuft and entero-
endocrine cells do not share the same transcription factor re-
quirements for differentiation.
Furthermore, within the crypt progenitor cell compart-
ment, Neurog3 expression was restricted to enteroendocrine
progenitor cells, in which SOX9 is barely detectable, in contrast
to tuft cells where SOX9 is highly expressed (Fig. 7 C). These
data, and the lack of evidence for an endocrine function of tuft
cells, lead to the conclusion that tuft cells do not represent a
subset of enteroendocrine cells.
Distinct genetic requirements for
differentiation between tuft and Paneth or
Goblet and Paneth cells share some transcription factor require-
ments for differentiation, for instance GFI1 (Shroyer et al.,
2005; Bjerknes and Cheng, 2010) and SPDEF (Gregorieff et al.,
2009; Noah et al., 2010). To investigate the relationship between
these two cell types and tuft cells, we first analyzed tuft cell dif-
ferentiation in Gfi1-deficient mice (Shroyer et al., 2005). In such
mice (n = 5), we observed the expected increased representation
of Neurog3+ and/or ChgA+ cells (Fig. S4 A), at the expense of
goblet and Paneth cells (Shroyer et al., 2005). As previously re-
ported (Bjerknes and Cheng, 2010), we also observed de novo
Neurog3 expression and the presence of ChgA+ granules in
some Gfi1-deficient lysozyme+ Paneth cells (Fig. S4 B). In con-
trast, the tuft cell population was not affected and Neurog3 ex-
pression was never detected in tuft cells (n = 126) from
Gfi1-deficient mice (Fig. S4, A and B). This indicates that, un-
like Paneth and goblet cells, tuft cells do not require GFI1 to re-
press Neurog3 expression.
Second, we examined whether tuft cell differentiation
requires SPDEF function. Similar numbers of tuft cells were
identified after staining sections of intestine from Spdef-deficient
and control mice (Fig. S4 C). This indicates that tuft cells can
still differentiate in the absence of Spdef, which again distin-
guishes them from goblet and Paneth cells.
Finally, it was shown previously that the SOX9 transcription
factor is expressed in terminally differentiated Paneth cells (Blache
et al., 2004) and is required for their differentiation (Bastide et al.,
2007; Mori-Akiyama et al., 2007). The finding that post-mitotic tuft
cells also express SOX9 prompted us to test whether it is required
for their differentiation. Tamoxifen was injected to induce deletion
of Sox9 and the mice were analyzed after various times, ranging
from 1 wk to 4 wk after induction, the latest time corresponding to
at least four complete renewal cycles of the intestinal epithelium.
Independently of the time left between the tamoxifen injection and
the analysis, tuft cells were still present in Sox9LoxP/LoxP; Villin-
CreERT2 mice (n = 2), as evidenced by COX1 staining (Fig. 8 A),
This conclusion was based on the analysis of several markers
of tuft cells, including the SOX9 transcription factor and the
COX1 enzyme (Fig. 6, A and C) and the -tubulin and DCLK1
proteins (Fig. 6, B and D), which are related to the unique
morphology of tuft cells. Thus, together with goblet, Paneth,
and enteroendocrine cells, tuft cells depend on Atoh1 function
for their differentiation and belong to a secretory lineage of the
Distinct genetic requirements for
differentiation between tuft and
Tuft cells have recently been proposed to represent a subset of
enteroendocrine cells (Formeister et al., 2009; Kokrashvili
et al., 2009). To test this possibility, we checked whether
Neurog3, a transcription factor essential for all enteroendocrine
subtypes (Jenny et al., 2002; Mellitzer et al., 2010), is also
Figure 4. Tuft cells appear after birth. Immunofluorescent staining for
DCLK1 and PCNA in the developing small intestine of E18.5, P7, and P12
mice. Arrowheads point at DCLK1-expressing tuft cells. Nuclei are stained
with Hoechst (blue). Bars, 10 µm.
773Tuft cells: a new intestinal epithelial lineage • Gerbe et al.
Taken together, these data show that most of the well-known
genetic factors that control differentiation of goblet and Paneth cells
are not essential for differentiation of tuft cells, which suggests that
tuft cells are not closely related to either of these two cell types.
and still displayed their typical villin-immunoreactive apical tuft
(Fig. 8 B) and DCLK1 expression (not depicted). Thus, SOX9 is
not necessary for tuft cell survival and differentiation, nor for the
expression of the COX1 and DCLK1 proteins.
