1 Diabetes Center, Department of Medicine, University of California, San
Francisco, CA 94143, USA
2 Department of Pediatrics, Case Western Reserve University, Cleveland, OH
44106, USA
3 Program in Molecular Medicine, University of Massachusetts Medical School,
Worcester, MA 01605, USA
* Author for correspondence (e-mail: mhebrok{at}diabetes.ucsf.edu)
Accepted 29 March 2004
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SUMMARY |
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Key words: Pancreas, Cilia, Polycystic kidney disease, Polaris, orpk, Acinar-ductal metaplasia, Wnt signaling
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Introduction |
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Recent observations have indicated that primary cilia, cellular appendages
that are present on the surface of most cells
(Wheatley et al., 1996), play
a crucial role during the development of PKD
(Ong and Wheatley, 2003
).
Tg737, the gene mutated in the orpk mouse model of PKD
encodes polaris, a protein required for proper ciliary assembly
(Pazour et al., 2000
;
Taulman et al., 2001
;
Yoder et al., 2002b
).
Furthermore, both polycystin-1 and polycystin-2, the proteins encoded by
Pkd1 and Pkd2, respectively, localize to the cilium in mouse
and human kidney cells (Pazour et al.,
2002
; Yoder et al.,
2002a
). In addition, the genes mutated in the cpk and
inversin PKD mouse models encode the proteins cystin and inversin,
respectively, which also localize to the cilia
(Hou et al., 2002
;
Watanabe et al., 2003
).
Finally, Lin et al. have shown that the kidney-specific inactivation of
kinesin-II, a protein essential for cilia formation, leads to PKD in mice
(Lin et al., 2003
). Thus,
increasing evidence points to defects in cilia assembly and function as a
cause of PKD.
Although numerous studies have focused on the renal pathologies, less attention has been paid to the extrarenal abnormalities. Here, we present a detailed study of the pancreatic defects in one of the PKD models, the orpk mouse. Our findings show that a mutation in the Tg737 gene results in severe abnormalities in the pancreas, including massive acinar cell loss, formation of abnormal tubular structures, and appearance of endocrine cells in ducts. We demonstrate that pancreatic cells in orpk mice are marked by a reduction in cilia number and aberrant cilia architecture, which suggests that the pancreatic defects are caused by improper cilia assembly. We find similar pancreatic abnormalities in mice carrying mutations in the Pkd2 and inversin genes. Both Pkd2 and inversin proteins localize to cilia and mediate cilia function; however, in contrast to the orpk/polaris protein, they are not required for cilia formation. These observations indicate that both accurate cilia assembly and function are essential for maintenance of proper pancreatic tissue organization. Cell signaling properties of ductal cells are affected, as mislocalization of ß-catenin and increased expression of transcriptional activators of Wnt signaling are observed in orpk mice. Thus, our results suggest that pancreatic PKD phenotypes are at least in part mediated through the deregulation of Wnt signaling activity.
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Materials and methods |
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Tissue preparation, immunohistochemistry and microscopy
Isolated pancreata from E12.5, E15.5 and E18.5 embryos, newborn and adult
mice were fixed in 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered
saline (PBS) for 1 to 4 hours at 4°C. Histological analysis,
quantification of the tissue area and counting of cells, were performed as
described previously (Hebrok et al.,
2000). Hematoxylin/Eosin staining, immunohistochemical and
immunofluorescence analyses were performed on paraffin sections as described
previously (Kim et al., 1997
).
The following primary antibodies were used: guinea pig anti-insulin, diluted
1:500 (Linco); rabbit anti-glucagon, diluted 1:500 (Linco); rabbit anti-Pdx1,
diluted 1:3000 (gift from Dr Michael German); rabbit anti-Glut2, diluted
1:1000 (Chemicon); rabbit anti-amylase, diluted 1:700 (Sigma); rabbit
anti-cleaved caspase 3, diluted 1:100 (Cell Signaling Technology); anti-Pkd2,
diluted 1:1000 (Cai et al.,
1999
) (G.J.P., unpublished); anti-acetylated tubulin 611b1,
diluted 1:10,000 (Sigma); mouse anti-ß-catenin, diluted 1:100 (BD
Biosciences); rabbit anti-Ki-67, diluted 1:200 (Novocastra Laboratories); and
Armenian hamster anti-Muc1, diluted 1:200 (Neomarkers).
