|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 12 April 2006
doi: 10.1242/dev.02366
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Diabetes Center, Department of Medicine, University of California, San
Francisco, CA 94143, USA.
2 Department of Pharmacology, Graduate School of Medicine, Kyoto University,
Kyoto, 606-8501, Japan.
3 Department of Genetic Medicine and Development, University of Geneva Medical
School, Geneva CH-1211, Switzerland.
* Author for correspondence (e-mail: mhebrok{at}diabetes.ucsf.edu)
Accepted 16 March 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: ß-Catenin, FGF, Hedgehog, Organ size, Pancreas development, Pdx1, Wnt, Mouse, Pancreatomegaly
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In the absence of Wnt ligand, cytoplasmic pools of
ß-catenin are highly unstable because of multiple phosphorylations in the
N terminus of the protein that target the protein for degradation. However,
when the Wnt co-receptors Lrp and frizzled are engaged by ligand, the assembly
of proteins responsible for this phosphorylation state is inhibited.
Consequently, the unphosphorylated form of ß-catenin accumulates in the
cytoplasm, making it available for entry into the nucleus. Once in the
nucleus, ß-catenin interacts with Tcf/Lef transcriptional co-activators
to promote the expression of target genes
(Nelson and Nusse, 2004).
Previous work has established that the third exon of the ß-catenin
gene encodes the N-terminal phosphorylation sites necessary for degradation of
the protein via ubiquitylation (Harada et
al., 1999). Therefore, the removal of this exon in transgenic mice
using Cre/loxP technology results in a constitutively stabilized, or
activated, form of the ß-catenin protein
(Harada et al., 1999). These
ß-catactive mice have proven to be useful in probing
the effects of ß-catenin signaling on embryonic stem cell
differentiation, progenitor cell expansion in the nervous system,
epithelial-mesenchymal transition in the epiblast and other phenomena
(Kemler et al., 2004;
Kielman et al., 2002;
Zechner et al., 2003).
Previous studies have demonstrated that Wnt signaling components are
dynamically expressed within the developing pancreas, suggesting that
canonical Wnt signaling may be involved in pancreas organogenesis
(Dessimoz et al., 2005;
Heller et al., 2003;
Murtaugh et al., 2005;
Papadopoulou and Edlund,
2005). Two independent laboratories recently reported divergent
phenotypes resulting from the conditional deletion of ß-catenin within
the pancreatic epithelium. In one instance, loss of ß-catenin resulted in
a reduction in pancreatic endocrine cell numbers, whereas the gross morphology
of the organ appeared normal at birth
(Dessimoz et al., 2005).
However, a separate report demonstrated that loss of ß-catenin did not
affect pancreatic endocrine cell mass, despite the almost complete loss of the
exocrine compartment. Here, we have used the
ß-catactive mouse to help clarify how ß-catenin
stability affects pancreas development and organ maturation.
In mice, pancreas morphogenesis begins by 9.5 days post coitum (E9.5) when
epithelial tissue fated to become the dorsal pancreas buds from the gut
endoderm within a mesenchymal cap (Kim and
Hebrok, 2001). Emergence of two distinct ventral pancreatic buds
occurs slightly later, by E10.25-10.5. Signaling by the mesenchyme is
essential for epithelial proliferation and branching. The epithelium
eventually gives rise to two distinct tissue compartments: exocrine cells that
produce digestive enzymes and endocrine cells that produce hormones essential
for regulating blood glucose levels.
Pdx1, a homeobox transcription factor, is one of the earliest
genes to be expressed within the developing pancreatic epithelium and is
essential for normal organ formation. Moreover, Pdx1-expressing pancreatic
progenitor cells have been shown to give rise to all three types of pancreatic
tissue: endocrine, exocrine and duct (Gu
et al., 2002). A number of independent lines of transgenic mice
that express Cre recombinase under the control of Pdx1 promoter
fragments have been generated (Gannon et
al., 2000; Gu et al.,
2002; Herrera,
2000). Our characterization of two of these strains indicated that
the temporal and spatial activity of Cre-recombinase differed. This allowed us
to determine how ß-catenin stabilization in these distinct
temporal/spatial domains of the pancreatic epithelium affected organogenesis
and adult organ function. Interestingly, we observed significantly different
pancreatic phenotypes depending on the Cre strain employed. Using the
PdxCre mice generated in the laboratory of D. Melton that displayed
early and robust Cre recombinase activity (PdxCreearly),
we observed a nearly complete loss of pancreatic tissue
(Gu et al., 2002). Conversely,
slightly delayed and more mosaic Cre recombinase expression in Pdx mice
generated in one of our laboratories (PdxCrelate) drives
outgrowth of pancreatic tissue, resulting in a grossly enlarged pancreas
(Gannon et al., 2000;
Herrera, 2000). Thus, in one
instance, ß-catenin stabilization drives tissue loss, and in the other
culminates in an increase in organ size relative to body mass. Therefore,
ectopic stabilization of ß-catenin blocks/deregulates the normal
mechanisms that control embryonic pancreas formation and postnatal organ
growth.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) were
crossed with strains expressing Cre-recombinase under the control of the
Pdx1 promoter (PdxCreearly, PdxCrelate,
PdxCreER) (Gannon et al.,
2000; Gu et al.,
2002; Herrera,
2000), all of which were maintained in a mixed background. In all
experiments presented in this work, the floxed exon 3 allele of ß-catenin
was maintained as a heterozygote to insure that the level of ß-catenin
signaling pathway activation was consistent. Thus, the
ß-catactive nomenclature used refers to mice
containing one wild-type and one floxed exon 3 allele of ß-catenin. To
verify the expression pattern of Cre-recombinase, a lacZ reporter
line was used (R26R) (Soriano,
1999).
