Cell and Developmental Biology Program, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA
* Author for correspondence (e-mail: zaret{at}fccc.edu)
Accepted 3 November 2003
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SUMMARY |
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Key words: Pancreas, Endothelial, Endoderm, Transcription factor, Ptf1a
![]() |
Introduction |
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The dorsal and ventral pancreatic buds arise between embryonic days 8.5 and
9.5 of gestation (E8.5-E9.5) of the mouse and eventually combine to generate
the mature organ (Wessels and Cohen,
1967; Rugh, 1968
;
Pictet et al., 1972
). The
initial fields of prospective pancreatic endoderm consist of progenitors of
endocrine and exocrine cells (Deltour et
al., 1991
; Percival and Slack,
1999
; Herrera,
2000
; Gu et al.,
2002
). These cells express the homeobox containing transcription
factor Pdx1, which is also expressed in duodenal progenitors and is necessary
for pancreatic and duodenal differentiation after the formation of the dorsal
and ventral pancreatic buds (Wright et
al., 1989
; Ohlsson et al.,
1993
; Jonsson et al.,
1994
; Ahlgren et al.,
1996
; Offield et al.,
1996
). The dorsal and ventral endodermal expression of the
homeodomain factor Hnf6 (Rausa et al.,
1997
; Landry et al.,
1997
) is necessary for the timely initiation of Pdx1 expression
(Ohlsson et al., 1993
;
Ahlgren et al., 1996
;
Li et al., 1999
;
Gannon and Wright, 1999
;
Jacquemin et al., 2003
). The
dorsal and ventral endodermal expression of the basic helix-loop-helix
transcription factor Ptf1a, originally discovered by its function in the
exocrine pancreas (Krapp et al.,
1996
; Rose et al.,
2001
), was recently shown to be necessary for the development of
endocrine, exocrine and duct cell lineages both dorsally and ventrally
(Krapp et al., 1998
;
Kawaguchi et al., 2002
).
Although Pdx1, Hnf6 and Ptf1a function in both dorsal and ventral pancreatic
endoderm, the mesodermal inducers of these factors in the embryo remain
unknown.
Despite the commonalities, the dorsal and ventral pancreatic buds have some
different mesodermal inducers and transcriptional effectors. The homeodomain
protein Hb9 is necessary for dorsal, but not ventral, pancreatic bud emergence
(Li et al., 1999;
Harrison et al., 1999
). The
notochord, a mesodermal derivative, is required to inhibit the expression of
sonic hedgehog in the dorsal endoderm, thereby allowing pancreatic
development (Kim and Melton,
1998
; Hebrok et al.,
1998
; Hebrok et al.,
2000
). Deleting notochord has no effect on ventral pancreatic
development (Kim et al.,
1997
). Genetic mutations that deplete dorsal mesenchyme cells
cause deficiencies in dorsal but not ventral pancreatic bud development
(Ahlgren et al., 1997
;
Esni et al., 2001
). The
ventral pancreas emerges near the liver, and tissue explant studies showed
that pancreatic induction occurs in ventral foregut endoderm distal to
cardiogenic mesoderm (Deutsch et al.,
2001
; Kumar et al.,
2003
), the normal inducer of the liver
(Le Douarin, 1975
;
Fukuda, 1979
;
Houssaint, 1980
; Gualdi et
al., 1966). These and other studies show that dorsal pancreatic endoderm is
induced by notochord and mesenchyme, whereas ventral pancreatic endoderm
patterning is permitted by the absence of cardiogenic mesoderm and perhaps the
presence of local mesenchyme cells (Rossi
et al., 2001
; Kumar et al.,
2003
).
In addition, endothelial cells, another mesodermal derivative, provide
organogenic stimuli for the pancreas
(Cleaver and Melton, 2003).
Lammert et al. (Lammert et al.,
2001
) showed that at E9.0-E10 in the mouse, the aorta and the
vitelline veins are near the emerging dorsal and ventral pancreatic buds,
respectively. Using tissue recombination experiments, they showed that
fragments of aorta induce the expression of Pdx1 and insulin in dorsal
endoderm cultures from mouse embryos. Removal of the aorta from
Xenopus embryos causes a failure to induce certain pro-endocrine
transcription factors and insulin, in vivo. They also showed that transgenic
mice over-expressing vascular endothelial growth factor (Vegfa) in pancreatic
endocrine cells causes hypertrophy of the pancreatic islets. These studies
showed that endothelial cells provide inductive signals for pancreatic
development, apart from the ability of blood vessels to serve as conduits for
nutrients, oxygen and soluble signaling molecules.
