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First published online 17 July 2008
doi: 10.1242/dev.013722
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1 Department of Internal Medicine, Technical University of Munich, Ismaniger
Strasse 22, 81675 Munich, Germany.
2 Institute of Immunology, Friedrich-Loeffler Institut, Paul-Ehrlich Strasse 28,
72076 Tuebingen, Germany.
3 ISREC, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.
4 GSF-National Research Center for Environment and Health, Institute for
Clinical Molecular Biology and Tumor Genetics, Marchioninistrasse 25, 81377
Munich, Germany.
Author for correspondence (e-mail:
roland.schmid{at}lrz.tum.de)
Accepted 17 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Notch, Rbpj, Conditional knockout mice, Pancreas, Development
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Fujikura et al., 2006;
Fujikura et al., 2007;
Jensen et al., 2000). While
these studies have provided evidence for an important role of Notch signaling
in endocrine development, the dependence of the exocrine compartment on
specific Notch signaling members is not well understood. Recently,
RBPJ
, the transcriptional mediator of Notch signaling, was found to be
a binding partner of PTF1A in the PTF1 complex, suggesting a Notch-independent
function during pancreatic development
(Beres et al., 2006;
Masui et al., 2007). Moreover,
because of the existence of multiple receptors, ligands and target genes, and
because of the embryonic lethality of mice null for Notch signaling
components, the precise role of individual Notch signaling components in early
and late pancreatic organogenesis is not well defined. During pancreatic
organogenesis, Notch1 and Notch2 expression has been described in the
pancreatic epithelium, whereas Notch3 and Notch4 are expressed in mesenchymal
and endothelial cells (Lammert et al.,
2000).
In order to clarify the role of the epithelially expressed receptors Notch1
and Notch2 versus the abrogation of RBPJ signaling, we analyzed
conditional Notch1/Notch2 double-knockout and Rbpj knockout
mice by using Ptf1a+/Cre(ex1) mice for targeting
pancreatic progenitor and exocrine cells.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-DASHII phage library (Stratagene) and subcloned into pBluescript SK+
(Stratagene). A neomycin resistance cassette, flanked by two loxP
sites, was inserted into intron 7, and a single loxP site was
integrated into intron 5. A herpes simplex virus thymidine kinase (HSV-tk)
cassette was cloned at the 5' end of the gene-targeting construct
(Fig. 1A). The
NotI-linearized vector was transfected into murine 129/SvJ ES cells,
where it recombined with the host genome. The homologous recombination event
occurred at a frequency of 1:602 and was verified by PCR and Southern blot
analysis. The floxed neo-resistance cassette was removed by transient
transfection of a vector expressing the Cre-recombinase. Blastocyst injection
and germline transmission of the mutant allele were done as described
previously (Tanigaki et al.,
2002). Genotyping of mice was performed on DNA isolated from tail
biopsies using a PCR kit (Qiagen). For the detection of floxed (0.55 kb
fragment) and wild-type (0.5 kb) alleles of Rbpj, PCR amplification
(1 minute at 94°C, 30 seconds at 58°C and 30 seconds at 72°C, for
40 cycles) was carried out using primers 5'-AGT TTA GGC TTT CCA AAA
GGC-3' (forward) and 5'-GTA TTG CTA AGA ACT TGT TGC-3'
(reverse). All mice were housed in pathogen-free conditions. All mouse
protocols were approved by the Centre of Animals Research, the Faculty of
Medicine, Technical University of Munich.
X-gal staining
β-gal activity was determined on whole-mount preparations as described
previously (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
intraperitoneally into pregnant mice 2 hours before sacrifice.