Tamoxifen preparation and injection
Tamoxifen (10 mg/ml) (Sigma, T5648) was dissolved in corn oil (Sigma,
C8267) following 30 minutes of incubation at 37°C and vigorous vortexing.
Intraperitoneal injections (100 µl, 1 mg/mouse) were made into the pregnant
female at the indicated developmental timepoint using a 21-gauge needle.
Tissue preparation, immunohistochemistry, and microscopy
Embryonic tissues were fixed and paraffin wax imbedded as previously
described (Kawahira et al.,
2003). Hematoxylin/Eosin staining, immunohistochemical and
immunofluorescence analyses were performed as previously described
(Kim et al., 1997). The
primary antibodies used in this study are listed in
Table 1. For
immunohistochemistry, a biotinylated anti-goat (Vector; BA-9500) was used at a
dilution of 1:200. Staining for diaminobenzidine (DAB) was performed with the
ABC Elite immuoperoxidase system (Vector). The Alexa series of secondary
antibodies from Molecular Probes was used for the immunofluorescent analysis
performed in this study. However, in order to amplify the signal from the
ß-galactosidase antibody, we found it necessary to use the TSA Plus
Fluorescence system (Perkin Elmer, fluorescein NEL741). Slides were mounted
with Vectashield mounting media containing the nuclear stain, DAPI (Vector).
Bright-field images were acquired using a Zeiss Axio Imager D1 scope;
fluorescent images were captured using a Leica DMIRE2 SP2 confocal
microscope.
|
). Stained tissues were photographed with a Leica MZ FL3
dissecting microscope equipped with a Leica IM500 system.
Morphometric quantification of proliferation and cell density
Pancreatic paraffin wax-embedded sections (6 µm) were cut from both the
dorsal and ventral pancreas. Following immunofluorescent staining for the
proliferation marker phospho-histone H3, positive cells were then scored from
20 non-overlapping fields at 20x magnification from the dorsal and
ventral sections of four control and four PdxCrelate
ß-catactive mice. The average number of cells per
field was then normalized against the control. In order to determine endocrine
and exocrine cell density, Hematoxylin/Eosin-stained tissues were used to
count cell nuclei from 20 non-overlapping fields at 40x magnification
isolated from four control and four PdxCrelate
ß-catactive mice. The average number of nuclei
present in the field was then normalized against control.
For P0 pancreata, the whole pancreas was sectioned and aliquoted as
described previously (Kawahira et al.,
2003), in order to obtain representative results. Islet area was
assayed at P0 as described previously
(Hebrok et al., 2000;
Perez et al., 2005). Error
bars represent s.e.m., and confidence intervals were determined using a
Student's t-test analysis.
Glucose tolerance testing
Six control and six PdxCrelate
ß-catactive mice were fasted for 14 hours before
intraperitoneal injection of a 20% glucose (w/v) solution at a dose of 2 g per
kg body mass. Venous blood glucose readings were then taken at the indicated
intervals using a Bayer Ascensia Elite XL to analyze samples collected from
tail nicks. Error bars represent standard error of the mean.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In this
manner, we were able to map the timing and expression of pancreatic
Cre-recombinase. By E10.5, robust lacZ staining was detected in the
pancreatic epithelium of PdxCreearly R26R animals at the
gross and histological levels (Fig.
1C,F). Moreover, expression was detectable in the majority of
Pdx1+ cells observed (Fig. 1F).
Interestingly, no lacZ staining was observed in the
PdxCrelate R26R animals at this time point
(Fig. 1B,E). By E11.5, a few
lacZ-positive cells were found in the pancreatic epithelium of
PdxCrelate R26R, a delay of 
24 hours (data not shown)
from the onset of expression in the PdxCreearly.
Furthermore, comparison of lacZ/Pdx1 co-stained pancreatic tissue revealed
that even at E12.5, the Cre expression in the
PdxCrelate strain was more mosaic than in the
PdxCreearly (Fig.
1H,I). Analysis of lacZ stained pancreas sections in adult PdxCrelate R26R mice suggested that Cre expression within the terminally differentiated exocrine and endocrine cells remained mosaic in the PdxCrelate strain (Fig. 1K). Interestingly, ß-galactosidase can rarely be detected within the adult pancreatic ducts in PdxCrelate R26R mice (Fig. 1N). By comparison, the majority of endocrine, exocrine and ductal cells in PdxCreearly R26R mice exhibit ß-galactosidase activity (Fig. 1L,O). The higher number of lacZ+ cells in the adult PdxCreearly R26R mice strongly indicates that the onset of Cre expression was not only earlier in timing, but also targeted a greater portion of the pancreatic epithelium. Control tissue was stained for each of the time points assayed to demonstrate the specificity of the lacZ staining reaction/immunofluorescence (Fig. 1A,D,G,J,M). Therefore, the PdxCrelate and PdxCreearly mice were used in this study as tools to probe the effect of increased ß-catenin signaling on distinct temporal/spatial populations of cells in the early embryonic pancreas.