The following secondary antibodies were used for immunofluorescence: FITC-conjugated anti-guinea pig (Molecular Probes); Cy3-conjugated anti-rabbit (Molecular Probes); FITC-conjugated anti-Armenian hamster (Jackson ImmunoResearch); Alexa488-congugated anti-rabbit; and Alexa594- or Alexa693-conjugated anti-mouse (Molecular Probes). Fluorescence was visualized and photographed with a Zeiss Axiphoto2 plus and a Leica TCS SP2 confocal microscope.
Terminal transferase-mediated dUTP nick end labeling (TUNEL) analysis
Immunohistochemical analysis for apoptosis was performed following the
manufacturer's recommendations (Invitrogen). In brief, the sections were
deparaffinized, rehydrated in graded alcohols and incubated with 20 µg/ml
proteinase K for 2 minutes. They were then incubated with 3% hydrogen peroxide
in methanol for 30 minutes to inactivate endogenous peroxidase, and treated
with terminal deoxynucleotidyl transferase and digoxigeniuridine triphosphate
(dUTP) for 60 minutes at 37°C. To visualize the nick-end labeling with a
light microscope, the sections were treated with anti-digoxigenin HRP
conjugate (Roche) for 30 minutes and then incubated with
diaminobenzidine-tetrahydrochloride (Vector). Apoptosis was quantified by
averaging the number of stained nuclei per field.
Staining for ß-galactosidase activity
Pancreata isolated from heterozygous
Tg7372-3ßgal mutant mice, in which the
lacZ gene was inserted into the Tg737 locus
(Murcia et al., 2000
), were
fixed for 4 hours at 4°C in 4% paraformaldehyde and then incubated
overnight in phosphate-buffered saline (PBS) supplemented with
5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal; 400 µg/ml) at
4°C. The dissected organs were photographed on a Leica MZ FL3 equipped
with a Leica IM500 system.
Morphometric quantification of islet areas
Morphometric analysis was performed as described previously
(Hebrok et al., 2000). In
short, a portion of the whole pancreas was used for quantification to obtain
representative results. The first five consecutive sections of P9 pancreatic
tissue were mounted on the first of a series of microscope slides, followed by
the next five sections placed on the second slide. A total of five individual
slides (1a-5a) were filled with consecutive sections. When necessary,
additional series of five (1b-5b, etc.) slides were prepared until all
pancreatic sections were mounted. After immunohistochemistry, pancreatic
epithelial areas were outlined and measured with the OpenLab software.
Insulin- and glucagon-positive areas of the P9 pancreas were measured on every
twenty-fifth section (every 150 µm) from one set of slides (1a-1e). Data
analysis was performed with Excel software (Microsoft). Statistical
significance was assessed by employing the Student's t-test.
Quantification of cilia length
Immunofluorescent images of cilia stained for acetylated tubulin were
acquired using a Leica TCS SP2 confocal microscopy system with Leica Confocal
Software (LCS). Images were acquired in 50 confocal z-stack slices and
composite images were prepared using LCS, from which µm measurements of
cilia length were obtained. Data analysis was performed with Excel
software.
RNA preparation and RT-PCR analysis
Dissected pancreata were dissolved in Trizol (Gibco-BRL) and total RNA was
prepared according to the manufacturer's instructions. RT-PCR was performed as
described in Wilson and Melton (Wilson and
Melton, 1994). PCR was performed under the following conditions: 1
cycle of 95°C for 10 minutes; followed by 35 cycles of 94°C for 45
seconds, 60°C for 45 seconds and 72°C for 1 minute. Mouse actin was
used as the internal control. Initial experiments were carried out to ensure
that conditions for PCR were within the linear range of amplification.