Figure 5. Tuft cells are found in mouse and human intestinal tumors. Immunofluorescent staining for tuft cells in K-RasV12G mouse adenoma (A and B);
Apc14 mouse adenoma (C–F), human adenoma (G), and human adenocarcinoma (H). Large fields (A and C) show clusters of tuft cells within the lesions.
The lesion is delimited by PCNA (A) or -catenin staining (C). DCLK1+ tuft cells coexpress the COX1 enzyme (B and D), show nuclear translocation of
-catenin (E), and are not in a proliferative state (F). Using HPGDS staining, tuft cells can also be detected in human adenomas (G) and, in rare cases, in
restricted areas of human adenocarcinomas (H). For fluorescent staining, overlay (left) and individual signals of the indicated markers (right) are shown.
Yellow dotted circles in E highlight tuft cell nuclei. Arrowheads point at tuft cells identified by DCLK1, COX1, and HPGDS expression. Nuclei are stained
with Hoechst (blue) or hematoxylin (G and H). Bars: (A–F) 10 µm; (G and H) 100 µm.
JCB • VOLUME 192 • NUMBER 5 • 2011 774
Intestinal tuft cells have long been refractory to functional analy-
ses, and until now could only be formally identified by electron
microscopic analysis. In this paper we report several molecular
markers that allow unambiguous identification of tuft cells of
the intestinal epithelium, as well as in other organs (see Fig. S1),
thus paving the way to their characterization.
Development of tuft cells
Tuft cells can be identified by DCLK1 expression from 1 wk
postnatal in the mouse intestine. This is consistent with a
study in the rat stomach, in which tuft cells are detected from
the weaning stage (4 wk postnatal; Iseki et al., 1991). How-
ever, it cannot be excluded that immature tuft cells that do
not express differentiation markers such as DCLK1 exist
at earlier stages. For instance, tuft cells have been identi-
fied in the 20-wk-old fetal human small intestine, but their
Tuft cells are responsible for opioid
production by the intestinal epithelium
Endogenous opioids mediate multiple functions in the regula-
tion of the gastrointestinal mucosa physiology, including regu-
lation of gastric emptying, gut motility, intestinal secretion, and
pain (Holzer, 2009), and their production was recently reported
to rely on a subpopulation of enteroendocrine cells (Kokrashvili
et al., 2009) expressing the TRPM5 ion channel, but not ChgA, a
marker of most enteroendocrine cells. As tuft cells are some-
times considered as enteroendocrine cells, and Trpm5 expression
has been reported in tuft cells (Bezençon et al., 2008), Kokrashvili
et al. (2009) hypothesized that opioid-producing cells could be
tuft cells. We tested this hypothesis using our tuft cell markers.
Indeed, costaining of intestinal villi for -endorphin, COX1, and
villin expression confirmed that -endorphin production is re-
stricted to tuft cells (Fig. 9). Importantly, all tuft cells express
-endorphin (n = 70) in the intestinal epithelium, thus validating
the first tuft cell–specific functional property identified so far.
Figure 6. Atoh1 is required for tuft cell differentiation. Immunofluorescent staining for the SOX9 transcription factor (A and C), the COX1 enzyme (A–D),
and for the structural- and morphological-related tuft cells markers DCLK1 and -tubulin (B and D) in intestines from control (A and B) and Atoh1-deficient
mice (C and D), 3 wk after tamoxifen injection. Each panel contains the merged image on the left, and separate pictures of the indicated markers corre-
sponding to the yellow inset on the right. Yellow arrowheads point at tuft cells revealed by SOX9 and COX1 or DCLK1, -tubulin, and COX1 expression.
Nuclei are stained with Hoechst (blue). Bars, 10 µm.