Stabilization of ß-catenin results in disruption of pancreas formation in PdxCreearly ß-catactive
In order to assess the consequences of increased ß-catenin signaling
on pancreas organogenesis, we crossed ß-catactive
animals with the PdxCrelate or
PdxCreearly mice. Because previous studies have shown that
the loss of the third exon of one allele of ß-catenin is sufficient to
drive strong increases in Wnt pathway activity, all experiments were carried
out using mice that were heterozygous for the floxed ß-catenin allele
(Harada et al., 1999;
Kemler et al., 2004;
Zechner et al., 2003).
Analysis of gross morphology and pancreas architecture at E18.5 in the
PdxCrelate ß-catactive mice did
not reveal any overt changes compared with control littermates
(Fig. 2A,B,D,E). By contrast,
the PdxCreearly ß-catactive
animals displayed near total pancreas agenesis, and the pancreatic remnant
contained multiple large cysts (Fig.
2C,F). Histological examination revealed a significant reduction
in the epithelial derived exocrine and endocrine tissues. Consequently,
PdxCreearly ß-catactive survive
on average only 7 days after birth, whereas PdxCrelate
ß-catactive are viable and reproductively active.
Nuclear ß-catenin localization was abundant and easily detected by
confocal microscopy in both PdxCrelate
ß-catactive and PdxCreearly
ß-catactive pancreata at E18.5
(Fig. 2H,I). In control
samples, ß-catenin was detected only at the plasma membrane
(Fig. 2G). Therefore, as has
been shown in other tissues (Jamora et
al., 2003; Miller and Moon,
1997; Tolwinski and Wieschaus,
2004), stabilization of ß-catenin leads to increased nuclear
ß-catenin signaling and, presumably, hyperactivation of the canonical Wnt
signaling pathway in the pancreas.
In addition to its role in the canonical Wnt signaling pathway, the
ß-catenin protein also participates in cell adhesion at adherens
junctions. ß-Catenin links the cytoplasmic domain of transmembrane
cadherins to the actin cytoskeleton via its association with the adaptor
protein 
-catenin (reviewed by Bienz,
2005). Therefore, ß-catenin stabilization may also impact
cell adhesion. However, despite clear evidence of nuclear localization of
ß-catenin in PdxCrelate
ß-catactive and PdxCreearly
ß-catactive mice, E-cadherin remained properly
localized to the plasma membrane (Fig.
2K,L inset), suggesting that adhesion has not been disrupted.
Similarly, other studies using the ß-catactive mouse
strain have not found adhesion defects in cells expressing the stabilized form
of ß-catenin (Gounari et al.,
2002; Harada et al.,
1999). Using E-cadherin as a marker of epithelial cells,
immunohistochemical analysis revealed the extensive loss of epithelial tissue
mass in the PdxCreearly
ß-catactive organ remnant. As expected, the
developing cysts are E-cadherin positive, indicating that the cysts were
epithelial in origin (Fig.
2L).
|
|
-cells (Fig.
2M). Although the PdxCrelate
ß-catactive mutants displayed islet architecture
(Fig. 2N) and islet area (data
not shown) equivalent to control, PdxCreearly
ß-catactive mutants had few insulin+ or
glucagon+ cells (Fig.
1O). In addition, these endocrine cells were found scattered
throughout the remaining organ, rather than organized into discrete islet
structures (Fig. 2O). Thus, early and widespread upregulation of the ß-catenin signaling pathway in the PdxCreearly ß-catactive mouse strain prevents normal formation of the exocrine and endocrine compartments of the pancreas. However, the delayed and more mosaic upregulation of the ß-catenin signaling pathway in the PdxCrelate ß-catactive mutants appears to be well tolerated and does not result in any obvious developmental defect.
Stabilization of ß-catenin at E11.5, but not E13.5, results in pancreas hypoplasia
Other differences between the PdxCreearly and
PdxCrelate mouse strains, beyond the delay in onset of Cre
expression, might account for the difference in phenotypes observed. For
example, differences in the specific subset of cells targeted by each Cre
strain after E12.5 when both lines are active might be responsible for driving
the pancreas hypoplasia observed. Therefore, we directly assessed the temporal
dependence of the phenotype seen in the PdxCreearly
ß-catactive mice by crossing the
ß-catactive mouse to an inducible PdxCre
strain (PdxCreER) (Gu
et al., 2002) containing the same 5.5 kb promoter fragment as the
PdxCreearly mouse. In this system, the Cre
recombinase is expressed as a fusion protein with the estrogen receptor, and
remains inactive in the cytoplasm in the absence of tamoxifen ligand. However,
once bound to tamoxifen, the Cre recombinase enters the nucleus where it can
catalyze recombination (Gu et al.,
2002).
Injection of tamoxifen at E11.5 induced accumulation of ß-catenin in the nucleus of a large number of pancreatic epithelial cells in PdxCreER ß-catactive mice (Fig. 3J), resulting in a severe reduction of pancreas mass in all mutants observed (n=9, Fig. 3B). Nuclear localization of ß-catenin was not observed in control pancreata (Fig. 3I). Moreover, cystic structures similar to those seen in PdxCreearly ß-catactive are apparent in histological sections of the pancreas (Fig. 3F, indicated by arrows).
