First published online 27 July 2004
doi: 10.1242/dev.01280
Development 131, 4213-4224 (2004)
Published by The Company of Biologists 2004
Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas
Farzad Esni1,*,
Bidyut Ghosh1,*,
Andrew V. Biankin1,
John W. Lin1,
Megan A. Albert,
Xiaobing Yu2,
Raymond J. MacDonald3,
Curt I. Civin2,
Francisco X. Real4,
Michael A. Pack5,6,
Douglas W. Ball2 and
Steven D. Leach1,2,
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
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Fig. 1. Notch pathway activation assessed by Hes1 expression in developing mouse
pancreas. (A,D,G) Single channel and merged confocal images demonstrating
co-expression of Hes1 (red) and Pdx1 protein (green) in E13.5 mouse pancreas.
(B,E,H) Single channel and merged confocal images demonstrating co-expression
of Hes1 (red) and Ptf1-p48 protein (green) in E13.5 mouse pancreas. (C,F,I)
Double fluorescent in situ hybridization demonstrating absence of
Hes1 expression in Ngn3-positive endocrine precursors. Cells
with cytoplasmic Hes1 transcripts (red) do not contain transcripts
for Ngn3 (green). (J) Immunostaining for Hes1 and E-cadherin on E14.5
pancreas reveals nuclear Hes1 protein in central undifferentiated epithelium
as well as in epithelial branches and centroacinar cells (arrow). (K)
Magnified view of area outlined in J, demonstrating nuclear Hes1 protein in
cells comprising peripheral epithelial branches (arrow), but exclusion of Hes1
from acinar cluster, indicated by broken line. (L) Immunostaining for Hes1 and
amylase, demonstrating Hes1 protein in nuclei of amylase-negative terminal
ductal/centroacinar cells, but not in differentiating amylase-positive acinar
cells. Dashed lines indicate contour of representative Hes1-negative acinar
cell, identified by basal nuclei and apical collection of amylase-positive
zymogen granules. Dotted lines indicate contour of representative
Hes1-positive, amylase-negative terminal ductal/centroacinar cell. Images in
C,F,I and J-L generated by 3D image deconvolution. Scale bars: 10 µm.
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Fig. 2. Effect of ectopic Notch activation on endocrine and exocrine
differentiation in explant cultures of E10.5 dorsal pancreatic buds. E10.5
dorsal epithelial buds were infected with lentiviral vectors encoding either
GFP alone, GFP and Hes1, or GFP and Notch1-IC, then recombined with mesenchyme
and allowed to differentiate in vitro. (A-C) Examination of intact buds
demonstrates mosaic GFP expression on day 1 following lentiviral infection.
(D-O) Deconvolution fluorescent microscopy demonstrating expression of
endocrine and exocrine markers in infected E-cadherin-positive epithelial
cells marked by GFP expression, assessed on day 6 following lentiviral
infection. Cells infected with GFP alone are able to complete either endocrine
or exocrine differentiation programs, as assessed by co-expression of GFP with
insulin (D), glucagon (G), amylase (J), or Ptf1-P48 (M). Cells infected with
GFP in combination with either Hes1 or Notch1-IC maintain the capacity for
alpha-cell differentiation, as evidenced by co-expression of GFP and glucagon
(H,I), but are unable to undergo beta-cell differentiation, indicated by
absence of cells co-expressing GFP and insulin (E,F). Cells infected with GFP
in combination with either Hes1 or Notch1-IC are also unable to undergo acinar
cell differentiation, as evidenced by absence of cells co-expressing GFP and
amylase (K-L). In contrast, expression of Ptf1-P48 is preserved in cells
expressing GFP in combination with either Hes1 or Notch1-IC (N,O). Arrowheads
in D,G,H-J,M-O indicate examples of individual cells co-expressing GFP and
indicated marker. Scale bars: 10 µm.
