Primary cilia regulate Gli/Hedgehog activation
Sara Cervantes1, Janet Lau1, David A. Cano1,2, Cecilia Borromeo-Austin, and Matthias Hebrok3
Diabetes Center, Department of Medicine, University of California, San Francisco, CA 94143
Edited* by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved April 20, 2010 (received for review September 6, 2009)
Previous studies have suggested that defects in pancreatic epithelium
caused by activation of the Hedgehog (Hh) signaling pathway are
secondary to changes in the differentiation state of the surrounding
pancreatic epithelium, both during development and in adult tissue
during neoplastic transformation. To determine the consequences of
epithelial Hh activation during pancreas development, we employed
a transgenic mouse model in which an activated version of GLI2,
in the pancreatic epithelium. Surprisingly, efficient Hh activation was
not observed in these transgenic mice, indicating the presence of
Hh activation. Additional studies revealed that primary cilia regulate
is sufficient to cause significant up-regulation of the Hh pathway in
pancreata of mice overexpressing GLI2. As a consequence of overt Hh
activation, we observe profound morphological changes in both the
exocrine and endocrine pancreas. Increased Hh activity also induced
the expansion of an undifferentiated cell population expressing pro-
genitor markers. Thus, our findings suggest that Hh signaling plays
a critical role in regulating pancreatic epithelial plasticity.
progenitor cells|pancreatic duct|islet|embryonic development|
studies in mice have revealedthat deregulationofHhactivity affects
pancreas morphogenesis and function (reviewed in ref. 1). In these
studies, Hh activity was deregulated by either manipulation of Hh
ligands, which can signal both to the pancreatic epithelium and
mesenchyme, or by the use of conventional mutant mice (1–5).
Therefore, these studies do not differentiate between epithelial and
mesenchymal Hh signaling. Hh signaling is known to mediate com-
addressed the differential requirement for Hh specifically in the
pancreatic epithelium and mesenchyme. Epithelial cell-specific ac-
tivation of Hh signaling appears not to have any major effect on
pancreatic development (6, 7), andHh elimination in epithelial cells
Conversely, pancreatic mesenchyme appears to be the predominate
receiving tissue of Hh signaling, and inappropriate stimulation in-
terferes with the epithelial–mesenchymal crosstalk essential for
inhibition of Hh signaling by inactivation of the Hh transducer
Smoothened (Smo) in epithelial cells does not affect pancreatic ad-
enocarcinoma formation (8), whereas Smo inactivation in stromal
cells results in growth inhibition in pancreatic cancer xenograft
signaling plays the major role during pancreas development and
pancreatic cancer, and suggest that the developing pancreatic epi-
thelium is insensitive to deregulation of Hh signaling.
However, previous studies from our group have shown that
pancreas-specific overexpression of a version of GLI2 lacking the
N-terminal repressor domain (GLI2ΔN) in pancreatic epithelial
progenitor cells in Pdx1-Cre;CLEG2 mice results in the forma-
tion of undifferentiated tumors (7). These findings suggest an
he Hedgehog (Hh) signaling pathway plays a critical role in
pancreas development and function. Loss- and gain-of-function
additional, cell-autonomous role of activated Hh signaling within
the mature pancreas epithelium. To determine whether activa-
tion of Hh signaling in the pancreatic epithelium also affects
pancreas formation, we have analyzed pancreas organogenesis in
Surprisingly, we find that ectopic expression of GLI2ΔN fails
to efficiently up-regulate Hh pathway within the pancreas epi-
thelium. This observation suggests that mechanisms exist in
pancreatic epithelial cells that block inappropriate activation of
the pathway. Recent studies have shown that primary cilia, cel-
lular organelles, are critical regulators of the Hh pathway during
embryonic development, organ function, and in cancer (11–15).
Specifically, cilia ablation increases Hh activation mediated by
GLI2ΔN during medulloblastoma and basal cell carcinoma
(BCC) formation (11, 15). Our findings indicate that concomi-
tant elimination of cilia in the presence of GLI2ΔN in mice
results in overt Hh activation in pancreatic epithelium and,
consequently, impaired pancreas formation. These pancreata
display a significant loss of both exocrine and endocrine tissue
accompanied by the appearance of undifferentiated epithelial
cells expressing pancreatic progenitor cell markers. Thus, our
study reveals a role for primary cilia in regulating Hh signaling
during pancreas formation and demonstrates that excessive Hh
activation results in unique phenotypes in the pancreas, under-
scoring a potential role for Hh signaling in modulating the dif-
ferentiated state of pancreatic cells.