Histology and immunohistology
Dissected tissues were fixed in ice-cold 4% paraformaldehyde,
paraffin-embedded and cut into 2-3 µm sections. Immunohistochemistry was
performed using the following primary antibodies: rabbit anti-PTF1A (1:500,
kind gift from Raymond J. Macdonald, University of Texas, USA); rabbit
anti-PDX1 (1:10,000, kind gift from C. V. Wright, Vanderbilt University
Medical Center, Nashville, USA); guinea pig anti-insulin (1:1000, Linco);
rabbit anti-glucagon (1:1000, Linco); guinea pig anti-glucagon (1:500, Linco);
rabbit anti-β-galactosidase (1:500, ICN); rabbit anti-somatostatin
(1:1000, ICN); rabbit anti-pancreatic polypeptide (1:500, BioTrend); rabbit
anti-amylase (1:1000, Sigma) rabbit anti-phosphohistone H3 (1:500, Upstate);
rabbit anti-carboxypeptidase A (1:500, BioTrend); mouse anti-CK19 [1:500,
Developmental Studies Hybridoma Bank (DSHB), University of Iowa]; rabbit
anti-HES1 (1:100, kind gift from T. Sudo, Toray Industries, Tokyo, Japan);
rabbit anti-cyclin B1 (1:500, Millipore); rat anti-BrdU (1:250, Serotec);
mouse anti-Neurogenin (1:500, DSHB). For immunoperoxidase detection,
Vectastain ABC kit (Vector Labs) was used according to the manufacturer's
instructions. 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). The number of islets was calculated,
with the definition of an islet being a group of β cells surrounded by
cells. 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 neurogenin 3-positive
cells, whole pancreatic buds from three control and three knockout embryos
were cut into 3 µm serial sections. Every fifth section was stained and the
number of PHH3+, neurogenin 3+ and insulin+
cells were counted and calculated relative to the whole area of
PDX1+ pancreatic epithelium in every section
Laser capture microscopy
Acini and pancreatic buds were dissected from 5- to 6-µm sections using
a Leica and P.A.L.M microlaser system, respectively. Cells were incubated
overnight at 37°C in 20 µl of TE buffer [1 mM EDTA, 20 mM Tris (pH 8)]
containing 0.5 mg/ml proteinase K, after which the proteinase K was heat
inactivated by incubation at 95°C for 15 minutes. For detection of the
floxed (0.33 kb) and deleted (0.30 kb) alleles of Notch1, PCR
amplification (94°C for 20 seconds, 55°C for 30 seconds, and 72°C
for 30 seconds, for 40 cycles) was performed using primers P1 (5'-AAC
TGA GGC CTA GAG CCT TGA AG-3'), P2 (5'-GTG GTC CAG GGT GTG AGT GTT
C-3') and P3 (5'-ACC TGT TCG CAG GCA TCT CCA G-3'). Floxed
(0.29 kb) and deleted (0.37 kb) alleles of Notch2 were detected using
primers P4 (5'-GGA GAA GCA GAG ATG AGC AGA TGG-3'), P5
(5'-CAC ATG TGC GTG CGT GTG CAT G-3'), P6 (5'-CAG AGA TGA
GCA GAT GGG CAT A-3') and P7 (5'-GAG GCC AGA GGA CGA CTC
TGT-3'). For Rbpj, a 2-kb (floxed) and a 0.75-kb (deleted)
fragment were obtained using primers P8 (5'-TAT TGC TAA GAG CTT GTT
GC-3') and P9 (5'-ACT GAG TGT GTA TCT TAA GC-3').
Western blot analysis
Whole-cell lysates were prepared and western blots were performed as
previously described (Siveke et al.,
2007).
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DISCUSSION
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). For
analysis of Cre recombinase expression, Ptf1a+/Cre(ex1)
mice were crossed to Rosa26RlacZ (R26R) reporter
mice (Soriano, 1999). In
pancreata of newborn mice, Cre-induced recombination measured by X-gal
staining occurred as expected from previous studies
(Kawaguchi et al., 2002) (data
not shown). To abrogate Notch receptor signaling, an approach generating
either Rbpj or Notch1/Notch2 double-knockout mice was
chosen. For simplicity, Ptf1a+/Cre(ex1);Rbpjf/f
will be termed RbpjKO and
Ptf1a+/Cre(ex1);Notch1f/f;Notch2f/f
will be termed Notch1/2KO mice; heterozygote littermates and
littermates not expressing Cre recombinase will be termed
Rbpj+/-, Notch1/2+/- and WT mice,
respectively.
