1 Department of Surgery, Johns Hopkins University School of Medicine, Baltimore,
MD 21287, USA
2 Department of Oncology, Johns Hopkins University School of Medicine,
Baltimore, MD 21287, USA
3 Department of Molecular Biology, University of Texas Southwestern Medical
Center, Dallas, TX 75390-9148, USA
4 Unitat de Biologia Cellular i Molecular, Institut Municipal
d'Investigació Mèdica, Universitat Pompeu Fabra, 08003
Barcelona, Spain
5 Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104,
USA
6 Department of Cell and Developmental Biology, University of Pennsylvania,
Philadelphia, PA 19104, USA
Author for correspondence (e-mail:
stleach{at}jhmi.edu)
Accepted 25 May 2004
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SUMMARY |
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Key words: Zebrafish, Mouse, Pancreatic, Exocrine, Embryo, p48
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Introduction |
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The mammalian pancreas is comprised of endocrine and exocrine lineages,
both derived from a common progenitor pool in foregut endoderm
(Gu et al., 2003;
Gu et al., 2002
;
Kawaguchi et al., 2002
;
Kim and MacDonald, 2002
).
Based on studies involving targeted inactivation of Notch pathway components
in the mouse, it appears that Notch is required to prevent excessive precursor
commitment to the endocrine lineage
(Apelqvist et al., 1999
;
Jensen et al., 2000b
).
However, these gene targeting studies have been less informative with respect
to a possible additional role for Notch in regulating exocrine lineage
commitment. More recent studies have suggested that ectopic Notch activation
may prevent acinar cell differentiation during murine pancreatic development
(Hald et al., 2003
;
Murtaugh et al., 2003
), and
also induce dedifferentiation of exocrine cell types in adult pancreatic
epithelium (Miyamoto et al.,
2003
). However, it remains to be determined whether these
observations reflect a similar role for endogenous Notch pathway activation
during development, and the mechanism for this effect remains unknown. In the
current study, we find that Notch signaling is indeed active within a
committed exocrine progenitor pool in developing mouse pancreas, and further
demonstrate that Notch blocks terminal acinar cell differentiation, but not
initial commitment to the exocrine lineage. In addition, we take advantage of
altered lineage relationships in developing zebrafish pancreas to demonstrate
a biologic role for endogenous Notch in developing exocrine pancreas.
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Materials and methods |
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Double in situ hybridization on tissue sections
For double fluorescent in situ hybridizations, embryonic mouse pancreas was
fixed in 4% PFA and embedded in OCT. Sections were hybridized simultaneously
with fluorescein-labeled Hes1 and digoxigenin-labeled Ngn3
riboprobes. Hybridized probes were sequentially detected using AP-conjugated
anti-fluorescein antibody visualized with Fast Red (Roche), followed by a
30-minute postfixation in 4% PFA and inactivation of anti-fluorescein-AP by
acid quenching with 100 mM Glycine-HCl pH 2.2, 0.1% Tween-20. Following
additional washes, sections were incubated with AP-conjugated anti-digoxigenin
antibody, which was then visualized using ELF®97 In Situ Hybridization
Kits (Molecular Probes). Probes against Hes1 and Ngn3 were
kindly provided by Mahendra Rao (National Institute on Aging) and Douglas
Melton (Harvard University).
Construction of dual promoter lentiviral vectors and lentivirus production
cDNAs encoding mHes1 (gift from Mary Hatten, Rockefeller University)
(Solecki et al., 2001) and
hNotch1-ICD (gift from Warren Pear, University of Pennsylvania) were cloned
into the EF.v-CMV.GFP lentiviral vector
(Yu et al., 2003
) and sequence
verified. Lentivirus production and concentration was performed as previously
described (Yu et al., 2003
).
Briefly, VSV.G-pseudotyped recombinant lentiviruses were produced by transient
transfection of the transducing vector into 293T cells, along with two
packaging vectors: pMD.G, a VSV.G envelope-expressing plasmid, and
pCMV
R8.91, containing the HIV-1 gag/pol, tat and
rev genes (1.5 µg: 2.0 µg: 0.5 µg ratio of these three
vectors). Viral supernatants were collected at 24, 48 and 72 hours after
transfection, and concentrated using filtration columns (Centricon Plus-20,
molecular weight cutoff=100 kD; Millipore, Bedford, MA, USA). Additional
lentiviral vector validation was accomplished by transduction of COS7 cells,
with Hes1 protein expression confirmed by immunofluorescence and Notch1-ICD
protein expression confirmed by both immunofluorescence and by activation of a
Notch/Su(H)-responsive luciferase reporter (see Fig. S1 at
http://dev.biologists.org/supplemental).
