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First published online 20 August 2008
doi: 10.1242/dev.017137
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Research Report |
Department of Internal Medicine, Technical University of Munich, 81675 Munich, Germany.
* Author for correspondence (e-mail: roland.schmid{at}lrz.tum.de)
Accepted 1 August 2008
SUMMARY
Recent studies have shown that Wnt/β-catenin signaling is essential for development of the exocrine pancreas, but the role of β-catenin-dependent target genes such as Myc during pancreatic development is not well known. Here, we show that tissue-specific deletion of Myc causes a slightly accelerated differentiation of pancreatic epithelial cells into endocrine cells and perturbs the proliferation of pancreatic progenitors and acinar precursor cells during early development, resulting in a severe reduction of the epithelial cell mass of pancreatic buds and an extensive acinar hypoplasia. Loss of Myc does not affect the expression of the tissue-specific transcription factor PTF1a, which is required for the differentiation of acinar cells. In contrast to its role for exocrine cell growth, the development of endocrine cell lineages is not significantly disturbed. These data suggest that Myc is required for the expansion of the exocrine pancreas. Our observations are consistent with the findings in β-catenin-deficient pancreas, suggesting that Wnt/β-catenin signaling affects the proliferation of pancreatic epithelial cells and acinar precursors through its target gene Myc.
Key words: Myc, Pancreas, Development, Mouse
INTRODUCTION
Myc (previously known as c-Myc) is a transcription factor of the basic
helix-loop-helix (bHLH) leucine zipper that has been extensively studied as a
proto-oncogene but is also essential for normal cell cycle progression.
Although Myc promotes cell growth and proliferation in several
tissues, it induces or sensitizes cells to apoptosis in others
(Dang, 1999
).
Myc is a key target gene of the Wnt/β-catenin pathway
(He et al., 1998) and is
activated during pancreatic development
(Dessimoz et al., 2005). In
adult pancreata, accumulation of β-catenin through conditional
inactivation of Apc leads to hyperplasia of acinar cells with
increased expression of Myc (Strom et al.,
2007). β-Catenin itself is a component of the
E-cadherin-mediated cell-cell adhesion system
(Lin et al., 2000) and a key
effector of the Wnt signaling pathway, which plays a crucial role in growth,
cell division and cell fate decisions during organogenesis
(Orford et al., 1999;
Peifer and Polakis, 2000;
Polakis, 2000). In response to
Wnt signals, β-catenin complexes with T-cell factor/lymphoid-enhancing
factors (Tcf/Lef) and p300 to induce transcription of target genes known to be
important in cell proliferation such as Myc
(He et al., 1998) and cyclin D1
(Shtutman et al., 1999).
Here, we show that Myc is essential for the proliferation of pancreatic epithelial and acinar precursor cells. Inactivation of Myc during pancreatic development results in the decreased size of pancreatic buds and severe pancreatic hypoplasia during organogenesis.
MATERIALS AND METHODS
Generation of genetically modified mice
Mice containing a floxed allele of Myc were a generous gift from
Moreno de Alboran. The Myc allele in these animals contains LoxP
sites in the first and third intron as described previously
(de Alboran et al., 2001). To
obtain conditional deletion of Myc within the pancreas, we used a
Ptf1a-cre(ex1) knock-in mouse
(Nakhai et al., 2007),
previously reported to mediate efficient recombination in the developing mouse
pancreas by embryonic day 10.5 (E10.5)
(Nakhai et al., 2008). All
mouse protocols were approved by the Faculty of Medicine, Technical University
of Munich.
X-gal staining
β-gal activity was determined on whole-mount preparations according to
Kawaguchi et al. (Kawaguchi et al.,
2002).
BrdU labeling
In vivo pulse labeling with 5-bromo-2-deoxyuridine (BrdU) was used to mark
newly synthesized DNA. BrdU (20 mmol/l, 5 ml/kg body weight) was injected i.p.
into pregnant mice 2 hours before sacrifice.