To generate pancreas-specific, Rbpj-knockout mice, loxP
sites were inserted flanking exons 6 and 7 of Rbpj in embryonic stem
cells (ES) by homologous recombination
(Fig. 1A). ES cell clones with
a floxed (f) Rbpj locus were used to generate
chimeric mice. The mutant mouse line (Rbpj+/f) was
established through germline transmission. Cre-recombinase-mediated deletion
of exon 6 and 7 of Rbpj resulted in a mutant RBP-J protein
lacking a functional DNA-binding domain. By crossing
Rbpjf/f mice with a Nestin-Cre deletor line, we were not
able to obtain Rbpj-deficient newborns, and this provides strong
evidence for a functionally null Rbpj transcript after Cre-induced
recombination (data not shown). For pancreas-specific targeting, we next
crossed Rbpjf/f mice with
Ptf1a+/Cre(ex1) mice. Ptf1a+/Cre(ex1)
mediated deletion of the Rbpj gene was verified by Southern blot
analysis with DNA from different tissues of newborn
Ptf1a+/Cre(ex1);Rbpj+/f offspring
(Fig. 1B). For conditional
knockout of Notch1 and Notch2, previously described
Notch1f/f and Notch2f/f mice were used
(Radtke et al., 1999;
Schouwey et al., 2006). As we
did not find qualitative defects in pancreatic organogenesis, nor major
abnormalities in the unstimulated adult pancreata of conditional Notch1,
Notch2 or combined Notch1/Notch2 knockout mice over an
observation period of 18 months (Siveke et
al., 2008) (data not shown), combined Notch1/2KO were
chosen for the analysis of pancreatic development. In Notch1 and
Notch2 single receptor knockouts, as well as in double-knockout
pancreata, Notch1 and Notch2 protein and transcripts were decreased to less
than 10%, as analyzed by western blot and RT-PCR (data not shown). Both,
Rbpj and Notch1/Notch2 knockout lines were tested for the
possibility of mosaic Cre-induced recombination using Rosa26R
reporter mice as a surrogate for recombination-induced deletion of
Rbpj or Notch1/Notch2, respectively. Using X-gal staining,
we did not observe X-gal-negative exocrine cells in adult pancreata
(Fig. 1G-I). Regarding
recombination in the endocrine compartment, we found approximately 50% of
endocrine cells to be X-gal positive, a recombination pattern that was very
similar to that observed in
Ptf1a+/Cre(ex1);Rosa26RlacZ pancreata
(Fig. 5M-U).
Although heterozygous Rbpj+/- and homozygous Notch1KO, Notch2KO and Notch1/2KO mice showed no gross abnormalities and developed normally, RbpjKO mice survived only until 4-5 days postpartum. Although moderate signs of growth retardation were observable at birth (Fig. 1C), the early death was caused by insufficient postnatal growth with impaired milk digestion. We were able to raise some RbpjKO mice to adulthood by feeding them with pancreatic enzyme-enriched animal food (data not shown). Examination of the RbpjKO;R26R mice at day 1 postpartum (dpp) revealed a small and severely altered pancreas (Fig. 1F,I). In the duodenal part of the mutant pancreas, weakly branched ducts were observable (Fig. 1F, arrowhead), whereas the splenic part of the pancreas showed no branching (Fig. 1F, arrows). Histological examination demonstrated a lack of acinar tissue with large duct-like structures being present in the splenic and duodenal portion of the pancreas (Fig. 1I, blue). Interestingly, Notch1/2KO mice did not reveal striking abnormalities in pancreatic tissue organization or cell lineage distribution, suggesting a non-essential role for Notch1 and Notch2 during pancreatic development. However, the mutant pancreas was noted to be slightly smaller than that of wild type when analyzed at 1 dpp (Fig. 1 E,H). To further clarify the role of ablated Notch signaling, early stages of pancreatogenesis were investigated.