Tuft cells: a new intestinal epithelial lineage • Gerbe et al.
quiescent stem cells, at least in healthy conditions. However,
self-renewing stem cells and early differentiated precursor cell
populations may not be sharply and definitely distinguished dur-
ing the dynamics of epithelial turnover. For instance, tracing the
progeny of Neurog3-expressing cells with a LacZ reporter gene
could, in some rare events, identify complete -galactosidase+
crypt–villus axes, indicating that Neurog3-controlled Cre
DCLK1 expression status has not been analyzed (Moxey and
Early differentiation of tuft
cells in crypt bottoms
In the mouse small intestine, most tuft cells are found on villi or
near the crypt–villus junction, but some are present in intestinal
crypts where they can be detected at virtually any position above
the Paneth cell compartment. Even when localized in crypts,
identifiable tuft cells are postmitotic, always express differenti-
ation markers such as DCLK1, HPGDS, and strong SOX9 ex-
pression, have elevated F-actin immunoreactivity, and display a
gradient expression of COX1 and COX2 (exemplified for COX1
in Fig. 8 A). The presence of DCLK1+, COX1low in crypt bot-
toms suggests that, like for the enteroendocrine cell lineage
(Fig. S5 and Bjerknes and Cheng, 2006), tuft cell fate commit-
ment may occur at an early stage, in the close vicinity of or even
within the stem cell compartment.
DCLK1, quiescent stem cells,
and tuft cells
The fact that crypt base–located post-mitotic tuft cells express
none of the common markers of the other, best known, lineages
probably explains why DCLK1-expressing cells have previ-
ously been proposed as quiescent stem cells (Giannakis et al.,
2006; May et al., 2008, 2009; Dekaney et al., 2009; Jin et al.,
2009; Sureban et al., 2009). However, the majority of DCLK1+
cells are found in the villi, not in the stem cell compartment. In
this study we confirmed DCLK1 as a specific marker of tuft
cells. This permanently renewed cell population with a high
turnover rate can hardly be considered as a likely source of
Figure 7. Neurog3 is dispensable for tuft cell differentiation. (A and B)
Immunofluorescent staining for DCLK1 and ChgA expression in intestines
from 6-mo-old control or Neurog3-deficient mice. Yellow and green arrow-
heads point at tuft and enteroendocrine cells, respectively. (C) Immuno-
fluorescent staining for SOX9 and Neurog3 in wild-type mouse intestine.
The arrowhead points at a Neurog3+, SOX9 cell. Nuclei are stained with
Hoechst (blue). Bars, 10 µm.
Figure 8. Normal tuft cell differentiation in Sox9-deficient intestine.
(A) Immunofluorescent staining for SOX9 and COX1 in intestines from
control or Sox9-deficient mice, 1 mo after the first tamoxifen injection.
Yellow arrowheads point at tuft cells identified by SOX9 and/or COX1
expression. The right inset shows the gray level picture of the COX1 stain-
ing, which is hardly visible in the merged image. (B) Immunofluorescent
staining for SOX9, villin, and COX1 in the intestines of wild-type and Sox9-
deficient mice. Each panel contains the merged image on the left, and
individual fluorescent signals of the indicated markers corresponding to the
yellow inset on the right. Arrowheads point at tuft cells identified by SOX9
and/or COX1 and villin expression. The tuft cell nucleus shown in the
Sox9-deficient tissue is highlighted by the yellow circle. Nuclei are stained
with Hoechst (blue). Bars, 10 µm.
JCB • VOLUME 192 • NUMBER 5 • 2011 776
reports included tuft cells in the enteroendocrine lineage
(Formeister et al., 2009; Kokrashvili et al., 2009) prompted us
to investigate whether one or several differentiation pathways
are shared between enteroendocrine and tuft cells. We found
that in contrast to the enteroendocrine cell lineage, which strictly
depends on Neurog3 function (Jenny et al., 2002; Mellitzer
et al., 2010), tuft cells are still produced in the absence of Neurog3.
In addition, COX1low tuft cells that are still in the process of
terminal maturation express high levels of SOX9 and never
express Neurog3, whereas SOX9 is barely detectable in
Neurog3-expressing enteroendocrine precursor cells (Bjerknes
and Cheng, 2006). Tuft cells and Ngn3-dependent enteroendo-
crine cells thus constitute distinct cell types. In the future, how-
ever, it is conceivable that an endocrine type of secretion is
demonstrated for tuft cells. In this case, they could be consid-
ered as type 2 enteroendocrine cells, which do not require Ngn3
function for their differentiation.