The pioneering studies of endothelial cells in pancreas development did not address the following questions. (1) Although blood vessels are adjacent to where both dorsal and ventral pancreatic buds develop, do endothelial cells similarly promote organogenesis in both contexts? (2) What are the transcription factor gene targets required for early pancreatic development that are induced by endothelial cell interactions?
To address these issues, we used Flk1 (Kdr - Mouse Genome
Informatics) null mice (Shalaby et al.,
1995). Flk1 encodes a receptor for Vegfs and is expressed
in endothelial cells (Millauer et al.,
1993
; Quinn et al.,
1993
; Oelrichs et al.,
1993
; Yamaguchi et al.,
1993
). A homozygous null Flk1 mutation blocks endothelial
cell development at the angioblast stage, preventing the formation of mature
endothelial cells and blood vessels
(Shalaby et al., 1995
). The
genetic ablation approach is important because we previously found that during
tissue bud development, pockets of endothelial cells and nascent capillaries
can be detected within the nascent hepatic mesenchyme, making it difficult to
completely dissect away endothelial cells for tissue explant studies
(Matsumoto et al., 2001
).
For the present study, we performed a detailed temporal and functional analysis of blood vessel interactions with prospective pancreatic endoderm in the mouse embryo. We find that endothelial cell interactions are more critical for dorsal than ventral pancreatic development and that endothelial cells induce different pancreatic transcription factors in the endoderm than those induced by other mesodermal cell types. The studies described here provide a link in understanding how mesodermal interactions with the endoderm lead to specific transcription factor inductions within the network required for pancreatic organogenesis.
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Materials and methods |
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Flk1lacZ expression, immunohistochemistry and in situ hybridization
For assessing expression of the Flk1lacZ
allele, embryos were fixed, stained in X-gal solution, and embedded in
paraffin as described previously (Rossi et
al., 2001). For immunostaining, embryos were fixed in 4%
paraformaldehyde buffered at pH 7.4 with phosphate-buffered saline (PBS) at
4°C overnight, washed twice with 1x PBS at 4°C for 10 minutes,
dehydrated through a PBT (1x PBS + 0.1% Tween 20)-methanol gradient,
embedded in paraffin, and sectioned at 7 µm by standard procedures. The
following primary antibodies and dilution were used: rabbit polyclonal
anti-Pdx1, 1:5000 (gift from C. Wright), rabbit polyclonal anti-glucagon,
1:180 (Maine Biotechnology Services, Portland, ME). Briefly, after
deparaffinization and rehydration, sections were boiled in citrate buffer (10
mM sodium citrate, pH 6.0) for 10 minutes and endogenous peroxidase activity
was blocked by immersing sections in 0.3% H2O2.
Following treatment with blocking buffer (10% heat inactivated goat serum, 1%
ovalbumin in PBT), sections were reacted with primary antibodies in 0.1x
blocking buffer at 4°C overnight. Primary antibodies were detected with
Vectastain Elite ABC kit (Vector, Burlingame, CA) using either DAB (Sigma, St.
Louis, MO) or Vector SG (Vector, Burlingame, CA). Sections were counterstained
with Eosin Y. Whole embryos were immunostained for Pecam as described
previously (Sato and Bartunkova,
2000
) using 30 µg/ml of antibody (BD Pharmingen, San Diego,
CA), embedded in paraffin, sectioned and counterstained. In situ hybridization
was performed as described previously (Ang
et al., 1993
; Jung et al.,
1999
) with Foxa2, Hnf6 and Shh riboprobes reported previously
(Ang et al., 1993
;
Echelard et al., 1993
;
Rausa et al., 1997
); then
embryos were sectioned.