Explant culture of embryonic pancreas and lentiviral transgene gene delivery
Isolation, infection and culture of pancreatic rudiments were performed as
previously described (Ahlgren et al.,
1996; Esni et al.,
2004
). Prior to infection, intact dorsal pancreatic buds were
dissected and incubated for 1 minute at room temperature in 0.6 U/ml dispaseI
(Roche). After separating the epithelium from the surrounding mesenchyme,
naked buds were submerged in 200 µl culture medium (BioWhittaker Medium
199, 10% fetal calf serum, 50 U/ml penicillin G-streptomycin, 1.25 µg/ml
fungizone) and infected with appropriate lentiviral vectors overnight at
37°C in the presence of 8 µg/ml polybrene. For each E10.5 dorsal
epithelial bud, 1x106 transfection units were utilized.
Infected buds were then transferred onto Millicell-CM 0.4 µm inserts,
recombined with E11.5 dorsal mesenchyme and cultured for six additional days.
In the course of five independent experiments, a total of 20 dorsal bud
explants were successfully infected, recombined with mesenchyme, and cultured
(n=7 for GFP alone, n=6 for GFP;NotchIC, n=7 for
GFP;Hes1). Following harvest, cryosections were prepared and stained for
E-cadherin in combination with either insulin (examined on sections of five
buds infected with GFP alone, four buds infected with GFP;NotchIC, and five
buds infected with GFP;Hes1), glucagon (examined on sections of five buds
infected with GFP alone, four buds infected with GFP;NotchIC, and five buds
infected with GFP;Hes1), amylase (examined on sections of seven buds infected
with GFP alone, six buds infected with GFP;NotchIC, and seven buds infected
with GFP;Hes1), Ptf1-p48 (examined on sections of three buds infected with GFP
alone, three buds infected with GFP;NotchIC, and three buds infected with
GFP;Hes1), or nestin (examined on sections of three buds infected with GFP
alone, three buds infected with GFP;NotchIC, and three buds infected with
GFP;Hes1). To determine cell-autonomous effects of Notch1-IC and Hes1, the
number of E-cadherin-positive epithelial cells expressing GFP in combination
with insulin, glucagon, amylase, Ptf1-p48 or nestin was determined by direct
counting of multiple bud sections, and expressed as a fraction of the total
number of E-cadherin/GFP-expressing cells. Two-sample Z-tests comparing GFP
versus GFP:Notch1-IC and GFP versus GFP;Hes1 were performed using Stata 8.0
software (StataCorp LP).
Zebrafish stocks and embryo care
Wild-type (Scientific Hatcheries), mibta52b
(Itoh et al., 2003),
hsp70:Gal4, and UAS:notch1a-ICD
(Park and Appel, 2003
;
Scheer et al., 2001
) zebrafish
strains were raised according to standard protocols
(Westerfield, 2000
). Embryos
were raised at 28.5°C in E3 with 0.1 ppm methylene blue until 24 hpf, at
which point they were transferred to E3 with 0.003% phenylthiourea (2 mM) to
inhibit pigmentation and facilitate whole-mount examination. For Notch
gain-of-function analyses, homozygous hsp70:Gal4 females were mated
to UAS:notch1aICD heterozygote males. Notch1aICD
overexpression was induced by transferring embryos to 40°C media for 30
minutes. After in situ hybridization and phenotypic classification, embryos
were genotyped by proteinase K digestion and subsequent PCR for the
UAS:notch1aICD transgene using the following primers:
5'-CATCGCGTCTCAGCCTCAC and 5'-CGGAATCGTTTATTGGTGTCG.
Capped mRNA injection
Full-length, capped Su(H) dominant-negative messenger RNA was
generated by applying the SP6 mMessage kit (Ambion) to MluI-linearized
pCSGSuDN (Lawson et al.,
2001), which encodes a fusion protein comprised of EGFP and a
dominant-negative DNA binding mutant of Xenopus Su(H). The EGFP
coding region from pEGFP-1 (Clontech) was subcloned into the pCDNA3 expression
vector (Invitrogen) to enable generation of control full-length, capped EGFP
mRNA (T7 mMessage, Ambion). Both mRNAs were diluted to a final working
concentration of 100 ng/µL in Danieau's buffer
(Westerfield, 2000
). For
injection, single-cell stage embryos were transferred to a molded agarose
injection dish, and approximately 200 pg of mRNA was microinjected into the
yolk of each embryo. For both constructs, expression was confirmed by GFP
fluorescence beginning at 4-6 hpf and persisting until at least 32 hpf.