Immunohistochemistry and morphometric analysis
Dissected tissues were fixed in ice-cold 4% paraformaldehyde,
paraffin-embedded and cut into 2-3 µm sections. Immunohistochemistry was
performed using primary antibodies (details can be provided on request). For
immunoperoxidase detection, Vectastain ABC kit (Vector Labs) was used
according to the manufacturer's instruction. For double-immunofluorescence
staining, the primary antibodies were followed by incubation with secondary
antibodies conjugated with fluorescent Alexa 488 or Alex 568 (Molecular
Probes). Sections were mounted with Vectashield mounting medium (Vector
Laboratories) and examined using an Axiovert 200M (Zeiss) fluorescent inverse
microscope equipped with the Axiovision version 4.4 software (Zeiss). For
morphometric analyses, the pancreatic buds were immunostained with anti-PDX1
and analyzed using the AxioVision Image analysis software (Zeiss). To
calculate the number of PHH3- and neurogenin3-positive cells, the whole
pancreatic buds of three control and three PMycKO embryos were cut
into 3 µm serial sections. Every fifth section was stained and the number
of PHH3+ cells and glucagon+ cell area were counted and
calculated relative to the whole area of PDX1+ pancreatic
epithelium in every section. The endocrine cell mass at E18.5 was calculated
as the ratio of each hormone-positive cell area to the total area of the
pancreas section using AxioVision Image analysis software (Zeiss, Germany).
For each group three mice were used. The measurement and calculation were
carried out with three sections from each mouse. The sections were far apart
from one another. The whole area of each section was investigated, and the
insulin- or glucagon-positive cell area relative to the whole pancreatic area
was determined. All values are expressed as mean±s.e.m. Statistical
significance was tested using Student's t-test.
RESULTS AND DISCUSSION
Tissue-specific deletion of Myc results in severe pancreatic hypoplasia and neonatal lethality
We achieved pancreas-specific deletion of Myc by breeding a
Ptf1a-cre(ex1) strain (Nakhai et
al., 2007) with a strain containing a floxed Myc allele.
Recombination between the two loxP sites mediated by the Cre recombinase leads
to the complete inactivation of the Myc gene via deletion of exons 2
and 3 (de Alboran et al.,
2001). Ptf1a-cre(ex1) mice heterozygous for the floxed
Myc allele displayed no apparent abnormalities in either the
embryonic or adult pancreas. However, mice with conditional inactivation of
both Myc alleles in the pancreas
(Mycf/f;Ptf1a+/Cre(ex1)) die at birth, most
probably to defects in the neural system. For simplicity,
Mycf/f;Ptf1a+/Cre(ex1) mice will be termed
PMycKO (pancreas specific Myc knockout) and heterozygote
littermates and littermates not expressing Cre recombinase will be termed
PMyc+/- and wild-type mice, respectively.
|
) to
generate a PMycKO;R26R strain. In these mice, Ptf1a-driven
Cre expression causes inactivation of Myc as well as expression of
β-galactosidase, which can be identified by X-gal staining. Using this
method, we did not observe X-gal-negative exocrine cells in embryonic
pancreata (Fig. 1B,D). At
E18.5, the latest stage characterized, pancreatic X-gal staining of
PMycKO;R26R and Myc+/-;R26R embryos revealed a
severe pancreatic hypoplasia in Myc-deficient but not heterozygous
littermates (Fig. 1A,B).
Histological analysis showed poorly branched pancreatic ducts and disruption
of exocrine pancreas formation in PMycKO;R26R embryos compared with
PMyc+/-;R26R littermates
(Fig. 1C,D). Immunohistological
analysis using antibodies against acinar cell markers such as amylase and
PTF1a conformed a severe reduction of acini in Myc-deficient
pancreata compared with heterozygous littermates
(Fig. 1E,F,H,I, arrows).
Although the majority of the acinar cells were absent, it was striking that
there were clusters of normal appearing acini in mutant pancreata.
Double-immunofluorescence staining for β-galactosidase and amylase
confirmed Cre activity in amylase-positive acinar cells
(Fig. 1G, arrow).
Depletion of pancreatic acini is caused by decreased cell proliferation and accelerated differentiation of epithelial cells into endocrine cells
PTF1a is an essential transcription factor for differentiation of
pancreatic precursors into acinar cells
(Kawaguchi et al., 2002;
Krapp et al., 1998). The
expression of this factor in Myc-deficient pancreata at E18.5
suggests that the loss of Myc activity may not impair acinar specification and
differentiation. To determine the stage at which the exocrine pancreatic
development is perturbed, we analyzed E10.5-E13.5 PMycKO embryos by
immunohistochemistry. At E12.5, immunolabeling of pancreatic buds for Myc
displayed expression in pancreatic epithelial cells of wild-type embryo
(Fig. 2A) and confirmed the
loss of Myc protein in PMycKO embryos
(Fig. 2B). At this age,
staining of pancreatic epithelial cells for PDX1 revealed a significantly
reduced epithelial mass with weakly branched structures in both buds of
PMycKO embryos and those of wild-type littermates
(Fig. 2C,D), suggesting that
Myc is essential for the expansion of the early pancreatic
epithelium. A possible cause for this reduction of pancreatic epithelium
(Fig. 2N) could be the
premature differentiation of pancreatic progenitor into endocrine cells. As
reported previously in Rbpj-deficient buds
(Fujikura et al., 2006),
accelerated differentiation of endocrine cells leads to an increase in
glucagon-expressing cells and a decreased number of neurogenin 3+
(Ngn3) endocrine precursor cells early in pancreatic development.