Primary Cilia Prevent Full Hh Activation upon GLI2ΔN Overexpression.
We have recently shown that, in Pdx1-Cre;CLEG2 transgenic mice,
GLI2ΔN accumulation is observed in a mosaic fashion within the
mice is fused to a myc-tag in its N terminus, thus allowing for
immunodetection by an anti-myc antibody (myc-GLI2ΔN, hereaf-
ter) (7). The restricted expression pattern of myc-GLI2ΔN is sur-
prising because the CLEG2 transgene should be transcribed in all
pancreatic cells due tothe efficient eliminationof the precedinglox-
β-actin (CAG) promoter (7). To determine whether expression of
the CLEG2 transgene in the pancreas indeed leads to activation of
the Hh signaling pathway, we crossed Pdx1-Cre;CLEG2 mice with
Ptch1lacZ/+mice. Ptch1 is a direct transcriptional target of Hh sig-
Author contributions: S.C., J.L., D.A.C., and M.H. designed research; S.C., J.L., D.A.C., and
C.B.-A. performed research; S.C., J.L., D.A.C., and M.H. analyzed data; and S.C., J.L., D.A.C.,
and M.H. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1S.C., J.L., and D.A.C. contributed equally to this work.
2Present address: Instituto de Biomedicina de Sevilla, Servicio de Endocrinología y Nutri-
ción, Hospitales Universitarios Virgen del Rocío, Sevilla 41013, Spain.
3To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| June 1, 2010
| vol. 107
| no. 22
Hh pathway activity (16). Analysis of β-gal activity in 3-week-old
Pdx1-Cre;CLEG2;Ptch1lacZ/+mice revealed few cells within the
pancreas displaying detectable activity (Fig. 1A), suggesting that
control mechanisms present in pancreatic epithelial cells prevent
complete activation of Hh signaling in Pdx1-Cre;CLEG2 mice.
Primary cilia regulate the level of Hh signaling during mouse
development in different organs and tissues (17, 18), and
therefore could also potentially regulate Hh signaling in the
pancreas. Importantly, cilia have been recently shown to repress
Hh activation mediated by myc-GLI2ΔN during medulloblas-
toma and BCC formation (11, 15). To address the role of cilia in
pancreatic epithelial Hh signaling, we generated compound
Pdx1-Cre;CLEG2;Kif3alox/lox;Ptch1lacZ/+mice characterized by
ectopic expression of myc-GLI2ΔN (CLEG2), loss of primary
cilia via elimination of the Kif3a gene (Kif3alox/lox), one of the key
components of the kinesin-2 complex that is required for cilio-
genesis (19), and expression of Ptch1-lacZ as a marker for Hh
activity (Ptch1lacZ/+). A significant increase both in the area and
intensity of β-gal staining of positive cells was observed in the
pancreata of Pdx1-Cre;CLEG2;Kif3alox/lox;Ptch1lacZ/+compared
with Pdx1-Cre;CLEG2;Ptch1lacZ/+mice during postnatal (Fig.
1B) and embryonic stages (Fig. S1). Interestingly, Hh activity
remained mostly localized to the main ductal branches, one of
the ciliated cell types in the pancreas (20), at e15.5 and e17.5, in
Pdx1-Cre;CLEG2;Kif3alox/lox;Ptch1lacZ/+mice. Of note, cilia ab-
lation in Pdx1-Cre;Kif3alox/lox;Ptch1lacZ/+mice resulted in de-
creased β-gal activity during embryonic stages compared with
Ptch1lacZ/+controls (Fig. S1), thus suggesting a role for primary
cilia in regulating endogenous Hh activity. Importantly, β-gal
assay conditions used at embryonic stages were more sensitive
than those used in 3-week-old mice (SI Methods). Thus, Hh
signaling in postnatal pancreata is active at low levels in ductal
cells and islets (21). Quantitative measurements of expression of
Hh target genes in whole pancreata revealed that whereas Gli1
and Ptch1 expression was marginally increased in Pdx1-Cre;
CLEG2 tissue, cilia ablation in Pdx1-Cre;CLEG2;Kif3alox/loxmice
resulted in a robust increase of Hh target genes at early postnatal
stages (Fig. S2).