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Fig. 3. Quantitative effects of ectopic Notch activation on endocrine and exocrine
differentiation in explant cultures of E10.5 dorsal pancreatic buds. The
frequency of endocrine and exocrine differentiation was determined by direct
counting of GFP-positive epithelial cells in epithelial buds infected with
lentiviral vectors encoding either GFP alone, GFP and Hes1, or GFP and
Notch1-IC. Top, graphical representation depicting frequency of amylase,
insulin, glucagon, nestin and Ptf1-P48 expression in cells infected with
indicated vector. Y-axis indicates fraction of GFP-positive,
E-cadherin-positive epithelial cells expressing given marker. Bottom,
numerical data generated by direct cell counts. Values indicate mean
(%)±s.e.m. (bold), as well as corresponding absolute cell counts. Both
acinar and beta cell differentiation are prevented by Notch pathway
activation, reflected by significant reductions in the frequency of cells
expressing either amylase or insulin following infection with either GFP;Hes1
or GFP;Notch1-IC compared with GFP alone. In contrast, the frequency of cells
expressing glucagon, nestin or Ptf1-P48 are affected to lesser degrees.
Asterisks indicate P<0.05 compared with GFP alone, as determined
by two-sample Z-test.
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Fig. 4. Ectopic Notch activation expands a population of Ptf1-p48-positive,
amylase-negative cells in explant cultures of E10.5 dorsal pancreatic buds.
E10.5 dorsal epithelial buds were infected with lentiviral vectors encoding
either GFP alone or GFP and Hes1, then recombined with mesenchyme and allowed
to differentiate in vitro. (A,C,E) Deconvolution fluorescent microscopy
demonstrating patterns of Ptf1-p48 and amylase expression following infection
with GFP alone. (B,D,F) Expression patterns following infection with GFP;Hes1.
Note apical amylase expression in majority of Ptf1-p48 cells infected with GFP
alone, but expansion of a Ptf1a-p48-positive, amylase-negative population in
cells infected with GFP;Hes1. Broken lines in A and B indicate contour of
representative GFP-positive, Ptf1-p48-positive cells either expressing (A) or
lacking amylase (B). A similar pattern is observed following infection with
GFP;Notch1-IC. Scale bar: 10 µm.
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Fig. 5. Notch pathway activation delays exocrine differentiation in developing
zebrafish pancreas. Heat-shocked embryos expressing a hsp:Gal4
transgene alone or in combination with UAS:notch1aICD were assessed
for initiation of ptf1a-p48 and trypsin expression, as well
as expression of insulin and pdx1. Following heat shock at
24 hpf, ptf1a-p48 expression is delayed in 32 hpf
hsp:Gal4;UAS:notch1aICD embryos (B) compared with hsp:Gal4
controls (A), but recovers by 34 hpf (data not shown). As assessed at 44 hpf,
both trypsin (E,F) and insulin (G,H) expression are reduced
in hsp:Gal4;UAS:notch1aICD embryos compared with hsp:Gal4
controls, even while ptf1a-p48 (C,D) and pdx1 (I,J)
expression remain normal. Following delayed heat shock initiated at 34 hpf
(after normal onset of ptf1a-p48 expression), ptf1a-p48
expression remains normal at all time points (M,N), whereas expression of
trypsin is delayed (K,L). Arrowheads in A,C,D,M,N indicate endodermal
domain of ptf1a-p48 expression, distinct from expression in
developing hindbrain. Arrowheads in I and J indicate pdx1-positive principal
islet; arrows indicate adjacent pdx1-positive intestine.
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Fig. 6. Defects in Notch pathway activation result in acceleration of exocrine
differentiation in developing zebrafish pancreas. (A-D) Mindbomb
(mib) mutant embryos (B) and embryos injected with RNA encoding a
dominant-negative Suppressor of Hairless DNA binding mutant
[GFPdnSu(H)] (D) show either accelerated (mib) or normal [GFPdnSu(H)] onset of
ptf1a-p48 expression compared with clutchmate controls (A,C). Arrows
indicate endodermal domain of ptf1a-p48 expression, distinct from
expression in developing hindbrain. Insets in A-D show magnified view of
endodermal ptf1a-p48 expression domain. (E-J) mindbomb (mib)
mutant embryos (F,H) and embryos expressing GFPdnSu(H) (J) show accelerated
acinar cell differentiation compared with clutchmate controls (E,G,I), marked
by early onset of trypsin expression. Note normal absence of trypsin
expression in control embryos at 40 hpf (E,I), but accelerated onset of
trypsin expression in mibta52/ta52 and GFPdnSu(H)-injected embryos
(F,J). At 80 hpf, the size and contour of established trypsin-positive
exocrine parenchyma is also altered in mibta52/ta52 embryos
compared with wild-type clutchmates (G,H). Wt indicates wild-type clutchmates
arising from mibta52/wt x mibta52/wt cross. GFP
indicates clutchmate control embryos injected with RNA encoding GFP alone.