Expression of Lef1, Tcf1, Tcf3, and Tcf4 mRNA in P11 pancreata was studied by RT-PCR. Primers were designed to span introns to exclude nonspecific genomic DNA amplification. Forward and reverse primer sequences used are listed 5' to 3'. Primers used were:
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Results |
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Analysis of Hematoxylin/Eosin-stained tissue did not reveal major changes in pancreas gross morphology at the end of gestation (Fig. 1D,E; changes in embryonic cell differentiation and morphology are discussed below). After birth, a progressive loss of exocrine cells, accompanied by increased dilation of duct-like structures, was observed in postnatal day 0 (P0) to P17 mice (Fig. 1F-I). The acinar architecture was poorly organized, marked by the loss of the typical round shape of the acini and the presence of duct-like structures composed of cuboidal cells. The substantial decrease in acinar tissue results in a severe reduction of total pancreatic mass, even when the reduced body size of the mutants is taken into account (Fig. 1A-C). These defects might contribute to the overall smaller size of the orpk mice by limiting their digestive capability.
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The early lethality and compromised health of orpk mice prohibited any physiological assays to determine whether cilia are necessary for mature ß-cell function. Glucose tolerance tests performed in heterozygous adult orpk mice revealed no differences compared with wild-type littermates (data not shown).
Acinar cell loss and expansion of duct-like epithelium in newborn orpk mice
Although the endocrine compartment appears to form normally, significant
loss of acinar tissue was observed in orpk mice
(Fig. 1F-I). The exocrine
portion of the pancreas is organized as a tubuloalveolar gland. Acini secrete
digestive enzymes into intracinar ducts that conduct to progressively larger
intercalated ducts, interlobular ducts and eventually to the main pancreatic
duct. To further characterize the exocrine defects, we performed
immunofluorescent staining with antibodies directed against amylase, a
digestive enzyme produced by acinar cells. At the end of gestation, E18.5
orpk embryos showed normal amylase staining
(Fig. 3A,B). However, shortly
after birth, amylase expression was significantly reduced throughout the
exocrine tissue in orpk mice (Fig.
3C-F). Concomitant with the progressive loss of acinar cells, we
observed an increase in ductal structures in pancreatic sections when stained
with antibodies directed against mucin 1 (Muc1), a membrane protein expressed
in epithelial cells lining glands and ducts in several organs
(Graham et al., 2001). In
wild-type pancreas the Muc1 antibody specifically labeled all cells of the
ductal system, including intracinar ducts and intercalated ducts. No cross
reactivity with blood vessels or any other cells type was observed with the
antibody (Fig. 3). By employing
this cell specific marker, we were able to detect defects in ductal morphology
in orpk pancreata starting at E18.5, with intracinar ducts displaying
small dilations not typically observed in wild-type pancreas
(Fig. 3A,B). Associated with a
reduction of amylase immunoreactivity, we detected a rapid expansion of
pancreatic ducts in mutant tissue after birth
(Fig. 3C-F). Interestingly,
insulin-expressing cells were frequently detected in the expanding duct-like
structures (Fig. 3H) soon after
birth. These insulin-positive cells also expressed mature ß-cell markers,
including Pdx1 and glucose transporter 2 (Glut2) (data not shown). At the
stages analyzed, we failed to detect any insulin-expressing cells in ducts of
wild-type pancreas (Fig.
3G).
|
Increased proliferation of duct-like cells
One of the hallmarks of PKD is the abnormal proliferation of immature
epithelial cells (Gabow, 1993;
Murcia et al., 1999
). To test
whether the expansion of the duct-like epithelium in orpk mice is due
to an increase in cell proliferation, we counted cells marked by the
expression of Ki-67, a nuclear non-histone protein present only in cells
progressing through the cell cycle. Ki-67-positive cells were detected in
endocrine, acinar and ductal tissue, with the highest levels found in
expanding ducts of orpk mice (Fig.