Moreover, whereas differentiation of both goblet and
Paneth cells is regulated by GFI1 (Shroyer et al., 2005; Bjerknes
and Cheng, 2010) and SPDEF (Gregorieff et al., 2009; Noah
et al., 2010), these transcription factors are not essential for
tuft cells. In addition, Paneth cells require SOX9 function to
differentiate (Bastide et al., 2007; Mori-Akiyama et al., 2007)
but tuft cells, which express high levels of SOX9 in their ter-
minally differentiated state, are still present in Sox9-deficient
In summary, although tuft cells are produced from the
same Lgr5+ stem cell as enterocytes, goblet, Paneth, and entero-
endocrine cells, we show here that they represent an indepen-
dent cell type: (i) they express a specific marker signature, and
(ii) except for ATOH1, none of the transcription factors known
to be required for differentiation of the other intestinal epithelial
cell types and that we tested here is essential for tuft cell differ-
entiation (Fig. 10).
Future studies will deepen the knowledge of the lineage
intermediates that are shared by precursors of several differenti-
ated cell types and refine the current understanding of the rela-
tionship between tuft cells and the other constituents of the
Understanding the role played by tuft cells in the physio-
pathology of the intestinal epithelium is currently hampered by
the absence of animal models specifically devoid of tuft cells.
As previously suggested by gene profiling experiments in intes-
tinal Trpm5-expressing cells (Bezençon et al., 2008), we con-
firm here that tuft cells are the only epithelial cells in the healthy
mucosa to express the COX1 and COX2 enzymes, whose ex-
pression is rate limiting for the biosynthesis of prostanoids. Tuft
cells also express Hpgds and thus represent a likely epithelial
source of prostaglandin-D2. This is important given the central
role played by prostanoids in mediating inflammation and
tumorigenesis in the intestinal epithelium (Stenson, 2008; Wang
and Dubois, 2010). Indeed, the presence of tuft cell clusters in
tumors from Apc- or K-Ras–mutated mice suggests a possible
contribution of tuft cells during tumorigenesis. The previous
observation that siRNA-mediated inhibition of Dclk1 expres-
sion in tumor xenografts resulted in reduced tumor growth
(Sureban et al., 2009) further supports this notion. Finally, we
expression occurred in pluripotent cells of the crypt, or that
Neurog3-expressing enteroendocrine precursor cells reverted
to a pluripotent state (Schonhoff et al., 2004). In our case,
DCLK1 expression was only detected in post-mitotic cells, but
it is not possible to definitively exclude that some early tuft
cells that are still in the vicinity of the stem cell niche might
have the capacity to de-differentiate and revert to a stem cell
phenotype under stress conditions, which could potentially ex-
plain the results found by others using in vitro culture assays
(May et al., 2009).
Tuft cells represent a fourth secretory cell
type of the intestinal epithelium
Although tuft cells were identified 50 years ago, they have
remained poorly characterized. As a result, the relationship
between tuft cells and the four other cell types of the intestinal
epithelium has remained elusive, and it is currently assumed
that only four main differentiated cell types constitute this epi-
thelium. Studies on the lineage of intestinal epithelial cells are
still scarce, but pioneering experiments established that multi-
potent Lgr5-expressing CBC stem cells (Barker et al., 2007)
produce several types of intermediate bipotent or monopotent
precursors from which enterocyte or goblet cells (Bjerknes and
Cheng, 1999) and enteroendocrine cells (Bjerknes and Cheng,
2006) arise. In addition, multiple gene deletion studies in mouse
models facilitated the decoding of transcription factor require-
ments for differentiation of these four cell types, but tuft cells
have never been considered in such studies.
Here, we evaluated the status of tuft cells in several of
these genetically engineered mouse lines. We found that tuft
cells are absent in Atoh1-deficient mice, which according to the
prevalent model of intestinal epithelial differentiation (van der
Flier and Clevers, 2009), characterizes them as a secretory cell
type. This is consistent with their capacity to produce (Fig. 9)
and release opioids through an exocrine–paracrine mechanism
(Kokrashvili et al., 2009). However, the fact that previous
Figure 9. Tuft cells are responsible for opioid production by the intesti-
nal epithelium. Whole-mount immunofluorescent staining for -endorphin,
COX1, and villin in dissociated fragments of villus epithelium. Arrowheads
point at tuft cells. Nuclei are stained with Hoechst (blue). Bars, 10 µm.
777 Tuft cells: a new intestinal epithelial lineage • Gerbe et al.
genetic background. For proliferation analyses, mice were injected with
0.1 mg BrdU (Roche)/PBS per gram of mouse body weight. Mice were
sacrificed 2 h after injection. For long-term BrdU incorporation studies,
BrdU was given ad libitum (0.5 mg/ml) during the indicated time.