Early pancreatic development in Rbpj- and Notch1/Notch2-deficient pancreata
To analyze pancreatic development at defined stages of pancreatic
organogenesis, we investigated pancreatic bud development at embryonic day 10
(E10) to E13.5 by immunohistochemistry and X-gal staining. The pancreatic buds
of E13.5 Rbpj+/-;R26R and Notch1/2KO;R26R embryos
displayed the typical branching of the pancreatic epithelium. The
Notch1/2KO buds appeared smaller and less branched than in control
littermates (Fig. 2A,B,D). By
contrast, RbpjKO;R26R embryos revealed a significantly reduced
epithelial mass with weakly branched structures in both buds
(Fig. 2C,D), suggesting that
Rbpj is essential for the expansion of the pancreatic epithelium.
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pancreatic sections at E11.5 were stained for X-gal and glucagon expression.
In Rbpj+/-;R26R embryos, glucagon-expressing cells formed
small clusters peripheral to and dispersed within the dorsal bud
(Fig. 2H, brown). Similar to
Rbpj+/-;R26R mice, Notch1/2KO mice showed no
increased number of glucagon-positive cells
(Fig. 2I, brown). By contrast,
we observed an increased number of glucagon-positive cells in
RbpjKO;R26R embryos at this time point, consistent with the premature
differentiation of pancreatic progenitors to endocrine cells. These cells were
found within and peripheral from the ventral and dorsal buds
(Fig. 2J, brown). As expression of the transcription factor neurogenin 3 (Ngn3) is a prerequisite for endocrine lineage development, E13.5 pancreata were analyzed for expression of NGN3. Consistent with previous results, we found decreased numbers of NGN3+ cells in RbpjKO mice at this stage (Fig. 2M,N), when compared with Rbpj+/-;R26R embryos (Fig. 2K,N), suggesting an early commitment of these cells to endocrine cell lineages. We also found more insulin+ β cells per PDX1+ area in RbpjKO pancreata (Fig. 2Q,R). Regarding the differentiation of endocrine cells in Notch1/Notch2-deficient pancreata, no significant reduction of NGN3+ cells (Fig. 2L,N) and no related β cell increase were notable at E13.5 (Fig. 2P,R). The reduced branching and epithelial mass in the Notch1/2KO and RbpjKO embryos was accompanied by a decrease in the number of proliferating cells in pancreatic epithelium, as detected by phospho-histone H3 (PHH3) and PDX1 double-immunostaining (Fig. 2S-U). While the relative number of the PHH3+ cells to PDX1+ cell area in Notch1/2KO buds was reduced by 25% in comparison with control mice (Rbpj+/-;R26R; Fig. 2S,T,V), the relative number in RbpjKO buds was decreased to 40% (Fig. 2U,V).
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In RbpjKO mice at E18.5, co-immunostaining for PDX1 and amylase showed that the majority of amylase+ cells were also PDX1+, and were mitotically active, as determined by BrdU labeling (Fig. 4M,N). Because a functional PTF1 complex is required for the expression of acinar genes, such as amylase, we determined the expression of PTF1A in RbpjKO pancreata. Here, we detected PTF1A+ cells surrounded by stromal cells outside the main duct and in the duodenal part of the rudimentary pancreas (Fig. 4O, arrows). To determine whether the Rbpj gene was actually deleted in amylase-expressing cells in the mutant pancreas, pancreatic sections from RbpjKO newborns were co-stained for X-gal and amylase, demonstrating the Cre-induced recombination of amylase+ cells (Fig. 4P). In addition, PCR analysis of DNA isolated from amylase+ cells by microdissection confirmed that the Rbpj gene was deleted in acinar cells from RbpjKO newborns (Fig. 4Q).
Endocrine cell development in Rbpj- and Notch1/Notch2-deficient pancreas
Most mature endocrine cells appeared after E14 in both Notch1/2KO
and RbpjKO embryos, similar to in littermate controls. At E18.5, we
could detect all endocrine cell lines, glucagon-producing cells,
insulin-containing β cells, somatostatin+ 
cells, and
pancreatic polypeptide+ (PP) cells in both Notch1/2KO and
Rbpj+/- embryos. In RbpjKO mice, these cells were
detectable in the rudimentary pancreas within the tubular duct wall and in the
protruding formations of the pancreatic tubule
(Fig. 5A-L).