4C,D). Measurement of the proliferation indexes, defined as the
number of Ki-67-positive cells per field, revealed reduced proliferation of
acinar cells and ß-cells in orpk mice
(Fig. 4F). By contrast,
orpk ductal cells showed a proliferation index similar to that found
in wild-type animals, resulting in a 3-fold relative increase of ductal cell
proliferation when compared with orpk acinar and ß-cells
(Fig. 4F). This is in stark
contrast with control animals in which only a small difference was detected in
proliferation indexes between acinar/ß-cells and ductal cells (1.4 fold).
Thus, orpk ductal cells are marked by a relative increase in
proliferation compared with other pancreatic cell types, a defect that results
in duct expansion and formation of large pancreatic cysts.
Impaired cilia formation in pancreatic cells of orpk mice
The murine Tg737 gene (and its homologs in the algae
Chlamydomonas and the worm C. elegans) is involved in
primary cilia formation (Pazour et al.,
2000). Several of the phenotypes associated with mutations in
Tg737, such as polycystic kidney disease, defects in left-right
patterning and retinal degeneration are associated with ciliary assembly
defects in these tissues (Murcia et al.,
2000
; Pazour et al.,
2000
; Pazour et al.,
2002
; Yoder et al.,
2002b
). As Tg737 expression is detected in all pancreatic
cell types known to express cilia
(Aughsteen, 2001
), we wanted to
know whether pancreatic cells in orpk mice displayed primary cilia
defects. To determine the effects of the orpk mutation on the
formation of pancreatic cilia, pancreatic sections were stained with an
antibody directed against acetylated tubulin, a protein that is preferentially
localized in the ciliary axoneme. In wild-type pancreas, primary cilia were
present in tissues marked by Tg737
2-3ßgal
expression, including the luminal surface of cells in interlobular,
intercalated and intracinar ducts, and in islets
(Fig. 2,
Fig. 5A,C,E). During embryonic
development primary cilia were found throughout the pancreatic epithelium as
early as E12.5 (data not shown). By contrast, primary cilia were almost absent
in orpk pancreata at all stages analyzed
(Fig. 5B,D,F). Morphometric
quantification of cilia lengths using confocal microscopy at E15.5, E18.5 and
P4 revealed that the remaining primary cilia in pancreatic cells of
orpk mice are significantly shortened when compared with wild-type
primary cilia (Fig. 5G). The
observation that cilia length is already reduced in E15.5 pancreatic tissue,
in addition to the observed changes in duct morphology at E18.5
(Fig. 3A,B), further supports
the hypothesis that pancreatic deficiencies in orpk mice result from
embryonic defects. Furthermore, these results indicate that polaris is
required for proper assembly of primary cilia in pancreatic cells.
|
Pancreatic abnormalities in inversin and Pkd2 mice
The observation that orpk mutants showed increased polycystin-2
localization in dilated ducts could suggest that defective cilia function, as
a consequence of impaired cilia formation, is responsible for the observed
pancreatic abnormalities. To determine whether ciliary dysfunction causes
similar pancreatic phenotypes, we decided to characterize pancreas morphology
in other PKD mouse models harboring mutations in the inversin and
Pkd2 genes. Although both genes encode proteins known to localize in
the cilia of kidney cells, their function appears not to be necessary for
cilia formation (Hou et al.,
2002; Watanabe et al.,
2003
).
In agreement with previous reports, and similar to orpk mutants
(Figs 1,
3), histological and
immunohistochemical analysis revealed that both Pkd2 and inversin
pancreata displayed a reduction of acinar tissue and an increase of ductular
structures (Fig. 6A-H) (Morgan et al., 1998;
Wu et al., 2000
). As seen in
orpk mutants, the loss of exocrine tissue in inversin mice correlates
with an increase in acinar cell apoptosis
(Fig. 6I,J). No increase in
cell apoptosis was observed in Pkd2 mutants
(Fig. 6K,L), probably because
these embryos die prematurely at E14.5. In comparison, significant levels of
acinar cell apoptosis were found in orpk mutants only after birth.
