Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA
Author for correspondence (e-mail:
dmelton{at}mcb.harvard.edu)
Accepted 26 August 2005
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SUMMARY |
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Key words: Pancreas, Exocrine, Acinar, ß-catenin, Wnt
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Introduction |
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Formation of the pancreas, as of other organs, requires the coordination of
proliferation, differentiation and morphogenesis. For decades, it has been
known that neighboring tissues, including the adjacent mesenchyme, provide
some of this coordination. When cultured alone, pancreatic bud epithelium
exhibits minimal growth and branching in vitro, and fails to generate acini,
whereas it undergoes robust growth and acinar differentiation following
recombination with mesenchyme (Golosow and
Grobstein, 1962; Wessells and
Cohen, 1967
). The mesenchyme may also produce distinct signals
that promote endocrine differentiation (Li
et al., 2004
). Understanding the morphogenetic signals active in
the pancreas is of practical interest, as well as academic, in that the
ability to mimic endogenous regulatory mechanisms will be necessary to convert
stem cells into ß-cells in vitro. To this end, it is particularly
important to identify those signaling pathways that affect cell fate
specification and differentiation from pancreatic progenitors.
Among the few signaling pathways implicated in pancreatic development by
knockout studies are the Notch (Apelqvist
et al., 1999; Jensen et al.,
2000
) and Fgf (Bhushan et al.,
2001
) pathways, both of which appear to negatively regulate
progenitor differentiation (Esni et al.,
2004a
; Hald et al.,
2003
; Hart et al.,
2003
; Murtaugh et al.,
2003
; Norgaard et al.,
2003
). Positively acting signals, such as the pro-acinar signal
provided by the mesenchyme, remain undefined by genetic experiments. In vitro
culture and transgene misexpression experiments have identified additional
signals that can affect pancreas development, although their genetic relevance
remains unclear. These include Egf family members, which appear to inhibit
both exocrine and endocrine differentiation
(Cras-Meneur et al., 2001
;
Esni et al., 2004b
), and
Tgfß family members, which can selectively promote endocrine
differentiation under some circumstances
(Miralles et al., 1998
;
Sanvito et al., 1994
). In
addition, the expression of several Wnt genes has been detected in the
developing pancreatic mesenchyme and epithelium; misexpression of Wnt1 and
Wnt5a in the early foregut results in agenesis or hypoplasia of the pancreas,
respectively (Heller et al.,
2002
; Lin et al.,
2001
). How these and other Wnts act during normal pancreas
development, however, remains unknown.
Wnt proteins play crucial roles in the development of multiple tissues. In
the intestine, for instance, Wnt signaling is required both for stem cell
maintenance and for endocrine development
(Ireland et al., 2004;
Korinek et al., 1998
;
Pinto et al., 2003
). In these
and many other instances, Wnts act via the so-called canonical pathway, in
which receptor activation leads to the stabilization of cytosolic
ß-catenin protein (Logan and Nusse,
2004
). Cytosolic ß-catenin is normally phosphorylated and
targeted for proteolysis by a complex of proteins, including Apc, axin and the
serine/threonine kinase Gsk3ß. Wnt activation of the Frizzled and Lrp
co-receptors results in dissociation of this complex, so that newly
synthesized ß-catenin is no longer phosphorylated and degraded.
Stabilized ß-catenin then enters the nucleus to activate target genes in
collaboration with Lef/Tcf family transcription factors, and possibly other
partners (Logan and Nusse,
2004
).
We show here that the early pancreatic epithelium exhibits a specific accumulation of unphosphorylated ß-catenin, a hallmark of active Wnt signaling. Furthermore, by tissue-specific knockout, we find that ß-catenin is absolutely required for the formation of exocrine acini, but is dispensable for endocrine differentiation and function. Moreover, ß-catenin protein is dispensable for the survival of mature acinar cells, and for the maintenance of their cell type-specific gene expression. Although ß-catenin is also required for the robust proliferation of early pancreatic progenitor cells, these cells do not prematurely differentiate or apoptose in the absence of ß-catenin. Our results suggest a requirement for canonical Wnt signaling in the specification or differentiation of acinar cells.