To ascertain whether the number of glucagon+ cells was increased
and the number of endocrine precursors was decreased in the absence of
Myc, pancreatic buds were respectively analyzed for glucagon- and
Ngn3-positive cells. At E10.5, double-immunofluorescence staining of
PMycKO and wild-type littermates for glucagon and PDX1 showed a 1.4
times increase in glucagon+ cell area in the dorsal bud of knockout
embryos (Fig. 2E-G). At E12.5,
pancreatic buds of PMycKO and wild-type littermates were
immunolabeled for Ngn3. This analysis revealed that the number of
Ngn3+ cells decreased by about 1.2 times (20%) in PMycKO
compared with control embryos, suggesting partial premature differentiation of
endocrine precursor cells (Fig.
2H-J). In order to determine whether the loss of Pdx1+
epithelial cells in PMycKO embryos was also related to a decrease in
proliferation of Myc-deficient cells, we performed immunostaining of
PMycKO and wild-type embryos at E12.5, using an anti-phosphohistone
H3 (PHH3) antibody and assessed the number of PHH3+ cells per
Pdx1+ pancreatic area (Fig.
2K,L, arrows). These studies revealed a significantly decreased
proliferation rate (to 
40%) in Myc-deficient versus wild-type
buds (Fig. 2M), suggesting that
Myc plays an important role in controlling proliferation of
pancreatic epithelial cells. Several Myc target genes involved in
cell proliferation are expressed in the wild-type pancreatic epithelium at
this stage, including cyclin D1 (Fernandez
et al., 2003) and CDK4
(Hermeking et al., 2000).
Although expression of cyclin D1 appeared to be unaffected in PMycKO
littermates (Fig. 2N,O), CDK4
was downregulated in the absence of Myc
(Fig. 2P,Q). Regarding the role
of Myc as regulator of apoptosis, we found no evidence to suggest
that apoptosis contributed to early pancreatic hypoplasia (data not
shown).
|
). By E14.5, cells in the periphery of the branching
epithelial tree begin to adopt a pyramidal shape and organize into discrete
clusters, representing nascent acinar structures. At this time, nuclear Myc
immunoreactivity remained evident in nascent acini of wild-type embryos, which
also expressed acinar cell markers such as amylase
(Fig. 3A,C, arrows). In serial
sections of PMycKO embryos, we detected some amylase+
acinar cells that did not express Myc (Fig.
3B,D, arrows). To assess nascent acinar cell proliferation
directly, we performed a BrdU-labeling experiment in E15.5 embryos,
administering BrdU to pregnant females 2 hours prior to sacrifice.
Double-immunofluorescence staining for amylase and BrdU showed an 25%
rate of proliferating acinar cells in wild type and only about 5% in
PMycKO embryos after 2 hours of BrdU labeling
(Fig. 3E-G).
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cells,
insulin-containing β cells, somatostatin+ 
cells and
pancreatic polypeptide+ (PP) cells, in Myc-deficient
embryos (Fig. 4D,F,H,J).
Although no mosaic pattern of recombination was apparent by X-gal staining
of acinar cells in pancreatic section from Ptf1a-cre(ex1);R262R and
PMycKO;R26R mice at E18.5, the endocrine cells were only partially
positive for X-gal in both pancreata (Fig.
4K-P). To quantify the endocrine cell mass in PMycKO and
wild-type pancreata, we estimated the hormone-positive area per total
pancreatic area in multiple pancreatic sections. The relative cell areas of
β and cells were not significantly altered in PMycKO
pancreata (Fig. 4Q,R).