Analysis of zebrafish embryos by whole-mount in situ hybridization
Whole-mount in situ hybridization of Danio rerio embryos was
performed as previously described (Lin et
al., 2004). Trypsin, pdx1 and ptf1a-p48 probes
were generated by PCR from adult zebrafish poly-A primed cDNA. Probes for
hhex and GATA6 have previously been described
(Wallace and Pack, 2003
),
while probes for insulin and glucagon were provided by
Victoria Prince (University of Chicago). Embryos were scored for the presence
or absence of ptf1a-p48 and trypsin expression by
whole-mount observation in 75% glycerol. A test for independent proportions
was performed to determine the statistical significance of differences in
expression between control and experimental groups.
Plasmid and adenoviral constructs
Myc epitope-tagged human Notch1-IC and Notch1-IC truncation mutants were
generated by PCR amplification of appropriate fragments followed by in-frame
assembly in pCMV-Tag1 (Stratagene). All constructs were sequence verified and
protein expression confirmed by western blot using anti-myc antisera (Santa
Cruz). A myc-tagged Hes1 cDNA was obtained from Mahendra Rao (National
Institute on Aging). Expression vectors encoding flagtagged Hey1 and Hey2 were
obtained from Larry Kedes (University of Southern California). An expression
vector encoding human E47 was obtained from Michael Chin (Harvard University).
Previously characterized Ptf1-responsive and Su(H)-responsive luciferase
reporter vectors were obtained from from Masashi Kawaichi (Nara Institute) and
Diane Hayward (Johns Hopkins University), respectively. The Ptf1-responsive
luciferase reporter (Ptf1-luc) contains four tandem repeats of the Ptf1
binding site from the rat chymotrypsinogen reporter
(Obata et al., 2001), whereas
the Su(H)-responsive luciferase reporter [Su(H)-luc] contains eight tandem
repeats of the Su(H) binding element
(Hsieh et al., 1996
). The
construction and characterization of adenoviral vectors encoding GFP alone or
in combination with either Notch1-IC, Hes1, Hey1 or Hey2 has been described
previously (Miyamoto et al.,
2003
; Sriuranpong et al.,
2001
).
Cell culture, adenoviral infection and transient transfection
The AR42J rat acinar carcinoma line was cultured in F12 media with 10% FBS
and infected with indicated adenoviral vectors at an MOI of 20:1. COS7 cells
were cultured in modified DMEM medium with 10% FBS. Transfection was performed
in 24-well plates at 90% confluence using Lipofectamine (GIBCO), according to
manufacturer's protocol.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were isolated from AR42J cells using a modification of the
procedure described by Dignam et al.
(Dignam et al., 1983). EMSA
was performed as previously described, using a 32P-labeled, 21-bp
double stranded oligonucleotide (sense strand,
5'-GTCACCTGTGCTTTTCCCTGC-3') spanning the A element of the rat
elastase1 enhancer (Rose et al.,
2001
). For supershift, 1 µl of Ptf1-p48 antiserum was
preincubated with the nuclear extract prior to oligonucleotide addition.
Luciferase assays
COS7 cells were transfected in 24-well plates with 0.5 µg of either the
Ptf1-luc or the Su(H)-luc firefly luciferase reporter plasmids in combination
with indicated expression vectors. In addition, 0.025 µg of the pRL-TK
Renilla luciferase reporter plasmid (Promega) was added to control for
transfection efficiency. Total DNA per transfection was kept constant at 1
µg per well by adding vector DNA as needed. After 24 hours, cells were
harvested using the Promega Dual-Luciferase Reporter Assay System. For each
condition, sequential measurement of firefly and Renilla luciferase was
performed, and normalized luciferase activity reported as the ratio of firefly
to Renilla luciferase activity. The reported data represent the mean results
from three different experiments, each performed in triplicate. In addition,
parallel samples were processed for western blotting using the following
antibodies and dilutions: rabbit anti-rat Ptf1-P48
(Adell et al., 2000), 1:1000;
rabbit anti-E47 (Santa Cruz), 1:1000; mouse monoclonal anti-myc (Invitrogen),
1:5000; goat anti-Notch1-IC (Santa Cruz C20), 1:1000; mouse monoclonal
anti-flag (Sigma) 1:1000.