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Materials and methods |
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The Ela-CreERT construct was generated by fusing the
acinar-specific enhancer of the elastase gene [a gift from G. Swift and R.
MacDonald (Swift et al.,
1989)] to a minimal hsp68 promoter (a gift from M. Gannon), and
placing the chimeric promoter upstream of CreERT [a gift from A.
McMahon (Danielian et al.,
1998
)]. Two founders were identified that, when crossed with Z/AP
mice (Lobe et al., 1999
),
showed acinar-specific expression of human placental alkaline phosphatase upon
tamoxifen injection, performed as described
(Dor et al., 2004
). One of
these lines exhibited more robust tamoxifen-induced recombination than the
other, and was maintained and expanded; all of the experiments described here
made use of this line.
For timed pregnancies, it is assumed that the morning of plug detection corresponds to E0.5. Embryo ages are rounded to the nearest half day.
Tissue processing
Mice were euthanized with isoflurane, and adult or embryonic tissues
dissected in ice-cold PBS. To label S-phase nuclei, some mice were injected
with BrdU (50 µg/g body weight) one hour prior to sacrifice. For paraffin
embedding, tissues were fixed for 1-2 hours at 4°C in zinc-buffered
formalin (Polysciences). Paraffin sections (6 µm) were collected in
ribbons, and subsequently subdivided serially across a series of 4-16 slides
(depending on age and tissue size), such that each slide would contain
semi-adjacent sections across the entire tissue of interest.
For frozen sections, tissues were fixed for 1-2 hours at 4°C in fresh 4% paraformaldehyde, washed in PBS and cryoprotected overnight in 30% sucrose (w/v) in PBS, before embedding and freezing in Tissue-Tek OCT compound. Cryosections (12-14 µm) were collected serially across 6-16 slides (depending on age and tissue size), as above.
Immunostaining
Primary antibodies used in this study are listed in
Table 1. Secondary antibodies,
raised in donkey and biotinylated or conjugated to fluorophores, were
purchased from Jackson ImmunoResearch. A biotin conjugate of the duct-binding
lectin Dolichos biflorus agglutinin
(Kobayashi et al., 2002) was
purchased from Vector Laboratories, and detected with streptavidin-conjugated
fluorophores (Jackson ImmunoResearch). Unless otherwise noted, all
photomicrographs shown are representative of at least three samples of the
indicated genotype or condition.
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In situ hybridization
Non-isotopic in situ hybridization was performed with DIG-labeled cRNA
probes, transcribed from full-length IMAGE consortium EST clones. In situ
hybridization on frozen or paraffin sections followed the protocol of Brent et
al. (Brent et al., 2003). For
quantitation of endocrine and exocrine volumes at E15.5, individual sections
were photographed, along with a stage micrometer to normalize area, and the
area occupied by stained cells was measured using ImageJ software (Wayne
Rasband, NIH;
http://rsb.info.nih.gov/ij/).
The relative volume for each sample was determined by multiplying the total
measured area across a single slide (`area') by the total number of slides
used to collect and analyze the sample (`depth'). Statistical significance was
assessed by two-tailed t-test, with Bonferroni correction for
multiple testing (comparing insulin+, proglucagon+ and
Cpa1+ volumes between wild type and knockout).
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Results |
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We compared staining with the dephospho-specific monoclonal antibody to that of a monoclonal recognizing all ß-catenin species, and confirmed enrichment of dephospho-ß-catenin in known sites of canonical Wnt signaling, such as the ventricular zone of the E11.5 spinal cord (Fig. 1C,C') and the crypts of the neonatal intestine (Fig. 1D,D'). We then stained sections of the pancreas, at various developmental stages, with each antibody. Total ß-catenin protein is seen throughout the pancreatic epithelium and mesenchyme at all stages (Fig. 1E-I). However, dephospho-ß-catenin staining is considerably more specific; at E11.5-E13.5 it is robustly detected in the pancreatic epithelium and excluded from the mesenchyme (Fig. 1E'-I'). Dephospho-ß-catenin levels in the pancreas decline between E15.5-E17.5, and by birth are undetectable in differentiated endocrine and exocrine cells (Fig. 1I'). Dephospho-ß-catenin is similarly undetectable in the adult pancreas (data not shown).