Cilia formation appeared to be unaffected in both Pkd2 and inversin
mutants as revealed by immunohistochemistry against acetylated tubulin. In
addition, no increase in polycystin-2 expression was observed in inversin
mutants (data not shown). These results indicate that either impairment of
cilia formation in orpk mutants or cilia function in
Pkd2/inversin mutant mice results in acinar cell apoptosis and ductal
dilation in pancreatic tissue.
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Discussion |
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Pancreatic abnormalities were first detected in orpk mice at the end of gestation. At this time point, the intracinar ducts in orpk mice showed signs of dilation (Fig. 3B). The fact that defects first occur during embryonic development is consistent with the apparent reduction in cilia length at early embryonic stages (Fig. 5E-G). Dilation of pancreatic ducts becomes increasingly severe after birth, most likely caused by enhanced proliferation of the ductal epithelium (Fig. 1, Fig. 4D,F). Although the ducts are expanded, the overall size of the mutant pancreas is reduced. The decrease in pancreas size appears to be due to acinar cell atrophy, marked by increased TUNEL staining and reduced amylase expression (Figs 3, 4). The fact that an increase in acinar cell apoptosis is observed after dilation of pancreatic ducts suggests a causal relationship between these processes. Thus, two of the hallmarks of PKD, increased apoptosis and formation of tubular complexes by increased proliferation of epithelial cells, are present in pancreatic tissue in orpk mice.
The negligible effect of the orpk mutation on endocrine tissue
architecture and cell differentiation is somewhat surprising considering that
Tg737 expression is highest in islets and that endocrine cells are
highly ciliated (Figs 2,
5)
(Aughsteen, 2001).
Unfortunately, the early neonatal lethality of this line prevented us from
analyzing the importance of polaris and cilia function in adult islets.
Furthermore, orpk mice contain a hypomorphic mutation within the
Tg737 gene, and residual cilia function might mask potential defects
during endocrine cell differentiation or function. At this point we can only
speculate about the role of primary cilia in ß-cells but an attractive
hypothesis is that they also act as sensory organelles. For example,
somatostatin and serotonin receptors have been found on primary cilia in
neuronal cells in particular regions of the brain
(Brailov et al., 2000
;
Handel et al., 1999
). Future
experiments could address whether hormone receptors are localized on primary
cilia of endocrine cells and if their activity is required to sense and
respond to changes in external hormone concentration.
It is interesting to note that insulin-expressing cells were detected in
orpk dilated ducts, thus providing support for previous studies that
have described islet cell neogenesis in expanding ductal epithelium upon
tissue injury (Bonner-Weir et al.,
2000; Rosenberg,
1998
; Wang et al.,
1997
). The question of whether pancreatic progenitor cells reside
in ducts is still controversial
(Bonner-Weir and Sharma, 2002
;
Gu et al., 2002
), but
orpk mice display an increased proliferation of liver oval cells, a
population of cells considered to represent liver progenitor cells
(Richards et al., 1996
). Thus,
circumstantial evidence suggests that polaris function could regulate
progenitor cell proliferation in different organs.
The role of primary cilia in PKD
Tg737, the gene mutated in orpk mice, is required for
cilia formation and several reports have recently discussed the role of cilia
in various human diseases, including PKD
(Pazour and Rosenbaum, 2002).
Our studies show that primary cilia formation and polycystin-2 expression is
altered in pancreatic cells of orpk mice. Cells within dilated ducts
in orpk mice display a higher level of polycystin-2 protein,
indicating that defects in cilia formation elevate cytoplasmic levels of
proteins intended for delivery to the cilia. The increase in polycystin-2
expression appears to be specific to orpk mice as inversin mutants
did not show any obvious changes in protein expression when compared with
wild-type controls. The observation that inversin and Pkd2 mice, in
which cilia formation is not impaired, displayed similar pancreatic phenotypes
to those found in orpk mice indicates that both cilia assembly and
function are necessary for proper pancreas morphogenesis and maintenance of
tissue architecture.