At E18.5, most endocrine cells of the Rbpj+/- control
embryos aggregated with cells starting to organize around core
structures of β cells (Fig.
5P, arrowhead). Similar to control embryos, endocrine cells in
both Notch1/2KO and RbpjKO embryos also started to
aggregate; however, the number of formed islets was less than in control
embryos (Fig. 5V). Regarding
the morphological appearance of the formed islets, the endocrine epithelium in
Notch1/2KO embryos, and more prominently in RbpjKO embryos,
had a disturbed appearance. In most of the endocrine cell formations,
cells were not organized around β cells, and the morphology of these
islet-like structures appeared to be long rather than circular like in the
control mice (Fig. 5P,Q,R). In
adult pancreata of Notch1/2KO mice, however, the islets appeared
normal and were indistinguishable from wild-type controls (data not
shown).
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Recently, two different mouse models for conditional genetic inactivation
of Rbpj were described, using either transgenic Pdx1-Cre or
Ptf1a-Cre knockin mice for the targeting of pancreatic progenitor
cells (Fujikura et al., 2006;
Fujikura et al., 2007). During
early pancreatic development, both models, and our RbpjKO mice,
revealed an essential role for Rbpj with premature
glucagon+ cell development, a severe decrease in acinar cell
differentiation and disturbed ductal branching in mutant mice. Differences in
the phenotypical severity between these models during the early stages of
organogenesis are possibly due to differences in the onset, timing and rate of
Cre-induced genetic inactivation. In our Ptf1a+/Cre(ex1)
mice, X-gal staining in the buds was observable at E10.5 in all pancreatic
epithelial cells, similar to that previously reported
(Fujikura et al., 2007;
Kawaguchi et al., 2002).
However, even slight differences in targeting efficiency during early
pancreatic organogenesis in transgenic Pdx1-Cre mice, and between
different Ptf1a-knockin Cre lines, may have a large impact on the
development of the respective cell compartments.
Of interest is the appearance of late exocrine cells in all models;
however, phenotypical effects are notable. In our model, RbpjKO mice
do not survive more than 4-5 days after birth, most probably as a result of
the clinically apparent pancreatic insufficiency with impaired weight gain, a
high content of milk in the stomach of animals and no apparent neurological
phenotype. We favor this explanation over, for example, extra-pancreatic
causes, as we could not detect any defects in other PTF1A-expressing organs,
such as the retina or the CNS (data not shown). The reason why our
RbpjKO mice are not able to show the same ability to develop an
apparently normal adult exocrine compartment is not clear, but may possibly be
explained by a more rigorous deletion of early progenitors in our mice.
Nevertheless, the late appearance of acinar cells during organogenesis in our
and other Rbpj-deficient pancreata
(Fujikura et al., 2006;
Fujikura et al., 2007) is
surprising, and may occur through Rbpj-independent mechanisms
involving a recently identified regulator of acinar cell development, the
Rbpj homolog Rbpjl (Beres
et al., 2006). These authors showed that the initiation of the
acinar differentiation program by the PTF1 complex involves RBPJ
binding to PTF1A to form the PTF1-J complex. This complex then activates
RBPJL, which itself binds to PTF1A to form the PTF1-L complex. PTF1-L has been
shown to be the more active complex, activating acinar genes such as amylase
and elastase (Beres et al.,
2006). The finding of delayed expression of acinar genes such as
amylase at E18.5 in RbpjKO mice may be explained by two mechanisms.
First, Cre activation may not be complete in a few proacinar cells, which will
eventually form the exocrine pancreas. However, our results showing
Cre-induced recombination of both Rbpj alleles in microdissected
acini and in adult pancreatic tissue
(Fujikura et al., 2007) do not
support this hypothesis. Secondly, spontaneous activation of Rbpjl in
precursor cells expressing PTF1A may lead to the formation of PTF1-L and,
thus, to a positive-feedback loop activating the Rbpjl promoter. The
delayed appearance and the small initial population of acinar cells would be
consistent with a stochastic activation of Rbpjl, a hypothesis as yet
unproven however.