To determine whether the increase in Gli/Hh activity corre-
lates with accumulation of the myc-GLI2ΔN protein, we assessed
its level by immunohistochemical analysis of the myc-tag in
transgenic mice. As previously reported (7), myc-GLI2ΔN pro-
tein only accumulated in a small number of cells in Pdx1-Cre;
CLEG2 pancreata during postnatal stages (Fig. 1C). In contrast,
Pdx1-Cre;CLEG2;Kif3alox/loxmice displayed a significant increase
in the number of cells marked by wide expression of myc-
GLI2ΔN protein (Fig. 1D). Furthermore, we determined that
cilia ablation correlated with myc-GLI2ΔN accumulation, as well
as increased Hh activation (as measured by β-gal expression in
Pdx1-Cre;CLEG2;Kif3alox/lox;Ptch1lacZ/+mice; Fig. S3). Impor-
tantly, we found that, despite its modification, myc-GLI2ΔN is
able to locate to the primary cilia (Fig. 1 E and F).
Our findings indicate that cilia modulate myc-GLI2ΔN accu-
mulation in Pdx1-Cre;CLEG2 mice. In other contexts, cilia have
been shown to control Hh signaling activity by specifically regu-
lating the formation of processed Gli3 repressor (Gli3R). To de-
Gli3 in protein extracts from Pdx1-Cre;Kif3alox/loxand Pdx1-Cre;
CLEG2;Kif3alox/loxpostnatal pancreata.Surprisingly,we foundthat
total Gli3 levels (full length and processed) appeared dramatically
increased in both Pdx1-Cre;Kif3alox/loxand Pdx1-Cre;CLEG2;
Kif3alox/loxpancreata compared with control littermates (Fig. S4).
Thus, in contrast to recent studies in brain and skin (11, 15), these
data indicate that cilia function to curb Gli3 levels in the pancreas.
Cilia Regulate Hh Signaling Downstream of Smoothened. Next, to
determine whether cilia regulate Hh activity in pancreatic epi-
thelial cells at different levels of the signaling pathway, we used
a transgenic mouse harboring a constitutively active version of
Smoothened (SmoM2) that is expressed upon Cre-mediated
excision of an upstream lox-stop-lox sequence (22). Activation of
Hh signaling using this mouse model has been widely achieved in
multiple contexts (23, 24). In agreement with recent reports (7),
we found that Hh activity was not significantly increased in Pdx1-
Cre;SmoM2;Ptch1lacZ/+when compared with control Ptch1lacZ/+
pancreata (Fig. S5). To determine whether cilia prevented Hh
activation at the level of Smo, we eliminated cilia in the context
of SmoM2 expression. We found that Ptch1-lacZ activity re-
mained unchanged in Pdx1-Cre;Kif3alox/lox;SmoM2;Ptch1lacZ/+,
findings that are in stark contrast to the increased Hh signaling
activity observed in Pdx1-Cre;CLEG2;Kif3alox/lox;Ptch1lacZ/+mice
(Fig. S5 and Fig. 1B). To confirm these observations, we condi-
overexpressing mice. (A) Analysis of β-gal activity in 3-week-old Pdx1-Cre;
CLEG2;Ptch1lacZ/+mice revealed few cells within the pancreas displaying
detectable activity. (B) Ablation of primary cilia through Kif3a inactivation
results in a significant increase of both the area and intensity of β-gal
staining of positive cells in the pancreata of Pdx1-Cre;CLEG2;Kif3alox/lox;
Ptch1lacZ/+mice. (C) Myc-GLI2ΔN protein accumulates in a limited number of
cells in Pdx1-Cre;CLEG2 pancreata (arrowheads). (D) In contrast, the number
of pancreatic cells marked by strong expression of the myc-GLI2ΔN fusion
protein significantly increases in Pdx1-Cre;CLEG2;Kif3alox/loxmice. Note the
increased stromal compartment (asterisks) and the presence of epithelial cell
nests accumulating high levels of myc-GLI2ΔN (outlined). (E and F) Myc-
GLI2ΔN fusion protein localizes to primary cilia in ductal cells of Pdx1-Cre;
CLEG2 mice. Note that the areas of strong myc staining in the Pdx1-Cre;
CLEG2 sample correspond to one of the few duct areas in which the trans-
gene was active.