What is the normal role of primary cilia in pancreatic tissue? Recently it
has been shown that cilia can act as mechanosensors to measure luminal flow
(Praetorius and Spring, 2001;
Praetorius and Spring, 2003
),
and that polycystin-1 and polycystin-2 mediate this response
(Nauli et al., 2003
). Cilia in
pancreatic ductal cells may sense luminal pressure and control cell
proliferation to maintain appropriate luminal dimensions. In the case of
mutations in PKD loci (as in Pkd2 or inversin mice), or mutations
that affect ciliary assembly (as in orpk mice), the relay of the
sensory signals that control cell division would be lost, resulting in
uninhibited cell proliferation and progressive duct dilation. In agreement
with this, increased pressure caused by pancreatic duct obstruction leads to
ductal dilation and acinar cell death
(Scoggins et al., 2000
).
Although speculative at this point, this hypothesis would link the similar
phenotypes observed in human chronic pancreatitis, mouse models of pancreatic
duct obstruction, and models with defects in primary cilia structure and
function.
Cilia function regulates ß-catenin localization
The exact mechanism of how defects in cilia formation and/or function cause
the main manifestations of PKD is unknown. Some reports have previously
suggested an involvement of ß-catenin signaling in PKD
(Kim et al., 1999;
Saadi-Kheddouci et al., 2001
).
ß-Catenin is involved in cell adhesion and transcriptional regulation of
the Wnt signal transduction pathway (Barth
et al., 1997
; Polakis,
2000
; Willert and Nusse,
1998
). Recently, a connection between primary cilia and
ß-catenin signaling has been suggested. Inactivation of cilia caused by a
mutation of kinesin-II leads to increased levels of cytosolic and nuclear
ß-catenin in renal cells (Lin et al.,
2003
). In agreement with those results, we observed an increase in
cytoplasmic ß-catenin, as well as Wnt signaling components such Lef1 and
Tcf3, in pancreatic cells of orpk mice
(Fig. 6). Thus, the pivotal
role of ß-catenin in regulating decisions between cell proliferation and
differentiation (van de Wetering et al.,
2002
) is consistent with the phenotypes we have observed in
orpk mice.
PKD, acinar-to ductal metaplasia and pancreatic adenocarcinoma
The fact that Tg737 function is required to control intracellular
localization of ß-catenin, and that nuclear localized ß-catenin has
been found in several murine pancreatic tumors
(Kongkanuntn et al., 1999), is
intriguing as it provides further evidence for a previously suggested direct
link between PKD and pancreatic cancer formation
(Niv et al., 1997
;
Silverman et al., 2001
).
Acinar-to-ductal metaplasia, which is, at least in some cases, due to chronic
pancreatitis, has been proposed as one of the mechanisms that predispose for
the formation of pancreatic ductal adenocarcinomas
(Malka et al., 2002
). Some of
the phenotypes displayed by orpk mice are reminiscent of those found
in mouse models of pancreatic acinar-to-ductal metaplasia
(Scoggins et al., 2000
;
Wagner et al., 1998
). These
features include loss of acinar cell architecture, increased ductal
proliferation, stromal cell expansion and collagen deposition
(Fig. 1J,K). In combination
with ductal metaplasia, these defects are commonly observed in individuals
suffering from chronic pancreatitis
(Kloppel and Maillet, 1998
)
and, to some extent, in pancreatic cancer
(Kloppel, 1993
).
Unfortunately, due to early lethality of orpk mice, we were unable to
test whether the lesions observed in orpk mice progress to neoplasia.
However, mutations in the Tg737 gene have been found in liver tumors,
as well as in liver, kidney, and pancreatic human tumor cells lines,
suggesting that Tg737 functions as a tumor suppressor gene
(Isfort et al., 1997
).
Tissue-specific and temporal inactivation of cilia components might provide
evidence for a causal link between primary cilia dysfunction and pancreatic
adenocarcinoma.
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ACKNOWLEDGMENTS |
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