Conclusions
The generation of PMycKO mice via a Cre-mediated conditional
gene-targeted deletion strategy allowed us to define the role of Myc
during pancreatic development. Pancreata lacking Myc were smaller
than those of wild-type embryos, with dramatically fewer differentiated acinar
cells. This effect can be attributed mainly to a decrease in the proliferation
rate of early progenitors and partially to premature differentiation of these
cells to endocrine cells.
Although an accelerated premature progenitor cells conversion into
endocrine cells as a predominant effect has been observed in mice with
modulation of the Notch signaling pathway, this mechanism is not predominantly
involved into the reduced acinar cell mass in Myc-deficient
pancreata, as we observed only about 20% fewer Ngn3+ endocrine
precursor cells but a 40% reduction in proliferating epithelial cells in
PMycKO pancreata. Thus, the main reason for the impaired development
of the exocrine compartment in PMycKO pancreata is the reduced
ability of epithelial and acinar precursor cells to proliferate. The ability
of Myc to promote cell-cycle reentry is, in part, due to its ability
to directly induce the transcription of Cdk4
(Hermeking et al., 2000). The
loss of CDK4 expression in Myc-deficient pancreata reflects a direct
effect of Myc on progenitor and nascent acinar cell proliferation,
although no pancreatic exocrine phenotype was reported in mice lacking
Cdk4 (Mettus and Rane,
2003; Rane et al.,
1999). Thus, besides the loss of CDK4 expression, other
Myc-dependent factors probably contribute to the impaired proliferation. In
contrast to CDK4, the expression of cyclin D1 in PMycKO embryos was
not affected, suggesting that Myc-deficient epithelial cells do not
have a general defect in their mitogenic signaling cascades. Furthermore, the
expression of the tissue-specific transcription factor PTF1a in
Myc-deficient acinar precursor cells suggests that Myc is
required for expansion of exocrine cells, rather than for development of
progenitor into acinar cells.
Our findings are in agreement with those of other groups, who noted a
severe loss of exocrine pancreatic tissue following conditional ablation of
β-catenin (Dessimoz et al.,
2005; Murtaugh et al.,
2005; Wells et al.,
2007). Our results suggest that the loss of Myc
expression in β-catenin-deficient pancreata may be the causal defect,
leading to severe acinar hypoplasia. Additional support for Myc as a
main β-catenin target gene in the embryonic pancreas comes from a recent
study in which the effect conditional deletion of adenomatous polyposis coli
(Apc) was investigated (Strom et
al., 2007). Loss of Apc led to an accumulation of
β-catenin and to hyperplasia of acinar cells in adult
Apc-deficient pancreata accompanied by increased Myc expression. As
acinar hyperplasia could be reversed by additional conditional inactivation of
Myc, these results suggest that Myc is responsible for
acinar cell hyperplasia mediated by β-catenin. However, accumulation of
β-catenin was observed only postnatally, despite embryonic inactivation
of Apc and, consequently, reversal of the phenotype by Myc
ablation was reported in adult pancreata. In this study, no phenotype upon
deletion of Myc alone was reported. Our results are the first to
report the essential role of Myc during early pancreatic development
and substantiate a functional Wnt/β-catenin/Myc axis not only in the
adult but also in the embryonic exocrine pancreas.
Interestingly, inactivation of Myc did not affect development of
the endocrine pancreas, similar to results observed in pancreata that lack
β-catenin (Murtaugh et al.,
2005; Wells et al.,
2007). Although it has been reported that Wnt/β-catenin
signaling regulates pancreatic β-cell proliferation
(Rulifson et al., 2007), we
did not find a decrease in β cell numbers in Myc-deficient
pancreata. A reason for this could be the only partial activity of
Ptf1a-cre observed in endocrine progenitors from our
Ptf1a-cre(ex1) knock-in mouse. It is conceivable that the decreased
proliferation of Myc-deficient endocrine progenitors would have been
masked by the proliferation of Myc-expressing endocrine progenitor cells.
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ACKNOWLEDGMENTS
We are grateful to Moreno de Alboran and Klaus Rajewsky for generous gift of Myc floxed mice, and to Christopher V. Wright and Raymond J. MacDonald for PDX1 and PTF1a antibodies, respectively. We also thank Dr Brill, Dr Henke, Mrs Bergmeier and her colleagues at the Animal Research Facility of the Klinikum rechts der Isar for advising and supporting us. This work was supported in part by the Deutsche Forschungsgemeinschaft to R.M.S. (SFB 567).
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