Conversely, injection of tamoxifen at E12.5 induced more variable phenotypes. The majority of PdxCreER ß-catactive mutants exhibited an intermediate phenotype with some reduction of the ventral and dorsal pancreas obvious in examination of the gross morphology of the organ (Fig. 3C). The pancreas histology of these intermediate PdxCreER ß-catactive mutants (Fig. 3G) appeared equivalent to control (Fig. 3E). However, a smaller number of PdxCreER ß-catactive mutants from litters injected with tamoxifen at E12.5 displayed either pancreas hypoplasia similar to those in the E11.5 injection group (Fig. 3B) or appeared unaffected (exact proportion of mutants in each category summarized in Fig. 3M). Despite the variability in phenotype, significant numbers of cells with nuclear ß-catenin localization were present in the pancreatic epithelium in all mutants analyzed (Fig. 3K). Injection of tamoxifen at E13.5 did not disrupt pancreas formation in any of the eleven PdxCreER ß-catactive mutants observed (Fig. 3D,H), despite clear nuclear localization of ß-catenin in a large number of pancreatic cells (Fig. 3L).
|
Pancreatic defects in PdxCreearly ß-catactive correlate with changes in FGF and hedgehog signaling, and loss of Pdx1+ progenitor cells
In order to analyze the defects in PdxCreearly
ß-catactive mice in more detail, we performed a
series of histological and molecular assays aimed at characterizing the early
progression of the phenotype and identifying the underlying molecular
mechanisms involved. By E12.5, stabilization of ß-catenin within the
pancreas of PdxCreearly
ß-catactive mice caused the pancreatic epithelium in
some mutants to exhibit abnormal dilation not seen in control tissues,
representing the earliest histological change that we could detect (data not
shown). By E15.5, the rudimentary clusters of exocrine cells seen in control
embryos are almost completely absent in the PdxCreearly
ß-catactive (data not shown). In addition, abnormally
enlarged pancreatic ducts are frequently observed at this time point in
PdxCreearly ß-catactive
pancreatic tissues and it is likely that these dilated ducts later form the
cystic structures seen at E18.5 (data not shown).
Numerous studies have shown the importance of mesenchymal-epithelial
interactions for proper pancreas formation. One of the mesenchymal molecules
known to regulate expansion of pancreatic epithelial cells is fibroblast
growth factor 10 (Fgf10) (Bhushan et al.,
2001). Fgf10 expression is normally detectable within the
mesenchyme surrounding the pancreatic bud beginning at E9.5 and peaking at
E10.5. By E12.5, Fgf10 expression can no longer be detected. Loss of
Fgf10 in mice has been shown to disrupt pancreas formation
(Bhushan et al., 2001), a
phenotype similar to the one we observed in PdxCreearly
ß-catactive mice. Therefore, we asked whether
stabilization of ß-catenin resulted in changes in mesenchymal
Fgf10 expression. Whole-mount in situ hybridization at E10.5 revealed
that Fgf10 expression is decreased in the pancreatic mesenchyme of
PdxCreearly ß-catactive animals
(Fig. 4C) when compared with
control (Fig. 4A).
Fgf10 expression appeared normal in the
PdxCrelate ß-catactive mutants
(Fig. 4B). This observation
supports our R26R expression analysis, which indicated that
Cre recombinase is not active in PdxCrelate
ß-catactive mice at this time point. Thus,
ß-catenin stabilization in pancreas epithelium before E11.5 disrupts an
important component of the signaling exchange that occurs between the
mesenchyme and epithelium, which at least partially explains the dramatic
defects in organogenesis observed.
Wnt signaling has also been implicated in maintaining the expression of
hedgehog (Hh) ligands in both Drosophila wing discs and mouse limb
buds (Parr and McMahon, 1995;
Pinson et al., 2000;
Tabata and Kornberg, 1994). In
addition, ectopic Wnt signaling in the epidermis has been shown to upregulate
the expression of Hh signaling pathway components
(Silva-Vargas et al., 2005).
Hedgehog signaling, in turn, stabilizes Wnt expression in Drosophila
wing (Tabata and Kornberg,
1994), indicating that both pathways can crossregulate the
activity of one another. A previous study has shown that overexpression of the
Hh ligand, Shh, under the Pdx1 promotor results in pancreas
agenesis (Apelqvist et al.,
1997), a phenotype similar to that observed in the
PdxCreearly ß-catactive mice. To
determine whether stabilization of ß-catenin activates Hh signaling in
pancreatic tissue, we analyzed E12.5 pancreas tissue from wild-type,
PdxCrelate ß-catactive and
PdxCreearly ß-catactive mice. By
immunohistochemistry, protein levels of the Hh receptor Ptch1, a direct
transcriptional target of Hh signaling
(Goodrich et al., 1996), are
significantly upregulated within the pancreatic epithelium of the
PdxCreearly ß-catactive mice
(Fig. 4F) when compared with
control or PdxCrelate ß-catactive
(Fig. 4D,E). Protein levels of
Hh ligand, are also increased in the PdxCreearly
ß-catactive (Fig.
4I) when compared with control or PdxCrelate
ß-catactive mice
(Fig. 4G,H). Therefore,
stabilization of ß-catenin in the PdxCreearly
ß-catactive mice results in upregulation of the Hh
signaling pathway within the early pancreatic epithelium, which may also
partially account for the dramatic changes observed during organ
formation/morphogenesis.
|
).