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Results |
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Notch inhibits both endocrine and exocrine differentiation in developing mouse pancreas
In order to more directly evaluate the ability of Notch to regulate
exocrine differentiation in developing pancreas, we determined the effects of
forced Notch pathway activation in explant cultures of E10.5 dorsal pancreatic
buds. Using replication-incompetent lentiviral vectors encoding either GFP
alone, GFP plus the activated intracellular domain of murine Notch1
(GFP;NotchIC), or GFP plus murine Hes1 (GFP;Hes1), we effectively created
mosaic epithelial buds, in which a subset of cells undergo forced Notch
pathway activation. Infected buds were then recombined with dorsal mesenchyme
and allowed to differentiate in vitro. As demonstrated in
Fig. 2, developing pancreatic
epithelial cells expressing GFP alone were fully capable of completing either
endocrine or exocrine differentiation programs following six days of explant
culture, as determined by immunofluorescent detection of insulin, glucagon,
amylase and Ptf1-p48 in GFP-positive cells
(Fig. 2D,G,J,M). In contrast,
cells infected with either GFP;Hes1 or GFP;NotchIC were unable to
differentiate into insulin-producing beta-cells, but were able to
differentiate into glucagon-producing alpha cells
(Fig. 2E,F,H,I). In order to
quantify this effect, harvested epithelial buds were stained for E-cadherin to
allow identification of GFP-expressing epithelial cells, and the number of
cells expressing GFP in combination with markers of endocrine and exocrine
differentiation was determined by direct counting
(Fig. 3). Following infection
with GFP alone, approximately 9% of GFP-positive epithelial cells were
insulin-positive, compared with <1% following infection with either
GFP;Hes1 or GFP;NotchIC.
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|
Based on these observations, we next examined co-expression of Ptf1-p48 and amylase following infection with either GFP alone, GFP;Hes1 or GFP;NotchIC. In control explants, the vast majority of Ptf1-p48-positive cells completed an acinar cell differentiation program, marked by expression of amylase. In contrast, forced Notch pathway activation resulted in the abnormal accumulation of a Ptf1-p48-positive, amylase-negative population (Fig. 4). Following infection with GFP alone, 83% of all infected Ptf1-p48-positive cells demonstrated positive staining for amylase at the time of explant harvest, compared with 18% and 22% in explants infected with GFP;Hes1 or GFP;NotchIC, respectively. These results were especially striking, with mosaic acini frequently demonstrating inclusion of GFP-positive, Ptf1-p48-positive, amylase-negative cells immediately adjacent to GFP-negative, Ptf1-p48-positive, amylase-positive cells following infection with either GFP;NotchIC or GFP;Hes1 (Fig. 4). Thus, whereas early commitment to the exocrine lineage (marked by persistent Ptf1-p48 expression) is permitted, normal acinar cell differentiation was prevented in the setting of an active Notch pathway.
|
Effects of ectopic Notch activation in developing zebrafish pancreas
In contrast to mammalian pancreas, in which endocrine and exocrine cell
types are initially recruited from a common progenitor pool, zebrafish
pancreas development appears to be characterized by spatially segregated
endocrine and exocrine precursor populations
(Field et al., 2003;
Lin et al., 2004
). Zebrafish
pancreatic tissue is first apparent as bilateral rows of
pdx1-positive, insulin-positive endocrine cells which
coalesce in the midline to form the principal islet by 24 hpf
(Argenton et al., 1999
;
Biemar et al., 2001
;
Huang et al., 2001
). Committed
exocrine progenitors are not apparent at this time, but subsequently appear as
ptf1a-p48-positive cells arising in left lateral endoderm, separated
from developing endocrine pancreas (Lin et
al., 2004
). The relative independence of exocrine and endocrine
precursors in developing zebrafish pancreas has been further clarified by
examination of gene knockdown phenotypes following injection of antisense
morpholinos targeting either ptf1a-p48 or pdx1. ptf1a-p48
morphants fail to develop differentiated exocrine cells, but undergo entirely
normal development of the principal islet. Conversely, pdx1 morphants
display significantly disrupted islet development, even while the initial
expression of ptf1ap48 remains unaffected
(Lin et al., 2004
).