Slides of human intestinal biopsies were kindly provided by Drs. J.-F. Bourgaux
and C. Pignodel (Centre Hospitalier Régional Universitaire, Nîmes, France).
Fluorescent and bright-field immunohistochemistry
on paraffin-embedded tissue
Tissue dissection, fixation, and immunohistochemistry on thin sections of
paraffin-embedded tissue were performed essentially as described previ-
ously (Bastide et al., 2007). In brief, 5-µm-thick sections were dewaxed in
xylene and rehydrated in graded alcohol baths. Antigen retrieval was per-
formed by boiling slides for 20 min in 10 mM sodium citrate buffer, pH 6.0.
Nonspecific binding sites were blocked in blocking buffer (TBS, pH 7.4,
5% dried milk, and 0.5% Triton X-100) for 60 min at RT. Sections were then
incubated with primary antibodies diluted in blocking buffer overnight at
4°C. Primary antibodies used in this study were as follows: anti-SOX9
(AB5535; 1:1,000) and anti-villin (MAB1671; 1:500) were purchased
from Millipore. Anti-COX1 (sc-1754; 1:200), anti-COX2 (sc-1747; 1:200),
anti-PCNA (sc-56; 1:200), and anti-MUC2 (sc-15334; 1:200) were from
Santa Cruz Biotechnology, Inc. Anti-HPGDS (160013; 1:200) was from
Cayman Chemical. Anti–-tubulin (32–2500; 1:200) was from Invitrogen.
Anti-Neurog3 (F25A1B3; 1:100) was from the Developmental Studies
Hybridoma Bank (Iowa City, IA). Anti–-catenin (610154; 1:200) was
from BD. Anti–-endorphin (20063; 1:100) was from Immunostar. Anti-
BrdU was from Abcam (Ab1893; 1:300) or from the Developmental Stud-
ies Hybridoma Bank (G3G4; 1:300). Anti–-galactosidase (ab9361;
1:400) was from Abcam. Anti-lysozyme (RB-372; 1:500) was from NeoMark-
ers. Anti-DCLK1 was from Abcam (ab37994; 1:200) or Abgent (AP7219b;
1:200). Anti-ChgA was from Immunostar (20085; 1:1,000) or Santa Cruz
Biotechnology, Inc. (sc-1488; 1:400). Anti-Ki67 (ab15580; 1:500)
show here that tuft cells are the only intestinal epithelial cells to
produce -endorphin in healthy conditions, and thus likely con-
tribute to the regulation of vasoconstriction, peristaltic move-
ments, and pain in the intestine.
In conclusion, we present here molecular and genetic
evidences that tuft cells constitute a genuine fifth cell type in the
intestinal epithelium, as well as the first insights into functions
of these cells with their production of prostanoids and opioids.
Further studies will determine whether tuft cells from other
endoderm-derived epithelia share similar functions.
Materials and methods
Animals: mouse strains
The Sox9LoxP/LoxP; Villin-CreERT2 strain was obtained by mating Sox9LoxP/LoxP
mice (Kist et al., 2002) with Villin-CreERT2 animals (el Marjou et al., 2004).