Additionally, loss of Pdx1 in mice has been shown to cause pancreas agenesis
(Offield et al., 1996;
Jonsson et al., 1994).
Therefore, we examined Pdx1 expression within the E12.5 pancreatic epithelium.
Interestingly, PdxCreearly
ß-catactive mice exhibited a 76% reduction in
Pdx1+ progenitor cells (Fig.
4L). By contrast, PdxCrelate
ß-catactive mice had numbers of Pdx1+
progenitor cells (Fig. 4K) that
were equivalent to control (Fig.
4J). Moreover, only cells that did not display elevated levels of
ß-catenin retained Pdx1 expression (data not shown). Presumably, these
cells escaped recombination. Cells with a clear increase in
cytoplasmic/nuclear ß-catenin were rarely detected in the
PdxCrelate ß-catactive mutant
pancreatic epithelium at E12.5 (data not shown). Thus, the pancreatic hypoplasia and cyst formation in the PdxCreearly ß-catactive appears to be mediated by loss of Fgf10 signaling, concomitant with an increase in Hh signaling. Together, these perturbations in early pancreas specification may contribute to the loss of Pdx1+ pancreatic progenitors.
Stabilization of ß-catenin causes increased pancreas organ size in PdxCrelate ß-catactive mice
While the morphology and histological architecture of pancreata dissected
from PdxCrelate ß-catactive
appeared normal at the end of development
(Fig. 2B,E), pancreas mass was
not. Despite the fact that mutant pups were equivalent in body mass to control
littermates, pancreas mass at day 0 was increased by 53%
(Fig. 5B). Pancreas mass
continued to increase independent of animal mass after birth, resulting in a
grossly enlarged organ (Fig.
5A) that was 2.7-fold greater than control pancreata at 98 days
postnatally (Fig. 5B). In
transgenic mice older than 1 year, the pancreas had grown 4.6-fold larger than
those found in control littermates (Fig.
5B). Strong nuclear ß-catenin staining could be detected
within the adult exocrine pancreas, suggesting that increased Wnt activity in
the PdxCrelate ß-catactive mice
was responsible for the increase in organ size after birth. In support of this
hypothesis, we found a 2.5 fold increase in the number of proliferating adult
exocrine cells in the PdxCrelate
ß-catactive (Fig.
5I), as revealed by staining for the mitotic marker phosphohistone
H3 (Fig. 5E,F).
Interestingly, PdxCreER
ß-catactive mice injected with tamoxifen 4 weeks
after birth, also showed a twofold increase in pancreas mass by 1 year of age
(n=8, data not shown). Injection of PdxCreER R26R
mice with tamoxifen results in clear Cre recombinase activity (as indicated by
lacZ staining) in a significant proportion of the exocrine pancreas
at this stage (data not shown). Thus, as has been shown for other tissues
(Bierie et al., 2003;
Huelsken and Birchmeier,
2001), ß-catenin signaling, as a part of the canonical Wnt
signaling pathway, can act as a proliferative signal in mature exocrine
pancreas. In addition, the density of cells within the exocrine pancreas, as
determined by counting the number of nuclei present in a defined histological
field, is also increased 1.8-fold in PdxCrelate
ß-catactive when compared with controls
(Fig. 5G,H,J). By contrast, the
density of endocrine cells within the mutant is normal. Although these data
suggest that exocrine cell size is reduced in PdxCrelate
ß-catactive mutants, the mechanism driving this
phenomenon is unclear. Stabilization of ß-catenin within the adult
exocrine tissue may directly deregulate cell size. However, it is also
possible that the rapidly proliferating exocrine cells do not need to reach
their normal size before dividing again. Electron microscopy will be used in
future studies to further characterize how this mutation affects pancreatic
cellular ultrastructure.
|
ß-Cell differentiation and islet function appear normal in the PdxCrelate ß-catactive mouse at 3 months
As shown in Fig. 1K, islets
within the PdxCrelate strain were mosaic for Cre activity.
Therefore, only a subset of ß-cells were targeted for stabilization of
ß-catenin in the PdxCrelate
ß-catactive mouse. Immunofluorescence staining
indicated that PdxCrelate
ß-catactive adult mutants exhibit stereotypical
murine islet architecture, consisting of a central core of insulin producing
ß-cells and a periphery of glucagon-producing -cells
(Fig. 6A,B). Despite the
increase in overall organ size at birth, the total cross sectional area of
endocrine islets remained equivalent to control (data not shown), suggesting
that the ratio of endocrine content to animal mass was not perturbed. In
addition, analysis of relative hormone content in pancreas homogenates at
three months of age indicated that the total amount of insulin and glucagon
present in the PdxCrelate
ß-catactive is equivalent to control (data not
shown). Thus, in contrast to the vast expansion of the exocrine compartment of
the pancreas, the size of the endocrine compartment remains largely
unaffected.
As expected from the R26R expression data, the majority of ß-cells in adult PdxCrelate ß-catactive mutants exhibited plasma membrane localization of ß-catenin that appeared equivalent to control (Fig. 6C,D, higher magnifications are shown in Fig. 6F,G) and only a subset of ß-cells was marked by upregulated ß-catenin levels. However, unlike the exocrine compartment (Fig. 5D), confocal analysis revealed that Cre-mediated excision in ß-cells did not result in a significant increase in nuclear ß-catenin, but instead in an increase in cytoplasmic ß-catenin levels (Fig. 6E, higher magnification can be found in Fig. 6H).