To confirm that Notch pathway activation was similarly capable of
inhibiting exocrine differentiation in developing zebrafish pancreas, we
pursued gain-of-function analysis utilizing transgenic embryos expressing a
heat shock-inducible hsp70:Gal4 transgene, either alone or in
combination with a Gal4-responsive UAS:notch1aICD allele
(Scheer et al., 2001). The
UAS:notch1aICD transgene was induced by embryo transfer to 40°C
media for 30 minutes, and sustained by repeated heat shocks at 10-hour
intervals. In order to determine the effect of ectopic Notch pathway
activation on differentiation of exocrine pancreas, heat shock was initiated
at either 24 hpf (prior to onset of ptf1a-p48 expression) or at 34
hpf (after onset of ptf1a-p48 expression, but prior to onset of
trypsin expression). When heat-shock was initiated at 24 hpf,
hsp70:Gal4 and hsp70:Gal4;UAS:notch1aICD embryos remained
grossly indistinguishable (Fig.
5), and displayed normal development of liver and foregut as
assessed by examination of hhex and GATA6 expression (data
not shown). In contrast, subtle but highly reproducible effects on both
exocrine and endocrine differentiation were observed in the setting of forced
Notch pathway activation. As assessed at 32 hpf, shortly after the normal
onset of ptf1a-p48 expression, 4% (1/23) of
hsp70:Gal4;UAS:notch1aICD embryos demonstrated endodermal expression
of ptf1a-p48, compared with 68% (25/37) in hsp70:Gal4
controls (Fig. 5A,B;
P<0.001). This delay in ptf1a-p48 expression was
short-lived; by 34 hpf, endodermal expression of ptf1a-p48 was
uniformly present in both groups, and remained normal at later time points
(Fig. 5C,D). In spite of this
rapid recovery in ptf1a-p48 expression, the onset of trypsin
expression was significantly delayed in hsp70:Gal4;UAS:notch1aICD
embryos. As assessed at 44 hpf, only 42% (21/50) of
hsp70:Gal4;UAS:notch1aICD embryos showed detectable trypsin
transcripts at 44 hpf, compared with 89% (39/44) of hsp70:Gal4
sibling controls (Fig. 5E,F;
P<0.001).
|
Based on the ability of Notch to block acinar cell differentiation in
developing mouse pancreas even in the face of preserved ptf1a-p48
expression, we next examined the effects of delayed Notch pathway activation
initiated after the appearance of ptf1a-p48, but prior to the onset
of trypsin expression. For these studies, heat shock was applied at
34 hpf, at which point all embryos express ptf1a-p48 in developing
endoderm. Following heat shock at 34 hpf and harvest at 44 hpf, both
hsp70:Gal4 and hsp70:Gal4;UAS:notch1aICD embryos
demonstrated normal patterns of hhex, GATA6, pdx1 and
insulin expression (data not shown). The observed maintenance of
insulin expression following induction of notch1aICD at 34 hpf, but
not 24 hpf, suggests differential sensitivity between early and mature
endocrine cells with respect to ectopic Notch activation, similar to that
reported in the mouse (Murtaugh et al.,
2003). As expected, a normal pattern of ptf1a-p48
expression was also evident in both groups following heat shock at 34 hpf
(Fig. 5M,N). However, delayed
Notch activation again resulted in a significant delay in acinar cell
differentiation, with trypsin transcripts present in 88% (64/73) of
hsp70:Gal4 controls, but only 53% (32/60) of
hsp70:Gal4;UAS:notch1aICD embryos
(Fig. 5K,L;
P<0.001).
Loss of Notch signaling accelerates exocrine differentiation in developing zebrafish pancreas
In order to determine if the inhibitory effect of Notch on exocrine
differentiation reflected an influence normally exerted by endogenous Notch
pathway components, we utilized two different methodologies to generate Notch
loss-of-function phenotypes. Fish bearing homozygous nonsense mutations at the
mindbomb locus (mibta52b) lack a ubiquitin ligase
required for normal post-translational processing and trafficking of delta,
resulting in defective Notch pathway activation
(Itoh et al., 2003). A similar
phenotype can be generated by injection of single cell embryos with RNA
encoding a fusion protein comprised of GFP in frame with a dominant-negative
Suppressor of Hairless DNA binding mutant (GFPdnSuH)
(Wettstein et al., 1997
). To
determine the impact of defective Notch pathway activation on differentiation
of exocrine pancreas, we compared the timing of exocrine differentiation in
mibta52b/ta52b embryos, wild-type clutchmates,
GFPdnSuH-injected embryos, and control embryos injected with RNA encoding GFP
alone. In both mibta52b/ta52b and GFPdnSuH-injected
embryos, loss of Notch function was confirmed by generation of a neurogenic
phenotype revealed by both HuC staining (data not shown) and by gross
morphology. Embryos were collected across an extended time course designed to
span the period before and after the normal onset of endodermal
ptf1a-p48 expression (24-40 hpf), as well as the period before and
after the normal onset of trypsin expression (36-52 hpf).