The defective ductal branching observed in our, as well as in other, models
of Notch signaling ablation may be due to early reduction of the epithelial
progenitor pool, as has been suggested previously
(Fujikura et al., 2006;
Fujikura et al., 2007).
Interestingly, the ductal cells in RbpjKO and Notch1/2KO
mice expressed CK19, suggesting that the differentiation of progenitor cells
into ductal cells is not inhibited by inactivated Rbpj-dependent
Notch signaling. Future studies may help to determine the factors regulating
ductal differentiation in the pancreas.
Of unclear significance is our finding of amylase positivity in dorsal duct structures of RbpjKO pancreata. Although we detected amylase+ cells within the ducts, we did not detect expression of PTF1A in these cells (data not shown); however, expression of these acinar transcription factors may be below detection limits. Other explanations include artificial staining of amylase produced by extraductal acinar cells; however, we did not observe acini in the dorsal part of the organ. Whether or not inactivated Notch signaling contributes to acinar cell fate determination from ductal cells or within ductal structures needs to be determined.
Interestingly, we found differences between Notch1/Notch2 and
Rbpj ablated mice regarding the severity of impaired pancreatic
development. Whereas the buds of Notch1/2KO mice appeared smaller
than those of wild-type littermates, this reduction did not reach the extent
of that seen in RbpjKO mice. The reason for the reduced proliferation
of pancreatic progenitors may be due to the requirement of Notch signals for
the maintenance of actively proliferating pancreatic progenitor cells, as has
recently been shown for the transcription factor Sox9
(Seymour et al., 2007). In
this regard, we also found reduced, but not completely abolished, expression
of HES1 in Notch1/2KO and RbpjKO mice, as has been noted by
others (Fujikura et al., 2006;
Fujikura et al., 2007),
possibly as a result of the expression of factors such as Sox9, which
is necessary for the maintenance of HES1 expression. Studies with ectopic
overexpression of Notch1 showed the prevention of exocrine and endocrine
differentiation of pancreatic progenitor cells, leaving them in an
undifferentiated state (Esni et al.,
2004; Hald et al.,
2003; Murtaugh et al.,
2003). Despite technical issues, such as the transgenic expression
and potentially non-physiological Notch1 levels, these results, as well as the
aforementioned studies, point to a role for Notch in the regulation of
pancreatic progenitor cells, with one of the main conclusions being a
premature endocrine switch caused by insufficient Notch signaling.
Interestingly however, we found such a switch in RbpjKO but not
Notch1/2KO mice, possibly indicating the requirement of Rbpj
but not Notch1 or Notch2 for the regulation of premature
endocrine differentiation. However, we cannot rule out inefficient early
Cre-induced inactivation of Notch1 and Notch2. While we
could determine successful recombination of both Notch1 and
Notch2 alleles at E12.5 by PCR of microdissected epithelial cell,
incomplete earlier inactivation of both Notch genes might indeed be
responsible for the lack of effect in Notch1/2KO mice. The modest
phenotype of Notch1/2KO mice was unexpected and is in contrast to the
skin, where genetic inactivation of Rbpj and
Notch1/Notch2 leads to similar phenotypes
(Schouwey et al., 2006). While
early reports using mice null for Notch signaling family members, such as
Rbpj, Dll1 or Hes1, showed impaired growth and branching
defects (Apelqvist et al.,
1999; Jensen et al.,
2000), differences between null mice and conditional genetic
targeting approaches, such as the inactivation of targeted genes before
pancreatic development is started and the additional targeting of
extra-pancreatic cells in null mice, has a strong impact on the observable
phenotype.