This staining pattern is consistent with canonical Wnt signaling being
active in the early pancreas, during the transition from undifferentiated
progenitors to committed exocrine and endocrine cells. It should be noted that
we could not unambiguously detect nuclear ß-catenin in any tissue, with
any antibody, including the monoclonal anti-dephospho ß-catenin antibody,
the monoclonal pan-specific antibody, and two independent polyclonal antisera
(see Materials and methods). We assume this is because endogenous Wnt
signaling generates only low levels of nuclear ß-catenin, relative to the
high levels of ß-catenin constitutively localized to the cell membrane.
Indeed, previous studies have reported difficulty in detecting endogenous
nuclear ß-catenin protein in wild-type (as opposed to Apc
mutant) tissue sections (Anderson et al.,
2002), suggesting that unconventional sensitivity is required.
Pancreas-specific deletion of ß-catenin results in dramatic organ hypoplasia
To establish its function in the developing pancreas, we took advantage of
a conditional allele of ß-catenin (Catnblox)
generated by Brault et al., in which the first five coding exons are flanked
by loxP sites. Deletion of these exons removes codons 1-312 of
ß-catenin, resulting in an absence of functional protein
(Brault et al., 2001). We
achieved deletion of the ß-catenin gene by breeding
Catnblox mice with mice in which Cre is expressed under
control of the Pdx1 promoter (Pdx1-Cre), thus driving
recombination in all endodermal lineages of the developing pancreas
(Gu et al., 2002
).
We examined the guts of neonatal offspring from experimental crosses to
look for a gross pancreatic phenotype (Fig.
2A). In all cases where the pancreas had at least one undeleted
allele of Catnb (Catnblox/+; Pdx1-Cre,
Catnb+/+; Pdx1-Cre, or lacking Pdx1-Cre),
the organ was completely normal; for sake of simplicity, we refer to these
pancreata as `wild type'. By contrast, the majority of mice that inherited the
Pdx1-Cre transgene and were homozygous for the floxed allele
(Catnblox/lox; Pdx1-Cre) had considerably smaller
pancreata than their wild-type littermates, with particularly severe reduction
of the ventral lobe (Fig.
2B-E). Variability in this genotype results from a stochastic
inefficiency in Cre recombination (see below) (c.f.
Murtaugh et al., 2003); as
such, it was reduced by pre-deleting one allele of Catnb in the
germline (Catnb
; see Materials and methods). Below,
we designate both sets of genotypes (Catnblox/lox;
Pdx1-Cre and Catnb
/lox;
Pdx1-Cre) as `PBKO' (pancreatic beta-catenin knockout), unless otherwise
specified.
|
Maintenance of hypoplastic early progenitors in the absence of ß-catenin
Pancreatic hypoplasia is observed in several other mouse mutants, notably
those lacking components of the Notch signaling pathway, including Dll1,
RBPJk (Rbpsuh Mouse Genome Informatics) and
Hes1 (Apelqvist et al.,
1999; Jensen et al.,
2000
), as well as in Fgf10 mutants
(Bhushan et al., 2001
). In all
of these cases, the hypoplasia has been attributed to premature depletion of
Pdx1+ progenitor cells, which normally persist through
approximately E13.5 (Guz et al.,
1995
). To ascertain whether progenitors were similarly lost in the
absence of ß-catenin, we analyzed marker expression in wild-type and PBKO
pancreatic buds at E11.5, when the majority of the epithelium consists of
undifferentiated progenitor cells. Confocal immunofluorescence confirmed that
nearly all cells (>95%) of the PBKO pancreatic epithelium lacked detectable
ß-catenin (Fig.