For both ptf1a-p48 and trypsin, the onset of expression was accelerated in mibta52b/ta52b embryos compared with phenotypically wild-type (either mibta52b/wt or mibwt/wt) clutchmates, consistent with a normal inhibitory role for endogenous Notch in regulating exocrine differentiation (Fig. 6A,B,E-H, Fig. 7). The effect was especially pronounced for trypsin, with 100% of mibta52b/ta52b embryos showing expression at 40 hpf, whereas only 4% of wild-type fish had detectable trypsin transcripts at this time point (Fig. 6; P<0.001). In contrast, no acceleration of endocrine differentiation was apparent in mibta52b/ta52b embryos, as assessed by evaluating the onset of pdx1 and insulin expression (data not shown).
|
|
Notch inhibits activity of the Ptf1 transcriptional complex
In many systems, Notch-mediated inhibition of cellular differentiation
involves downregulated expression of lineage-specifying transcription factors
(Beatus and Lendahl, 1998;
Fisher and Caudy, 1998
;
Kuroda et al., 1999
;
Lee et al., 2001
). Based on
these precedents, we initially predicted that Notch pathway activation would
be associated with downregulated expression of Ptf1-P48. In both
mouse and zebrafish model systems, however, Ptf1-P48 expression was
preserved in the setting of forced Notch pathway activation, even while acinar
cell differentiation was prevented or delayed. Based on the additional
observation that Notch activation was capable of delaying exocrine
differentiation in zebrafish pancreas even when initiated after the normal
onset of Ptf1-P48 expression, we considered that Notch might inhibit
the functional activity of the Ptf1 transcriptional complex, comprised of the
Class II HLH Ptf1-p48 protein and a Class I E-box binding partner
(Obata et al., 2001
;
Rose et al., 2001
). As an
initial assessment of the effect of Notch on Ptf1 activity, we performed
electrophoretic mobility shift assays with nuclear extracts from the AR42J rat
acinar cell carcinoma line, using a labeled oligonucleotide corresponding to
the A element of the rat elastase 1 enhancer
(Rose et al., 2001
). In this
context, a positive gel shift results from endogenous Ptf1 DNA-binding
activity, with specificity further confirmed by supershift following addition
of anti-Ptf1-p48 antibody (Fig.
8A). Endogenous Ptf1 DNA-binding activity was inhibited following
forced Notch pathway activation, accomplished using bicistronic adenoviral
vectors encoding either GFP alone or in combination with Notch1-IC. In
addition, activation of selective Notch pathway components was accomplished
using adenoviral vectors encoding GFP in combination with individual
Notch/Su(H) target genes, including Hes1, Hey1 and Hey2. In these experiments,
activated Notch1-IC, Hes1 and Hey1 significantly reduced Ptf1 DNA-binding
activity, whereas Hey2 had no effect (Fig.
8A). Northern blot analysis demonstrated associated downregulation
of elastase-1 expression, but inconsistent changes in expression of
Ptf1-P48 (data not shown), suggesting that Notch pathway activation
may alter Ptf1 function, independent of associated changes in Ptf1-p48
expression.