Primary cilia prevent full Hh activation in pancreas of myc-GLI2ΔN-
| www.pnas.org/cgi/doi/10.1073/pnas.0909900107Cervantes et al.
tionally eliminated cilia along with the Hh repressor Patched1,
which blocks Hh signaling by inhibiting Smo transduction activ-
ity. We found that in Pdx1-Cre;Kif3alox/lox;Ptch1lox/lacZmice, Hh
activity remained similar to that found in the other mice har-
boring Hh-activating mutations upstream of Gli2 (Fig. S5). Thus,
our findings suggest that primary cilia regulate Hh signaling
downstream of Ptch1 and Smo, possibly at the level of Gli, in the
Full Hh Activation in the Pancreas Results in Loss of Pancreatic Tissue
in Pdx1-Cre;CLEG2;Kif3alox/loxMice. The finding that cilia ablation in
Pdx1-Cre;CLEG2;Kif3alox/loxmice results in overt activation of
Hh signaling allowed us to evaluate whether cell-autonomous
deregulation of Hh signaling in the pancreatic epithelium im-
pacts pancreas formation. Triple-transgenic Pdx1-Cre;CLEG2;
Kif3alox/loxmice were born at the expected Mendelian ratio and
appeared grossly normal. However, by 2–3 weeks of age, Pdx1-
Cre;CLEG2;Kif3alox/loxmice were smaller, failed to thrive, and
showed postnatal lethality. A profound loss of pancreatic tissue,
in both the exocrine and endocrine compartments, was observed
in Pdx1-Cre;CLEG2;Kif3alox/loxanimals (Fig. 2D). The severe
acinar cell loss was accompanied by expansion of the duct-like
epithelium and stromal compartment (Fig. 2D). In addition, the
duct-like epithelium present in triple-transgenic Pdx1-Cre;
CLEG2;Kif3alox/loxmice was composed of tall columnar cells
distinct from the normally cuboidal duct morphology (Fig. 2L
Inset). These cells are likely derived from the duct-like epithe-
lium, as they were contiguous with tissues staining for ductal
markers CK19 and mucin. However, the abnormal cells com-
pletely lost expression of these markers and accumulated high
levels of myc-GLI2ΔN protein, suggesting an inverse correlation
between Hh signaling levels and ductal marker expression (Fig. 2
H and L). Within or adjacent to ducts, we also found solid epi-
thelial cell nests embedded in a matrix of fibrous stroma (Figs. 1D
and 2 D and H). The epithelial cell nests were strongly positive for
myc-GLI2ΔN and did not express markers for any of the three
mature pancreatic cell lineages (amylase, acinar; synaptophysin,
endocrine; mucin, duct; Figs. 1D and 2 H and L and Fig. S6), in-
that is accompanied by a loss of cellular differentiation and ex-
pansion of abnormal cells. In contrast, and as previously reported
Cre;CLEG2 mice (Fig. 2 B, F, and J), whereas mild duct dilation
and reduction in acinar cells were noted in Pdx1-Cre;Kif3alox/lox
mice by 2–3 weeks of age (Fig. 2 C, G, and K).
Expression of Progenitor Cell Markers in Hh-Active Pancreatic
Epithelial Cells. The abnormal epithelial nests budding from the
ducts in Pdx1-Cre;CLEG2;Kif3alox/loxmice did not express markers
normally present in mature pancreatic cell types, and thus we de-
cided to characterize them in more detail. Expression of the cell-
adhesion protein E-cadherin confirmed the epithelial nature of the
high-myc-GLI2ΔN-expressing cells (Fig. 3B). Though markers typ-
did detect expression ofFoxA2 and Sox9 (Fig. 3 B and D), proteins
and C). However, these transcription factors are also expressed
during pancreatic organogenesis in progenitors before the second-
ary transition (26–28), suggesting that the high-myc-GLI2ΔN-
expressing cells might have acquired a progenitor-like state.