3A,A'; in this and in all subsequent figures, ß-catenin
was detected with pan-specific antibodies). The expression of Pdx1 in
ß-catenin-deficient cells was indistinguishable from that of wild type
(Fig. 3B,B'), and the
overall size and morphology of the early bud appeared similarly unaffected by
loss of ß-catenin. At these early stages, the only mature pancreatic
marker detectable in wild type was glucagon, expressed by a few scattered
cells in the E11.5 bud (Fig.
3C). A similar number and distribution of glucagon+
cells was seen in PBKO pancreata (Fig.
3C'), and no other endocrine or exocrine markers were
ectopically expressed (data not shown). In further contrast to Notch component
and Fgf10 mutants, PBKO pancreata retain normal Pdx1+
progenitors through E13.5 (Fig.
3E-F').
|
Several Wnt/ß-catenin target genes, likely to regulate proliferation,
are expressed in the wild-type pancreas at this stage, including cyclin D1
(Shtutman et al., 1999;
Tetsu and McCormick, 1999
),
Id2 (Rockman et al.,
2001
) and Myc (He et
al., 1998
) (Fig.
4B-D). Although cyclin D1 and Id2 expression appears to
be unaffected in PBKO littermates (Fig.
4B'-C'), expression of the proto-oncogene Myc
appears to be downregulated in the absence of ß-catenin
(Fig. 4D'). As
Myc mutants die before E10.5
(Davis et al., 1993
), their
pancreatic phenotype cannot be evaluated, but it is possible that Myc
mediates the effects of ß-catenin on proliferation. Finally, although Wnt
and Notch signaling appear to cooperate in several tissues
(Radtke and Clevers, 2005
), we
find that expression of the Notch target gene Hes1 is maintained in
PBKO pancreata (Fig.
4E,E'), consistent with the observed maintenance of early
progenitor cells.
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To address the possibility that deletion of the Catnb gene occurs
more robustly in one lineage than another, we performed confocal
immunofluorescence to detect ß-catenin co-expression with islet and
acinar markers. Whereas both insulin+ ß-cells and
glucagon+ -cells of wild-type E15.5 pancreata co-expressed
ß-catenin (Fig. 6A-F), in
PBKO pancreata, nearly all (>95%) of both cell types lacked detectable
ß-catenin (Fig.
6A-F'). Nevertheless, numerous ß-catenin+
cells were detected in PBKO pancreata, typically organized in epithelial
clusters separate from the ß-catenin endocrine cells.
These primarily represented the residual acinar cells, which were uniformly
(>99%) ß-catenin+ (Fig.
6G'-I'). Thus, there is a specific and nearly absolute
requirement for ß-catenin in acinar development.
|
The PBKO phenotype, with respect to differentiation, appears to be
selective for acinar cells. To further understand this phenotype, we examined
expression of the transcription factor Ptf1a/p48. Ptf1a directly regulates
many acinar-specific genes, and its expression in the mature pancreas is
restricted to acinar cells (Krapp et al.,
1996). Although Ptf1a is expressed broadly in the early pancreatic
primordium, in progenitors of all mature cell types
(Kawaguchi et al., 2002
), it
becomes specifically upregulated in peripherally epithelial clusters 1-2 days
prior to acinar differentiation, likely marking the nascent acini
(Esni et al., 2004a
). We
observe this upregulation in the wild-type E13.5 pancreas
(Fig. 7A-C), as expected, but
these Ptf1a+ clusters are rarer in PBKO pancreata, and, more
importantly, are restricted to residual ß-catenin+ regions
(Fig. 7D-F). Thus,
ß-catenin appears to be required for the upregulation of Ptf1a prior to
acinar differentiation, potentially providing a mechanism for the PBKO acinar
phenotype.