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Fig. 7. Frequency of ptf1a-p48 and trypsin expression in
developing wild-type and mibta52/ta52 zebrafish
embryos. (A) Graph depicting fraction of embryos expressing either
ptf1a-p48 or trypsin at indicated developmental time points,
as determined by whole-mount in situ hybridization. (B,C) Raw numerical data
generated by analysis of more than 600 embryos. Mib indicates
mibta52/ta52 embryos; WT indicates wild-type
clutchmate controls.
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Fig. 8. Notch pathway activation inhibits activity of the Ptf1 transcriptional
complex. (A) Electrophoretic mobility shift assay measuring endogenous Ptf1
DNA-binding activity in nuclear extracts of rat AR42J cells infected with
adenoviral vectors encoding GFP alone or in combination with either Notch1-IC,
Hes1, Hey2 or Hey1 as indicated. Upper band (arrow) represents specific
binding of oligonucleotide corresponding to the A element of rat elastase
promoter, confirmed by supershift in the presence of anti-Ptf1-p48 antiserum
(arrowhead). Cold indicates presence of excess unlabelled oligonucleotide.
Note inhibition of endogenous Ptf1 DNA-binding activity by Notch1-IC, Hes1 and
Hey1. (B) Western blot analysis of COS7 extracts following co-transfection of
Ptf1-P48 alone or in combination with E47 and either Notch1-IC, Hes1, Hey2 or
Hey1. Note constant level of Ptf1-p48 and E47 protein across conditions,
allowing analysis of Ptf1 activity (C) in the absence of associated changes in
component protein levels. (C) Corresponding determination of Ptf1 activity as
assessed by activation of Ptf1-responsive luciferase reporter (Ptf1-luc).
Notch1-IC, Hes1, Hey2 and Hey1 effectively inhibit Ptf1 activity, independent
of associated changes in protein levels of Ptf1-p48 or E47. (D) Schematic
depiction of Notch1-IC truncation mutants used in E and F, below. NLS
indicates nuclear localization signal. ANK indicates ankyrin repeats. (E)
Ability of Notch1-IC and Notch1-IC truncation mutants to activate
Su(H)-responsive luciferase reporter [Su(H)-luc]. (F) Corresponding ability of
Notch1-IC and Notch1-IC truncation mutants to inhibit Ptf1-p48/E47-mediated
activation of Ptf1-responsive luciferase reporter (Ptf1-luc). Ability to
inhibit Ptf1 resides within N-terminal domains. (-) in C,E,F indicates
transfection of reporter construct alone.
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Fig. 9. Notch regulates sequential recruitment of endocrine and exocrine cell types
in developing mouse pancreas. Simplified model summarizes current results
within the context of known lineage relationships
(Chiang and Melton, 2003 ;
Gu et al., 2003;
Gu et al., 2002;
Kawaguchi et al., 2002), as
well as prior studies of Notch signaling in developing mouse pancreas
(Apelqvist et al., 1999;
Hald et al., 2003;
Jensen et al., 2000a;
Jensen et al., 2000b;
Murtaugh et al., 2003).
Diagonal red line distinguishes cell types characterized by active Notch
signaling, indicated by Hes1-positivity, from those with an inactive
Notch pathway. Notch pathway activation inhibits early recruitment of
Ngn3-positive endocrine precursors from a common endocrine/exocrine
precursor pool, and also inhibits generation of differentiated acinar cells
from dedicated exocrine precursors. Between E10.5 and E12.5,
Ngn3-positive endocrine precursors are recruited from a
Ptf1-P48/Hes1-positive common precursor pool. Recruitment of
dedicated endocrine precursors in inhibited by Notch, thereby reserving an
undifferentiated cell population responsible for ongoing epithelial growth as
well as subsequent exocrine differentiation. By E13.5, ongoing
Ptf1-P48 expression marks a dedicated exocrine precursor pool.
Ongoing Notch activity within this pool results in an inactive Ptf1
transcriptional complex, allowing terminal acinar cell differentiation to be
avoided until Ptf1-independent influences of Ptf1-P48 on epithelial
morphogenesis are fully realized. Notch silencing at E14.5 allows onset of
Ptf1 activity and acinar cell differentiation. The persistence of
Notch-regulated precursor cells in adult pancreas remains uncertain.
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© The Company of Biologists Ltd 2004