Within the ß-cells marked by high levels of cytoplasmic ß-catenin protein, expression of insulin (Fig. 6H), adult ß-cell transcription factors Pdx1 (Fig. 6J) and Nkx6.1 (data not shown), and the glucose transporter Glut2 (data not shown) appear equivalent to control (Fig. 6F,I). These results demonstrate that the ß-cells are fully differentiated, despite the dramatic increase in cytoplasmic ß-catenin. Additionally, fasting glucose tolerance is normal in PdxCrelate ß-catactive animals (Fig. 6J), suggesting that either ß-cell function is normal in these mutants or that the number of ß-cells expressing the stabilized form of ß-catenin is insufficient to affect this physiological assay. Future experiments using cultured islets are necessary to determine what biochemical mechanisms might protect pancreatic endocrine cells from accumulating nuclear ß-catenin.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Wnt, Hh and FGF interaction during pancreas development
Our observation that stabilization of ß-catenin in
PdxCreearly ßcatactive mice
results in upregulation of Hh signaling components concomitant with a loss of
Fgf10 suggests that the activities of these three major embryonic signaling
pathways are intimately linked during this stage of pancreas development.
Although we cannot exclude the possibility that Wnt signaling in the
epithelium directly affects the expression of mensenchymal Fgf10, it is
interesting to note that upregulation of Hh signaling resulting from loss of
the Hh inhibitor Hhip is known to cause a delay in Fgf10
expression in the pancreatic mesenchyme
(Kawahira et al., 2003).
Therefore, our results are consistent with the idea that at least some of the
defects observed in epithelium and mesenchyme are mediated via increased Hh
activity. A reduction of pancreas size has also been observed in transgenic
mice expressing Wnt1 under the control of the Pdx1 promoter
(Heller et al., 2003). The
phenotype observed in the PdxCreearly
ßcatactive mice results from upregulation of
ß-catenin signaling in a cell-autonomous fashion within the epithelium.
However, transgenic expression of the soluble Wnt1 ligand presumably exerts a
direct effect on both the pancreatic mesenchyme and epithelium. Therefore, it
would be interesting to compare whether Hh and FGF signaling are also affected
in the Pdx-Wnt1 mice. Moreover, future studies are necessary to determine the
precise signaling hierarchy between the Wnt, Hh and FGF signaling pathways
during normal pancreas specification and organogenesis.
|
Interestingly, although the pancreas becomes grossly enlarged, the exocrine
cells that comprise it appear smaller. Modulation of Wnt signaling has been
implicated in increasing the size of skeletal muscle cells after mechanical
overload (Armstrong and Esser,
2005). In addition, Wnt activation via overexpression of a
canonical Wnt ligand has been shown to result in chondrocyte hypertrophy
(Day et al., 2005). Although
the opposite was observed in the PdxCrelate
ß-catactive mice, it is possible that deregulation of
Wnt signaling in pancreatic exocrine cells may directly impact cell size.
However, exocrine cells contain a large number of granules whose digestive
enzyme content is emptied into the gut in response to feeding. Because the
PdxCrelate ß-catactive mutant
pancreas contains significantly more exocrine cells than are present in the
wild type, a feedback mechanism might exist that causes a reduction in the
number of granules in each cell, thereby reducing cell size. Ultrastructural
analysis is required to precisely determine how ß-catenin stabilization
affects pancreatic exocrine cell morphology.
ß-Catenin activation and adult ß-cell function
The absence of a proliferative response or enlargement of the ß-cell
compartment, within PdxCrelate
ß-catactive mutants may reflect differences in the
way in which exocrine and endocrine cells respond to activation of
ß-catenin. Alternatively, ß-cells may have a mechanism that allows
for the active exclusion of stabilized ß-catenin from the nucleus, which
then prevents downstream activation of the canonical Wnt signaling pathway. A
recent study involving ß-catenin stabilization in preimplantation embryos
also observed similar cytoplasmic localization of ß-catenin
(Kemler et al., 2004). The
authors propose several mechanisms that might prevent localization of the
stabilized form of ß-catenin in the blastomere nuclei. Among these are
the presence of an alternative ubiquitylation pathway that can degrade even
the stabilized form of ß-catenin or the activity of endogenous inhibitors
that might prevent ß-catenin from exerting its nuclear activity.
Endocrine cells may lack, or have low levels of, nuclear transport proteins
required for normal translocation and retention of ß-catenin in the
nucleus. Experiments using cultured islets and known inhibitors of nuclear
transport might prove instructive in uncovering the mechanism underlying the
resistance of the pancreatic endocrine cell to nuclear accumulation of
ß-catenin.
One of the truly puzzling findings of our study is that a low number of cells with clear nuclear localization of ß-catenin can be found only in the islets of one year old PdxCrelate ß-catactive animals. Thus, it is possible that pancreatic endocrine cells possess some kind of innate mechanism to prevent nuclear localization of ß-catenin that was gradually circumvented over time. Alternatively, it might be a matter of the cellular dose of the stabilized ß-catenin; it might take time for enough of the protein to accumulate to affect cell function.