|
ß-Catenin is dispensable for the function and survival of differentiated islet and acinar cells
To determine whether ß-catenin-deficient islet cells survive and
function normally, we examined adult PBKO mice. The islets of adult PBKO mice
exhibited an almost complete lack of detectable ß-catenin protein
(Fig. 8A,A'), similar to
embryos, but their architecture was essentially normal. In addition, we
performed glucose tolerance tests on adult PBKO mice (age 12-15 weeks) to test
this cardinal function of ß-cells. As shown in
Fig. 8C, PBKO mice exhibited no
significant impairment in glucose clearance when compared with wild types,
indicating that ß-catenin is not required for glucose-stimulated insulin
release by ß-cells. This contrasts with the phenotype of mice hemizygous
or null for the canonical Wnt receptor Lrp5, which exhibit impaired
blood sugar homeostasis (Fujino et al.,
2003). That we do not observe such a defect in adult PBKO mice
suggests that Lrp5 may mediate ß-catenin-independent aspects of Wnt
signaling.
|
To test transgene function, we bred Ela-CreERT mice with
R26R indicator mice that carry a Cre-activateable lacZ
reporter (Soriano, 1999). As
expected, in the absence of Cre there is no detectable ß-galactosidase
(ß-gal) activity in the adult R26R pancreas (data not shown). In
the presence of Ela-CreERT, but without injected tamoxifen (TM), a
small proportion of acinar cells (
1%) are ß-gal+
(Fig. 9A), indicating a low
level of TM-independent recombination. When TM is administered to
double-transgenic mice (5 mg daily, 3x), robust ß-gal activity is
seen throughout the acini, with
50% being labeled two weeks after the
first injection (Fig. 9B).
Although no labeling is seen in the ducts
(Fig. 9B), a low level does
occur in islet cells (not shown), presumably reflecting inappropriate
transgene expression. Although this ectopic labeling, and the low level of
TM-independent activity, limit the utility of this transgenic line for
long-term lineage-tracing experiments (Dor
et al., 2004
), it remains useful for acute manipulation of adult
acinar cells.
To delete ß-catenin in adult acinar cells, we crossed Ela-CreERT onto a Catnblox/lox background, administered tamoxifen as above, and examined the mutant pancreata (n=3) two weeks after the initial injection. These pancreata were histologically unremarkable, and exhibited no increased apoptosis (data not shown). In the absence of either tamoxifen or the deletor transgene, expression of ß-catenin protein was observed in all acinar cells as expected (Fig. 9C, data not shown). By contrast, two weeks after tamoxifen administration to Catnblox/lox; Ela-CreERT mice, a widespread clearance of ß-catenin protein is seen in the acinar cells (Fig. 9C'). Confocal immunofluorescence revealed that ß-catenin-deficient acini retained normal expression of the digestive enzyme amylase (Fig. 9C-E'), indicating that their differentiation program remained intact. The scarcity of acinar cells in the PBKO pancreas is therefore unlikely to reflect an acute requirement for ß-catenin in their survival, or in the maintenance of cell type-specific gene expression; instead, it is most readily explained by a requirement for ß-catenin in the initial formation of acinar cells.
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Discussion |
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We find that pancreata lacking ß-catenin are dramatically smaller than
those of wild-type animals, and contain a striking paucity of acinar cells. In
part, this may be attributed to a decrease in the proliferation rate of early
progenitors, potentially reflecting loss of Myc expression.
Nonetheless, there is no evidence for premature differentiation or depletion
of the progenitor pool in PBKO pancreata. Indeed, the endocrine compartment
appears relatively unaffected in these pancreata, with - and
ß-cells differentiating in approximately normal numbers and with normal
timing. The acinar lineage, by contrast, appears to cell autonomously require
ß-catenin for its differentiation, but not for its maintenance or
survival.
What is the relationship between the hypoplasia and acinar depletion
phenotypes in the PBKO pancreas? A comparison with the Fgf10 mutant
phenotype may be instructive. In Fgf10/
pancreata, the Pdx1+ progenitor pool is lost to hypoplasia by
E12.5-E13.5, and the final organ is even smaller than that of PBKO mice.