Sox9 deletion in the intestinal epithelium was induced by a single daily
i.p. injection of 1 mg tamoxifen (Sigma-Aldrich) for 5 d. Mice were sacri-
ficed 1 wk and 1 mo after the first tamoxifen injection. The Apc14 (Colnot
et al., 2004), Villin-K-RasV12G (Janssen et al., 2002), Neurog3LoxP/LoxP; Villin-
Cre (Mellitzer et al., 2010), Gfi1/ (Shroyer et al., 2005), and Spdef/
(Gregorieff et al., 2009) strains have been described previously. The
Lgr5-EGFP-IRES-creERT2; Rosa26-LacZ strain has been described previously
(Barker et al., 2007); mice were induced at 4 wk of age by a single i.p. in-
jection of tamoxifen, and were analyzed 14 or 22 mo later. Atoh1LoxP/LoxP;
Villin-CreERT2 mice were obtained by crossing Atoh1LoxP/LoxP (Shroyer et al.,
2007) and Villin-CreERT2 animals (el Marjou et al., 2004). Atoh1 deletion
was induced by a single daily i.p. injection of 1 mg tamoxifen (Sigma-
Aldrich) for 4 d. Mice were sacrificed on d 5 or 22 after the first tamoxi-
fen injection. Wild-type animals used in this study had a C57BL/6
Figure 10. Updated model for the differentiation of the intestinal epithelial cell types. The scheme on the left represents a crypt–villus unit in the adult
mouse small intestinal epithelium. The main functions, including the recently discovered function of Paneth cells in maintaining the CBC stem cell population
(Sato et al., 2011), and representative molecular markers identifying each of the cell types and the intestinal stem cell are indicated. Opioid secretion is
known to occur in the gut lumen (blue arrows; see Kokrashvili et al., 2009). Strong evidence suggests that tuft cells can also act as an epithelial source of
prostanoids (Bezençon et al., 2008 and this paper), but the underlying secretion mechanism still has to be demonstrated. The diagram on the right sum-
marizes the genetic hierarchy of epithelial cell lineage commitment in the intestine. Intestinal CBC stem cells proliferate and produce progenitors. Choice
between absorptive or secretory cell fates is under the control of the hairy/enhancer of split 1 (Hes1) or atonal homologue 1 (Atoh1) gene. Within the
cells committed to secretory types, Neurog3 is required for enteroendocrine cell differentiation. Gfi1 is required for Paneth and goblet cell differentia-
tion, preventing the expression of Neurog3. Sox9 is essential for differentiation of Paneth cells. Spdef is required for both Paneth and goblet cell terminal
maturation. M-cells are known to derive from Lgr5+ CBC stem cells (Barker and Clevers, 2010), but knowledge of the molecular pathways leading to their
differentiation is still missing.
JCB • VOLUME 192 • NUMBER 5 • 2011 778
developed by Dr. O.D. Madsen and the BrdU antibody (G3G4) developed
by Dr. S.J. Kaufman were obtained from the Developmental Studies Hybrid-
oma Bank developed under the auspices of the NICHD and maintained by
The University of Iowa, Department of Biology (Iowa City, IA).
This work was supported by the Agence Nationale pour la Recherche
(ANR-09-BLAN-0368-01), Institut National du Cancer (INCa 2007-1-COL-6-
IC-1 to S. Robine and PLBIO09-070 to P. Jay), and Association pour la Recher-
che contre le Cancer (ARC 5050). F. Gerbe was supported by the Ligue
Nationale contre le Cancer (LNCC).
The authors disclose no conflict of interest.
Submitted: 26 October 2010
Accepted: 3 February 2011
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being incubated in 10 mM DTT for 15 min at room temperature to remove
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Fluorescent pictures were acquired at room temperature under a micro-
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[Sigma-Aldrich]) was used for fluorescent acquisition. Bright-field immuno-
histochemistry pictures were taken at room temperature with a microscope
(Eclipse 80i; Nikon) with Plan Fluor (10x, NA 0.3; 20x, NA 0.5; 40x, NA
0.75; and 60x, NA 0.5–1.25) lenses (Nikon) and a digital camera
(Q-Imaging Retiga 2000R with a Q-Imaging RGB Slider). Pictures were
captured with Q-Capture Pro software (Nikon). For wide-field acquisition
of bright-field images, slides were scanned at 20x with the Nanozoomer
Slide Scanner (Hamamatsu Photonics). Resulting images were visualized
and annotated with NDPview software (Hamamatsu Photonics). Post-treatment
of pictures, annotations, and panel composition were performed with
Photoshop software (Adobe).
Histograms and determination of standard deviations were calculated with Excel
software (Microsoft). Statistical analysis of proportions of -galactosidase+
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Online supplemental material
Fig. S1 shows identification of tuft cells in mouse and human tissues, using
the tuft cell signature. Fig. S2 shows that tuft cells are found all along the
intestinal tract. Fig. S3 shows that intestinal tuft cells do not share terminal
differentiation markers with the other secretory cell lineages. Fig. S4 shows
that the tuft cell population is not affected in Gfi-1– or Spdef-deficient mice.
Fig. S5 shows early enteroendocrine cell differentiation in intestinal crypts.
Online supplemental material is available at http://www.jcb.org/cgi/
The authors wish to thank Drs. Nick Barker and Alex Gregorieff for reagents,
Maaike van den Born for technical help, Daniel Fisher and Catherine Legraver-
end for critical reading and editing of the manuscript, and the staff of the
animal facility for excellent service. The Neurogenin3 antibody (F25A1B3)
779 Tuft cells: a new intestinal epithelial lineage • Gerbe et al.
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