Moreover, the location of these cells within the center of the islet raises the intriguing possibility that these are ß-cells that have undergone de-differentiation, after accumulating sufficient levels of nuclear ß-catenin. Genetic cell lineage tracing experiments would need to be performed to confirm this hypothesis. The absence of Pdx1 expression in these cells is consistent with the fact that the pancreatic epithelial cells with nuclear ß-catenin accumulation in the PdxCreearly ß-catactive also display a loss of Pdx1 expression at E12.5. However, the absence of normal pancreatic cell lineage markers makes it impossible to conclude what these cells were/are without further experimentation.
Given the fact that fewer than 30% of ß-cells are marked by cytoplasmic ß-catenin staining, it is also possible that potential ß-cell defects in ß-catenin-activated cells are masked by the remaining wild-type cells. Therefore, although the results presented here are intriguing, future studies using a transgenic strain that expresses Cre in a higher percentage of ß-cells are necessary to clarify these findings regarding the effect of ß-catenin stabilization on ß-cell differentiation, expansion and function.
Conclusion
Our findings further illustrate the dual nature of ß-catenin
signaling. In one context, ß-catenin activation prevents proper
differentiation and expansion of early pancreatic progenitor cells; in another
context, ß-catenin activation acts as a proliferative cue that induces
gross enlargement of the exocrine pancreas. Conversely, deletion of
ß-catenin has been shown to cause loss of exocrine cell mass
(Murtaugh et al., 2005) and,
in a different context, reduction of endocrine islet mass
(Dessimoz et al., 2005). Taken
together, these results highlight the complex roles ß-catenin signaling
plays in pancreas organ growth and the determination of the exocrine:endocrine
cell ratio.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Apelqvist, A., Ahlgren, U. and Edlund, H. (1997). Sonic hedgehog directs specialised mesoderm differentiation in the intestine and pancreas [published erratum appears in Curr. Biol. (1997) 7, R809]. Curr. Biol. 7, 801-804.[CrossRef][Medline]
Armstrong, D. D. and Esser, K. A. (2005).
Wnt/{beta}-catenin signaling activates growth-control genes during overload
induced skeletal muscle hypertrophy. Am. J. Physiol. Cell
Physiol. 289,C853
-C859.
Bhushan, A., Itoh, N., Kato, S., Thiery, J. P., Czernichow, P., Bellusci, S. and Scharfmann, R. (2001). Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 128,5109 -5117.[Medline]
Bienz, M. (2005). beta-Catenin: a pivot between cell adhesion and Wnt signalling. Curr. Biol. 15,R64 -R67.[CrossRef][Medline]
Bierie, B., Nozawa, M., Renou, J. P., Shillingford, J. M., Morgan, F., Oka, T., Taketo, M. M., Cardiff, R. D., Miyoshi, K., Wagner, K. U. et al. (2003). Activation of beta-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation. Oncogene 22,3875 -3887.[CrossRef][Medline]
Chuang, P. T., Kawcak, T. and McMahon, A. P.
(2003). Feedback control of mammalian Hedgehog signaling by the
Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching
morphogenesis of the lung. Genes Dev.
17,342
-347.
Day, T. F., Guo, X., Garrett-Beal, L. and Yang, Y. (2005). Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell 8, 739-750.[CrossRef][Medline]
Dessimoz, J., Bonnard, C., Huelsken, J. and Grapin-Botton, A. (2005). Pancreas-specific deletion of beta-catenin reveals Wnt-dependent and Wnt-independent functions during development. Curr. Biol. 15,1677 -1683.[CrossRef][Medline]
Gannon, M., Herrera, P. L. and Wright, C. V. (2000). Mosaic Cre-mediated recombination in pancreas using the pdx-1 enhancer/promoter. Genesis 26,143 -144.[CrossRef][Medline]
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A.
and Scott, M. P. (1996). Conservation of the hedgehog/patched
signaling pathway from flies to mice: induction of a mouse patched gene by
Hedgehog. Genes Dev. 10,301
-312.
Gounari, F., Signoretti, S., Bronson, R., Klein, L., Sellers, W. R., Kum, J., Siermann, A., Taketo, M. M., von Boehmer, H. and Khazaie, K. (2002). Stabilization of beta-catenin induces lesions reminiscent of prostatic intraepithelial neoplasia, but terminal squamous transdifferentiation of other secretory epithelia. Oncogene 21,4099 -4107.[CrossRef][Medline]
Gu, G., Dubauskaite, J. and Melton, D. A. (2002). Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129,2447 -2457.[Medline]
Harada, N., Tamai, Y., Ishikawa, T., Sauer, B., Takaku, K., Oshima, M. and Taketo, M. M. (1999). Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J. 18,5931 -5942.[CrossRef][Medline]
Hebrok, M., Kim, S. K., St Jacques, B., McMahon, A. P. and Melton, D. A. (2000). Regulation of pancreas development by Hedgehog signaling. Development 127,4905 -4913.[Abstract]
Heller, R. S., Klein, T., Ling, Z., Heimberg, H., Katoh, M., Madsen, O. D. and Serup, P. (2003). Expression of Wnt, Frizzled, sFRP, and DKK genes in adult human pancreas. Gene Expr. 11,141 -147.[Medline]
Herrera, P. L. (2000). Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Development 127,2317 -2322.[Abstract]
Huelsken, J. and Birchmeier, W. (2001). New aspects of Wnt signaling pathways in higher vertebrates. Curr. Opin. Genet. Dev. 11,547 -553.[CrossRef][Medline]
Jamora, C., DasGupta, R., Kocieniewski, P. and Fuchs, E. (2003). Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 422,317 -322.[CrossRef][Medline]
Jonsson, J., Carlsson, L., Edlund, T. and Edlund, H. (1994). Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371,606 -609.[CrossRef][Medline]
Kawahira, H., Ma, N. H., Tzanakakis, E. S., McMahon, A. P.,
Chuang, P. T. and Hebrok, M. (2003). Combined activities of
hedgehog signaling inhibitors regulate pancreas development.