Further evidence for this hypothesis comes from the observation
that the abnormal cells are highly proliferative (Fig. 3F) and display
against Hes1 (Fig. 3H), a Notch-target gene that is expressed in em-
bryonic progenitor cells (29). Hes1 was found only in centroacinar
3G). Interestingly, expression of Pdx-1, an embryonic pancreatic
transcription factor and β-cell marker (31, 32), was excluded from
Cre;CLEG2;Kif3alox/loxpancreata (Fig. 3J). Thus, these findings sug-
gest that cell-autonomous activation of Hh signaling in pancreatic
epithelium induces dedifferentiation and expansion of cells express-
ing a subset of progenitor markers.
Temporal analysis revealed that these undifferentiated cells
were first observed at P0. At this time point, a few abnormal
ductal structures could be found in Pdx1-Cre;CLEG2;Kif3alox/lox
pancreata (Fig. 3L). These structures displayed abundant myc-
GLI2ΔN and expressed low level of ductal mucin-1, which was
not appropriately localized to the apical membrane, further
suggesting that Hh activation interferes with the differentiation
state of duct cells (Fig. 3N). Furthermore, we also found unusual
cells that accumulated myc-GLI2ΔN and coexpressed amylase
and CK19 located within dilating ductal structures (Fig. 3 O and
P). The coexpression of both exocrine and ductal markers is
suggestive of a cell caught in an intermediate stage of trans-
differentiation that could contribute to the expansion of ductal
structures observed in older Pdx1-Cre;CLEG2;Kif3alox/loxmice.
Dysmorphic ducts displaying high Hh activity were detected as
early as e15.5 in Pdx1-Cre;CLEG2;Kif3alox/lox;Ptch1lacZ/+mice
(Fig. S1), thus suggesting impaired ductal formation during
embryogenesis in the context of up-regulated Hh signaling.
Endocrine Defects in Pdx1-Cre;CLEG2;Kif3alox/loxMice. In addition to
endocrine compartment in Pdx1-Cre;CLEG2;Kif3alox/loxmice. A
75% reduction in endocrine area in Pdx1-Cre;CLEG2;Kif3alox/lox
mice was found by postnatal day 5 (P5), in contrast to the 50% re-
duction observed in Pdx1-Cre;CLEG2 mice (Fig. 4K). Moreover,
the architecture of α- and β-cells in the remaining islets appeared
disorganized in Pdx1-Cre;CLEG2;Kif3alox/loxmice (Fig. 4D).
Hematoxylin/eosin-stained sections of 3-week-old mice reveal loss of pan-
creatic tissue and expansion of duct-like epithelium in Pdx1-Cre;CLEG2;
Kif3alox/loxmice. Note the increased stromal compartment in Pdx1-Cre;
CLEG2;Kif3alox/loxmice (asterisks in D). (E–H) Extensive acinar cell loss as
shown by the decrease of amylase staining and increased dilation of duct-
like structures marked by CK19 staining is observed in Pdx1-Cre;CLEG2;
Kif3alox/loxmice. (G) Mild ductal dilation is observed in Pdx1-Cre;Kif3alox/lox
mice. (I–L) Expression of the ductal marker mucin-1 is lost in cells expressing
high levels of myc-GLI2 in Pdx1-Cre;CLEG2;Kif3alox/loxmice (arrowheads). (J)
Myc-GLI2ΔN accumulation is observed in a subset of pancreatic cells in Pdx1-
Cre;CLEG2 mice. Epithelial cell nests are outlined in D and H.
Full Hh activation results in perturbed pancreas morphology. (A–D)
Cervantes et al. PNAS
| June 1, 2010
| vol. 107
| no. 22
To evaluate the timing of endocrine cell loss, we analyzed islet
area starting at e15.5, when endocrine cell neogenesis peaks, and
at P0, when islet cells begin reorganizing to achieve final islet
architecture. A substantial 50% reduction in the endocrine area
in Pdx1-Cre;CLEG2;Kif3alox/loxmice was observed at e15.5 and
P0, comparable to that seen in Pdx1-Cre;CLEG2 mice (Fig. 4 E–
J). Therefore, these data indicate that endocrine defects in Pdx1-
Cre;CLEG2;Kif3alox/loxmice deviate from Pdx1-Cre;CLEG2 mice
To further investigate the reduction in endocrine cells detected
at e15.5 in Pdx1-Cre;CLEG2 and Pdx1-Cre;CLEG2;Kif3alox/lox
mice, we performed immunofluorescence staining against neuro-
genin3 (ngn3), which marks all endocrine precursors, and found
that their number is similarly reduced in both Pdx1-Cre;CLEG2
and Pdx1-Cre;CLEG2;Kif3alox/loxmice (Fig. 4L). In addition,
reduction in Pdx1-Cre;CLEG2 and Pdx1-Cre;CLEG2;Kif3alox/lox
compared with control mice (Fig. 4M). Thus, though the ratio of
endocrine precursors to epithelial area is normal, the number of
endocrine precursors is reduced as a consequence of impaired
expansion of the pancreatic epithelium. Interestingly, CLEG2
transgene expression resulted in similar embryonic defects in the
presence and absence of cilia, suggesting that the level of Hh ac-
tivation achieved in Pdx1-Cre;CLEG2 pancreata is sufficient to
impair expansion of the embryonic pancreas epithelium.