The different impact of pancreatic Notch1/Notch2 and Rbpj
inactivation in our study strongly suggests a Notch-independent role of
Rbpj in pancreatic organogenesis. The near complete absence of acinar
cells until late gestation suggests that RBPJ is required for the
formation of the acinar lineage. Recent studies have shown that RBPJ is
the binding partner of PTF1A for formation of the early PTF1-J complex
(Beres et al., 2006;
Masui et al., 2007;
Obata et al., 2001). Our
results are in line with a Notch-independent role of RBPJ as an
obligate partner of PTF1A to form a functional PTF1 complex, a pivotal event
during early pancreatic development. Thus, RBPJ in Notch1/2KO
mice might still function as PTF1A-binding partner independently of its
transducer role in the Notch signaling pathway.
As expected from previous reports
(Apelqvist et al., 1999;
Jensen et al., 2000),
RbpjKO mice had earlier cells underscoring the relevance of
Notch signaling for the inhibition of premature differentiation of progenitor
cells into early cells. Our finding of less NGN3+ cells in
RbpjKO mice is similar to the results by Fujikura and colleagues
(Fujikura et al., 2006;
Fujikura et al., 2007), and
suggests that these endocrine progenitor cells are also compromised and forced
into premature differentiation by Rbpj deficiency. Notably,
Notch1/2KO mice revealed no significant decrease in NGN3+
cells and several mechanisms may account for this finding. First, RBPJ
might be activated independently and might lead to the activation of target
genes; second, the four Notch1 and Notch2 alleles might not
be inactivated in a timely manner to preserve the progenitor pool; or third,
the transition of the repressor into an activator state of RBPJ may be,
at least partially, Notch independent. Despite the premature differentiation
of pancreatic progenitors in Rbpj-deficient mice, we found that all
endocrine lineages develop in RbpjKO and Notch1/2KO mice,
consistent with the hypothesis of a dispensable role of Notch signaling in
late pancreatic development. However, we found fewer islets in both knockout
lines, which, more prominently in RbpjKO mice, had a partially
disturbed composition. One explanation for the development of endocrine cells
despite the genetic inactivation of Notch signaling is the later expression of
Cre recombinase in Ptf1a+/Cre(ex1) compared with in
Pdx1-Cre mice at E10.5. However, development of the endocrine
compartment and islet formation does not occur before E13.5, a time point at
which Rbpj and both Notch genes are inactivated. Our finding of
X-gal+ islets composed of all endocrine lineages at E18.5 in both
knockout lines is evidence for a non-essential role of Notch signaling in
promoting endocrine cell fate determination and differentiation, whereas the
lower amount of islets and the somewhat disturbed islet morphology, especially
in RbpjKO mice, may be an at least partial result of the severe
branching defect in these mice.
In conclusion, we demonstrate an essential role of Rbpj, but not of Notch1 and Notch2, in pancreatic organogenesis. Using a concomitant approach of Notch signaling inactivation, we show that the epithelially expressed Notch receptors 1 and 2 are not essential for pancreatic development, whereas lack of Rbpj leads to premature differentiation of pancreatic progenitors and a decrease in endocrine progenitor cells. During late pancreatic development, however, differentiated exocrine and endocrine lineages mature in both knockout lines. Although Rbpj seems to be an important regulator of the early pancreatic progenitor pool, our findings strengthen the hypothesis of as yet unknown and potentially Rbpj-independent mechanisms regulating the cell fate of adult pancreatic cell lineages. As we can show successful inactivation of Notch1 and Notch2 alleles at E12.5, this finding strongly suggests that these receptors, but not Rbpj, are dispensable for exocrine and endocrine development. Thus, at least in the pancreas, a Notch-independent role of Rbpj during development seems to be a likely mechanism.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Apelqvist, A., Li, H., Sommer, L., Beatus, P., Anderson, D. J.,
Honjo, T., Hrabe de Angelis, M., Lendahl, U. and Edlund, H.
(1999). Notch signalling controls pancreatic cell
differentiation. Nature
400,877
-881.[CrossRef][Medline]
Beres, T. M., Masui, T., Swift, G. H., Shi, L., Henke, R. M. and
Macdonald, R. J. (2006). PTF1 is an organ-specific and
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