Development 130,4871
-4879.
Kemler, R., Hierholzer, A., Kanzler, B., Kuppig, S., Hansen, K.,
Taketo, M. M., de Vries, W. N., Knowles, B. B. and Solter, D.
(2004). Stabilization of beta-catenin in the mouse zygote leads
to premature epithelial-mesenchymal transition in the epiblast.
Development 131,5817
-5824.
Kielman, M. F., Rindapaa, M., Gaspar, C., van Poppel, N., Breukel, C., van Leeuwen, S., Taketo, M. M., Roberts, S., Smits, R. and Fodde, R. (2002). Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling. Nat. Genet. 32,594 -605.[CrossRef][Medline]
Kim, S. K. and Hebrok, M. (2001). Intercellular
signals regulating pancreas development and function. Genes
Dev. 15,111
-127.
Kim, S. K., Hebrok, M. and Melton, D. A. (1997). Pancreas development in the chick embryo. Cold Spring Harb. Symp. Quant. Biol. 62,377 -383.[Medline]
Miller, J. R. and Moon, R. T. (1997). Analysis
of the signaling activities of localization mutants of beta-catenin during
axis specification in Xenopus. J. Cell Biol.
139,229
-243.
Murtaugh, L. C., Law, A. C., Dor, Y. and Melton, D. A.
(2005). Beta-catenin is essential for pancreatic acinar but not
islet development. Development
132,4663
-4674.
Nelson, W. J. and Nusse, R. (2004). Convergence
of Wnt, beta-catenin, and cadherin pathways. Science
303,1483
-1487.
Offield, M. F., Jetton, T. L., Labosky, P. A., Ray, M., Stein, R. W., Magnuson, M. A., Hogan, B. L. and Wright, C. V. (1996). PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122,983 -995.[Abstract]
Papadopoulou, S. and Edlund, H. (2005). Attenuated Wnt signaling perturbs pancreatic growth but not pancreatic function. Diabetes 54,2844 -2851.
Parr, B. A. and McMahon, A. P. (1995). Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb. Nature 374,350 -353.[CrossRef][Medline]
Perez, S. E., Cano, D. A., Dao-Pick, T., Rougier, J. P., Werb, Z. and Hebrok, M. (2005). Matrix metalloproteinases 2 and 9 are dispensable for pancreatic islet formation and function in vivo. Diabetes 54,694 -701.
Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J. and Skarnes, W. C. (2000). An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407,535 -538.[CrossRef][Medline]
Silva-Vargas, V., Lo Celso, C., Giangreco, A., Ofstad, T., Prowse, D. M., Braun, K. M. and Watt, F. M. (2005). Beta-catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells. Dev. Cell 9,121 -131.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs. Cell 76,89 -102.[CrossRef][Medline]
Tolwinski, N. S. and Wieschaus, E. (2004). A nuclear function for armadillo/beta-catenin. PLoS Biol. 2,E95 .[CrossRef][Medline]
Willert, K. and Nusse, R. (1998). Beta-catenin: a key mediator of Wnt signaling. Curr. Opin. Genet. Dev. 8,95 -102.[CrossRef][Medline]
Zechner, D., Fujita, Y., Hulsken, J., Muller, T., Walther, I., Taketo, M. M., Crenshaw, E. B., 3rd, Birchmeier, W. and Birchmeier, C. (2003). beta-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev. Biol. 258,406 -418.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
7 for each time point analyzed; control, blue;
PdxCrelate ß-catactive, orange).
(C,D) High levels of ß-catenin (green) can be detected in
the DAPI stained (blue) nuclei of adult PdxCrelate
ß-catactive exocrine cells (D), identified by amylase
staining (red). By contrast, ß-catenin is localized exclusively to the
plasma membrane of control cells (C). Scale bars: 10 µm.
(E,F) Staining of pancreatic sections for the proliferation
marker, phospho-histone H3 (PH3, red) and co-stained for DAPI (blue) reveals
an increase in the number of proliferating cells in the
PdxCrelate ß-catactive mutant (F)
when compared with control (E). Scale bars: 100 µm. (G,H)
Hematoxylin and Eosin staining of adult pancreas sections from
PdxCrelate ß-catactive mice (H)
reveals an increase in cell density within the exocrine pancreas, but not the
endocrine islet (at center) when compared with control (G). (I)
Quantification of PH3 positive cells (n=4 per genotype analyzed)
indicates that the relative number of proliferating cells in the
PdxCrelate ß-catactive mutant
(orange) is increased 2.5-fold over control (blue) 21 days postnatally.
(J) Quantification of the relative number of nuclei per field confirms
that the density of cells within the exocrine pancreas of
PdxCrelate ß-catactive mice
(orange) is increased 1.8-fold over control (blue), while the endocrine cell
density is normal (n=4). Confidence intervals calculated using
Student's t-test. #, not significant; *,
P<0.05; **, P<0.01. Error bars represent
s.e.m.