In contrast to embryonic expansion of endocrine cells via neo-
genesis, postnatal expansion of islet cells is predominantly achieved
via proliferation. To address the question of whether the postnatal
loss of endocrine tissue may be attributed to changes in islet cell
proliferation and/or apoptosis rates, we quantified phospho-Histone
3 and cleaved caspase-3-positive cells, respectively, in postnatal
pancreata. We found transient changes in proliferative capacity in
Cre;Kif3alox/loxpancreata at P0 and a marginal increase in Pdx1-Cre;
CLEG2;Kif3alox/loxislets at P5 (Fig. 4 N and O). In contrast, a signif-
CLEG2;Kif3alox/loxislets between P0 and P5 (Fig. 4 P and Q), in-
dicating that postnatal endocrine cells cannot sustain deregulated
To determine the role of epithelial Hh signaling on pancreas
formation, we have overexpressed an activated version of GLI2,
to activate Hh signaling in a cell-autonomous manner specifically
in the pancreatic epithelium. Despite the forced expression of
this Hh-activating allele, we did not observe efficient Hh acti-
vation. To determine whether primary cilia could regulate Hh
signaling in the pancreatic epithelium, we eliminated these
organelles in mice carrying distinct Hh gain-of-function muta-
tions. Ablation of primary cilia resulted in strong Hh activation
in mice harboring a GLI2-activating mutation but not in SmoM2
and Ptch1lox/LacZmutant mice, indicating that primary cilia reg-
ulate Hh activity downstream of Smo in the pancreas.
The intriguing fact that Hh activating mutations upstream of
Gli2 (i.e., SmoM2) do not efficiently activate Hh signaling sug-
gests that pancreatic epithelial cells are equipped with regulatory
mechanisms that prevent Hh activation. Our study shows that
primary cilia do not fulfill this role, because their ablation in the
context of SmoM2 or loss of Ptch1 do not lead to pancreatic
abnormalities observed in Pdx1-Cre;CLEG2;Kif3alox/loxmice.
progenitor markers in the ductal-like epithelium of Pdx1-
Cre;CLEG2;Kif3alox/loxmice. (A and B) Expression of the
embryonic pancreatic marker Sox9 in undifferentiated cells
in 2- to 3-week-old Pdx1-Cre;CLEG2;Kif3alox/loxmice. E-
cadherin expression confirms the epithelial nature of those
cells. (C and D) FoxA2 is expressed in undifferentiated cells
in Pdx1-Cre;CLEG2;Kif3alox/loxmice. (E and F) Myc-GLI2ΔN-
expressing cells in Pdx1-Cre;CLEG2;Kif3alox/loxmice are
highly proliferative as determined by staining with phos-
pho-Histone H3. (G and H) Activation of Notch signaling in
undifferentiated cells of Pdx1-Cre;CLEG2;Kif3alox/loxmice as
determined by Hes1 expression. (I and J) Pdx-1 expression is
excluded from undifferentiated cells expressing high levels
of myc-GLI2ΔN in Pdx1-Cre;CLEG2;Kif3alox/loxpancreata. (K
and L) Undifferentiated cells are first observed at P0 in
Pdx1-Cre;CLEG2;Kif3alox/loxpancreata. (M and N) Abnormal
mucin-1 expression in undifferentiated cells of P0 Pdx1-Cre;
CLEG2;Kif3alox/loxpancreata. Mucin-1 expression is de-
creased and not restricted to the apical membrane in myc-
GLI2ΔN-expressing cells of Pdx1-Cre;CLEG2;Kif3alox/loxmice
(arrows). Mucin-1 is properly localized to the apical mem-
brane in Pdx1-Cre; CLEG2 mice. Note the low number of
myc-GLI2ΔN-expressing cells observed in Pdx1-Cre; CLEG2
mice. (O and P) Coexpression of exocrine (amylase) and
ductal markers (Mucin-1) in myc-GLI2ΔN-expressing cells of
P0 in Pdx1-Cre;CLEG2;Kif3alox/loxmice. The area marked by
arrowheads is shown at higher magnification in inset in P.