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Discussion |
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Although the competitive and sequential recruitment of endocrine and
exocrine cell types from a common precursor pool in developing mouse pancreas
has made it difficult to discern the inhibitory influence of endogenous Notch
pathway components on exocrine differentiation, this influence is apparent in
developing zebrafish pancreas, where endocrine and exocrine precursors appear
to arise independently (Field et al.,
2003; Lin et al.,
2004
). Thus, while accelerated exocrine differentiation is not
observed following targeted inactivation of Notch pathway components in the
mouse (Apelqvist et al., 1999
;
Jensen et al., 2000b
), this
phenomenon is observed in developing zebrafish pancreas in the setting of
either inactivating mindbomb mutations or expression of
dominant-negative Su(H). These findings clearly define a role for
endogenous Notch pathway components in regulating zebrafish exocrine
pancreatic differentiation. Based on observed co-expression of Hes1 and
Ptf1-p48 in E13.5 mouse pancreas, the absence of Hes1 expression in
differentiated acinar cells, the ability of either Hes1 or Notch1-IC to
inhibit exocrine differentiation in a cell autonomous manner, and the ability
of either Notch1-IC or Notch/Su(H) target genes to inhibit Ptf1 activity, it
is likely that this influence is also active in the mouse.
In both mouse and zebrafish, we frequently observed ongoing expression of
Ptf1-P48 in the setting of either endogenous or ectopic Notch
activation, even while acinar cell differentiation was prevented. In addition,
we observed that activated Notch and Notch/Su(H) target genes were able to
downregulate Ptf1 activity even in the face of ongoing Ptf1-p48 protein
expression. Thus the respective mechanisms by which Notch inhibits endocrine
and exocrine differentiation appear to be fundamentally different, with
inhibition of endocrine differentiation achieved at least in part by blocking
Ngn3 expression (Hald et al.,
2003; Jensen et al.,
2000a
; Murtaugh et al.,
2003
), whereas inhibition of exocrine differentiation is achieved
by blocking Ptf1 function in the face of ongoing Ptf1-P48 expression.
This difference probably reflects the known requirement for Ptf1-P48
in early pancreatic development, prior to its role in promoting acinar cell
differentiation. In developing mouse pancreas, Ptf1-P48 expression is
detectable as early as E9.5 (Chiang and
Melton, 2003
; Kawaguchi et
al., 2002
; Obata et al.,
2001
), well before the presumed onset of Ptf1-dependent zymogen
expression. In addition, rigorous lineage tracing studies and further
characterization of the Ptf1-P48-null phenotype have demonstrated
that Ptf1-P48-positive precursor cells broadly contribute to both
exocrine and endocrine lineages in developing mouse pancreas, and that
Ptf1-P48 is required for critical events in early pancreatic
development (Kawaguchi et al.,
2002
). It is intriguing to consider that these early functions of
ptf1a-p48 may be mediated through mechanisms not involving activity
of the Ptf1 transcriptional complex, and that Notch may function not only to
reserve an undifferentiated precursor pool, but also to inhibit Ptf1-dependent
aspects of Ptf1-P48 activity until that point when Ptf1-P48
expression becomes restricted to dedicated acinar cells. Thus, while the
initial influence of Ptf1-P48 on early pancreatic morphogenesis
appears to occur in the setting of an active Notch pathway and low levels of
Ptf1 transcriptional activity, subsequent Ptf1-dependent functions require
silencing of Notch activity within the exocrine lineage. Based on this
interpretation, the identification of mechanisms underlying Notch-resistant,
Ptf1-independent aspects of Ptf1-P48 activity represents an important
area for future research.
In attempting to synthesize the current results with prior studies of
lineage relationships and Notch signaling in developing mouse pancreas
(Apelqvist et al., 1999;
Chiang and Melton, 2003
;
Gu et al., 2003
;
Gu et al., 2002
;
Hald et al., 2003
;
Jensen et al., 2000a
;
Jensen et al., 2000b
;
Kawaguchi et al., 2002
;
Murtaugh et al., 2003
), we
have generated the working model depicted in
Fig. 9. This model emphasizes
both the sequential and competitive recruitment of endocrine and exocrine cell
types from a common progenitor pool, as well as the iterative influence of
Notch signaling in regulating these events. Under this model, silencing of
Notch signaling within a common Ptf1-P48-positive precursor pool is
initially associated with endocrine lineage commitment, marked by the onset of
Ngn3 expression. In Ptf1-P48-positive cells with ongoing
Notch activity, the effects of Ptf1-P48 on pancreatic morphogenesis
continue to be realized, even while Ptf1-dependent acinar cell differentiation
is prevented. Upon subsequent silencing of Notch on E14.5, widespread acinar
cell differentiation ensues. Although the model assumes the predominantly
sequential recruitment of endocrine and exocrine cell types from a common
progenitor pool, it must be noted that these processes are characterized by
considerable temporal overlap, and that definitive markers indicating
`irreversible' commitment to either the endocrine or exocrine lineages have
not yet been identified.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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