Nonetheless, most of the remnant pancreas in the Fgf10 mutant is
composed of differentiated acini (Bhushan
et al., 2001). If the lack of exocrine cells observed in PBKO
pancreata reflected an indirect effect on progenitor proliferation, we would
expect that the acinar phenotype of Fgf10 mutants should be at least
as severe as that of PBKO mice. The fact that it is actually less so implies
that ß-catenin function extends beyond simply promoting
proliferation.
It is also possible that ß-catenin functions solely in the expansion of differentiated exocrine cells. We regard this as unlikely: at E15.5, shortly after acinar differentiation has commenced, we observe very few amylase+/ß-catenin cells (<1% of all amylase+ cells), whereas at E13.5, prior to differentiation, we find that Ptf1a expression is restricted to the residual ß-catenin+ cells. Therefore, we think that the PBKO exocrine phenotype is most readily explained by a direct requirement for ß-catenin in the development of progenitor cells into acini, rather than by an indirect effect of decreased proliferation.
We find that ß-catenin-deficient cells are capable of normal
contribution to islet endocrine cells. This contrasts with the intestine,
where canonical Wnt signaling is required for endocrine development
(Pinto et al., 2003), possibly
acting through a direct ß-catenin/Tcf regulation of hormone genes such as
proglucagon (Yi et al., 2005
).
Interestingly, and consistent with our results, ß-catenin/Tcf-binding
sites are dispensable for proglucagon expression in pancreatic
-cell
lines (Yi et al., 2005
). These
results identify an important difference in the developmental program of the
two classes of endocrine cells, which is in contrast to their shared
requirement for upstream regulators such as Ngn3
(Gradwohl et al., 2000
;
Jenny et al., 2002
) and the
Notch target Hes1 (Jensen et al.,
2000
).
Our finding that unphosphorylated ß-catenin is enriched in the early
pancreatic epithelium, as in tissues where the Wnt pathway is known to be
active, is strongly consistent with the possibility that Wnt proteins are
acting on the pancreas at these stages. Several Wnt genes are expressed in the
pancreatic mesenchyme (Heller et al.,
2002; Lin et al.,
2001
), and the mesenchyme has long been known to be required for
acinar differentiation in vitro (Golosow
and Grobstein, 1962
; Wessells
and Cohen, 1967
). Although transgenic expression of Wnt1
under control of the Pdx1 promoter leads to pancreatic agenesis
(Heller et al., 2002
), this
may reflect an alternative role for Wnt signaling specifically at the early
stages that Pdx1 expression initiates, for instance in specifying
intestinal fates (Okubo and Hogan,
2004
). Future work will address whether or not Wnt signaling
components upstream of ß-catenin are similarly required for pancreas
development, and with what downstream partners ß-catenin might
interact.
We are also keenly interested in determining the fate of those ß-catenin-deficient cells that would normally contribute to acini: are they are diverted to a non-acinar fate, such as islets? Although we found no expansion of the endocrine population in PBKO pancreata, it is possible that any increase would have been masked by the decreased proliferation of early ß-catenin-deficient progenitors. Resolving this issue in the future will require a Cre-deletor transgene specific to acinar precursor cells.
In summary, we have demonstrated that ß-catenin is required both for
the robust proliferation of pancreatic progenitor cells and for their
subsequent differentiation into acinar cells, potentially acting upstream of
the Myc and Ptf1a genes in these respective processes. As
ß-catenin is dispensable for islet endocrine development, it occupies a
unique place in the genetic cascade of pancreas development
(Murtaugh and Melton, 2003),
and its crucial role in the Wnt cascade suggests that Wnt proteins may prove
to be important intercellular signals in pancreatic organogenesis.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
---|
Consistent with our finding that loss of Wnt/ß-catenin signaling does
not impair pancreatic endocrine development, a recent publication
(Pedersen and Heller, 2005)
indicates that hyperactivation of Wnt/ß-catenin signalling actively
antagonizes endocrine differentiation induced by Ngn3 misexpression.
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/21/4663/DC1
* Present address: Department of Human Genetics, University of Utah, Salt
Lake City, UT 84112, USA
Present address: Department of Cellular Biochemistry and Human Genetics,
The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
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