Activation of Hh signaling results in expression of
| www.pnas.org/cgi/doi/10.1073/pnas.0909900107Cervantes et al.
Our findings suggest that overexpression of GLI2ΔN in CLEG2
mice bypasses these additional modulators, but that GLI2ΔN
activity is still subject to cilia-mediated regulation. Thus, elimi-
nation of cilia in Pdx1-Cre;CLEG2;Kif3alox/loxmice is necessary
for full activation of the Hh pathway. The presence of additional
regulators that curb signaling activity in the presence of Smo
activation might also partly explain the resilient response of
pancreatic epithelial cells to Hh ligands in vitro as previously
reported (8, 10). These observations are in contrast to recent
reports in medulloblastoma and BCC, where expression of
SmoM2 is sufficient to induce tumors in a cilia-dependent
manner (11, 15). The pancreas further differs from those tissues
in that expression of CLEG2 in Pdx1-Cre;CLEG2 mice only lead
to a modest increase in Hh signaling activity, which was dra-
matically boosted by cilia ablation. In cerebellum and skin,
CLEG2 expression with or without cilia ablation resulted in
similar levels of Hh activation, albeit cilia ablation accelerated
tumor formation. Furthermore, whereas cilia regulate generation
of Gli3R in cerebellum and skin (11, 15), they modulate total
Gli3 protein levels in the pancreas. Together, these differences
suggest that regulatory mechanisms other than cilia modulate Hh
activity in a tissue-specific manner.
The increased Hh activity achieved in Pdx1-Cre;CLEG2;
Kif3alox/loxmice revealed an unknown role of epithelial Hh sig-
naling during pancreas development and differentiation. One of
the most intriguing malformations observed in Pdx1-Cre;CLEG2;
Kif3alox/loxmice is the formation of cells with abnormal mor-
phology displaying high myc-GLI2ΔN levels. These cells
expressed pancreatic progenitor markers, such as Hes1, FoxA2,
and Sox9, suggesting that activation of Hh impairs the ability of
pancreatic cells to maintain a differentiated state. Interestingly,
it has been shown that inactivation of epithelial Hh signaling
blocks pancreas regeneration upon injury due to the inability of
metaplastic cells to redifferentiate back to exocrine cells, further
supporting a role for Hh signaling in modulating pancreatic cell
GLI2ΔN accumulation in Pdx1-Cre;CLEG2 mice, prolonged ex-
pression of myc-GLI2ΔN results in formation of undifferentiated,
pancreatic tumors. These undifferentiated tumors display loss of
mesenchymal transition. Although the undifferentiated cells ob-
served in Pdx1-Cre;CLEG2;Kif3alox/loxmice expressed E-cadherin,
we cannot rule out the possibility that these cells represent an early
transitional state toward tumor cells found in older Pdx1-Cre;
distinct from pancreatic adenocarcinoma, it is intriguing that pri-
mary cilia are also absent both from adenocarcinoma precursor
and BCC, which are ciliated tumors (11, 15). Unfortunately, the
compromised health of Pdx1-Cre;CLEG2;Kif3alox/loxmice prevents
analysis in older animals. Focal/temporal inactivation of cilia and
GLI2ΔN overexpression will be required to clarify whether un-
islet formation in Pdx1-Cre;CLEG2;Kif3alox/loxmice.
(A–D) Islets are reduced in number and their ar-
chitecture is affected in Pdx1-Cre;CLEG2;Kif3alox/lox
mice at P5. Islet morphology is shown at higher
magnification in insets. (E–H) Islet morphology
appears unaffected in Pdx1-Cre;CLEG2;Kif3alox/lox
mice at P0. (I–K) Quantification of islet area in E15.5
embryos and P0 and P5 mice. Note the dramatic
reduction of islet area in Pdx1-Cre;CLEG2;Kif3alox/lox
mice at P5. (L and M) Quantification of ngn3-posi-
tive cells and epithelial area in E15.5 embryos. Both
epithelial area and neurogenin3 endocrine pre-
cursors are reduced in Pdx1-Cre;CLEG2 and Pdx1-
Cre;CLEG2;Kif3alox/loxmice. (N and O) Quantifica-
tion of endocrine cell proliferation based on phos-
pho-Histone H3 expression at P0 and P5. (P and Q)
Quantification of endocrine cell death based on
cleaved caspase-3 expression at P0 and P5. Note the
sustained 2- to 3-fold increased cell death rate in
Pdx1-Cre;CLEG2;Kif3alox/loxmice. All values are rel-
ative to control littermates, whose averages were
considered to be one. Sample numbers represent
n ≥ 3. (See Table S1 for sample numbers.) Error bars
represent SD. *P < 0.05 and **P < 0.005.
Increased Hh activity results in disruption of
Cervantes et al. PNAS
| June 1, 2010
| vol. 107
| no. 22
differentiated cells in Pdx1-Cre;CLEG2;Kif3alox/loxmice represent Download full-text
early stages of pancreas neoplasia.
Increased Hh signaling also results in dramatic defects in endo-
exocrine compartment, transdifferentiation does not appear to play
a role in the postnatal loss of endocrine area. Rather, our findings
suggest that increased apoptosis is most likely responsible for this
process, indicating that deregulation of Hh signaling compromises
endocrine cell health. Previous reports from insulinoma cells have
shown a positive role for this pathway in regulating β-cell function
CLEG2;Kif3alox/loxmice has prevented us from analyzing the effects
of increased Hh signaling on mature endocrine cell function, and
In summary, we have shown that primary cilia regulate Hh
activity downstream of Smo and possibly at the level of the Gli
transcription factors in the pancreatic epithelium. Our findings
also show that increased Hh signaling in pancreatic epithelial
cells leads to loss of differentiated state and activation of em-
bryonic foregut and pancreas progenitor markers. These findings
suggest that modulation of epithelial Hh signaling controls dif-
ferentiated phenotypes and therefore might have important im-
plications for reprogramming of mature exocrine cells, a process
involved in pancreatic tumor formation and a potential source of
insulin-producing cells (36).
Mice used in this study were maintained in the barrier facility according to
protocols approved by the Committee on Animal Research at the University
of California, San Francisco. Pdx1-Cre, CLEG2, Ptch1lacZ/+, Ptch1lox/lox, SmoM2,
and Kif3alox/loxmice have been described previously (7, 16, 22, 32, 37, 38).
Reagents and procedures are described in detail in SI Methods.
ACKNOWLEDGMENTS. We thank Drs. Doug Melton (Harvard University,
Cambridge, MA), Andrzej A. Dlugosz (University of Michigan Medical School,
Ann Arbor, MI), Brandon Wainwright (University of Queensland, Brisbane,
Australia), and Lawrence S. Goldstein (University of California at San Diego,
La Jolla, CA) for providing us with mouse lines. We are indebted to Dr. Baolin
Wang (Weill Medical College of Cornell University, New York, NY) for the
anti-Gli3 antibody. We thank Dr. Young-Goo Han for critical reading of the
manuscript, Ramya Sundararajan for technical assistance, and the Genome
Analysis Core at the University of California, San Francisco (UCSF) Helen Diller
Family Comprehensive Cancer Center for providing the custom Taqman assay
sequences. S.C. was recipient of a research grant from the National Pancreas
Foundation. D.A.C. was supported by a postdoctoral fellowship from the
California Institute of Regenerative Medicine (CIRM). J.L. was enrolled in the
Biomedical Graduate Student Program at UCSF. Work in M.H.’s laboratory was
supported by National Institutes of Health Grants DK6053 and CA112537 and
the Brehm Coalition. Image acquisition was supported by the UCSF Diabetes
and Endocrinology Research Center Microscopy Core (P30 DK63720).
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| www.pnas.org/cgi/doi/10.1073/pnas.0909900107Cervantes et al.