RT-PCR analysis
RNA was isolated by cesium chloride gradient centrifugation, reverse
transcribed with oligo(dT) primers, and subjected to PCR as described
previously (Gualdi et al.,
1996). PCR was performed with 1.5 mM Mg2+ at 60°C
for annealing, unless indicated otherwise below. PCR cycle ranges for each
gene and tissue fragment were determined by first analyzing actin mRNA in each
sample at three cycle steps over a nine cycle range, quantitating
electrophoretic products with a phosphorimager, and determining cycle ranges
to use for other primer sets; the latter, in turn, were analyzed at multiple
cycle steps, usually differing by three cycles, to ensure that the reactions
were in the exponential range of PCR. The sequences of the primers (and
certain cases of specific conditions for PCR) were: Pecam sense
5'-GCAAAGAGTGACTTCCAGAC-3', antisense
5'-GTACCTCGTTACTCGACAGG-3'; Tal1 sense
5'-AACAACAACCGGGTGAAGAG-3', antisense
5'-ACTTGGCCAGGAAATTGATG-3'; Pdx1 sense
5'-CAGGAGGTGCTTACACAGC-3', antisense
5'-CCCGCTACTACGTTTCTTATCTTCC-3' (2 mM MgCl2, 2% DMSO,
annealing at 63°C); Ptf1a sense 5'-GGCCCAGAAGGTCATCATCTGC-3',
antisense 5'-AGGAAAGGGAGTGCCCTGCAAG-3'; Prox1 sense
5'-CCCAGCTGTTGAAAAATAAC-3', antisense
5'-TCTCAGGTGCTCATCACATA-3'; Ngn3 sense
5'-GGGATACTCTGGTCCCCCGTGC-3', antisense
5'-GAGCGCATCCAAGGGATGAGGC-3'; Neurod1 sense
5'-CTTGGCCAAGAACTACATCTGG-3', antisense
5'-GGAGTAGGGATGCACCGGGAA-3'
(Deutsch et al., 2001
);
glucagon sense 5'-CATTCACAGGGCACATTCACC-3', antisense
5'-ACCAGCCAAGCAATGAATTCCTT-3'
(Herrera et al., 1991
);
insulin sense 5'-CAGCCCTTAGTGACCAGCT-3',
5'-TGCTGGTGCAGCACTGATC-3'
(Krapp et al., 1998
); Hnf6
sense 5'-AGCCCTGGAGCAAACTCAAGTCG-3', antisense
5'-TGCATGTAGAGTTCGACGTTGGAC-3' (sequences from F. Lemaigre); Wt1
sense 5'-AACCACGGTATAGGGTACGA-3', antisense
5'-CGGCTATGCATCTGTAAGTG-3'; Hex sense
5'-GCACAAAAGGAAAGGCGGTCAAGT-3', antisense
5'-ACCTGTTTCAGTCTTCTCCATTTA-3'.
Tissue dissection and recombinant explants
Embryo tissues were dissected according to the method of Gittes and Galante
(Gittes and Galante, 1993).
The dissected tissues were cultured as described previously
(Lammert et al., 2001
) with
the following modifications. The tissues were recombined, embedded in
growth-factor reduced matrigel matrix (Becton Dickinson) and cultured with
DMEM medium supplemented with 10% calf serum, 1x penicillin-streptomycin
(Gibco-BRL) at 37°C with 5% CO2, for the indicated periods.
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Results |
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At 8-9S, just prior to induction of Pdx1 dorsally, the left and right
aortae were lateral to the notochord, somites and dorsal-medial endoderm
(Fig. 1A), and thus only in
partial contact with the dorsal pancreatic progenitor domain, which has been
mapped to the endoderm adjacent to the notochord and underlying the somites
(Matsushita, 1996;
Kumar et al., 2003
). By
12-15S, the aorta contacted the dorsal endoderm extensively, without
intervening cells (Fig. 1B,C;
green arrows), and Pdx1-positive cells appeared by 15S in the dorsal endoderm
near and beyond the area of contact with the aorta. At 26S, the fused aorta
was clearly separated from the dorsal bud
(Fig. 1D). We note that by 26S,
capillaries with Flk1-positive endothelial cells were detected in the
dorsal mesenchyme near the pancreatic bud
(Fig. 1E), emanating from the
aorta (data not shown). These observations show that endothelial cells of the
aorta gain a tight association with dorsal endoderm during the period when
Pdx1 is induced.
|
The presence of the aorta induces Ptf1a dorsally and the outgrowth of the pancreatic bud, but not the initial field of Pdx1-positive cells
To examine the roles of endothelial cells in pancreas development in vivo,
we studied Flk1 homozygous null mutant embryos, which lack
endothelial cells. Fig. 2A,B
shows the absence of the aorta in the dorsal region of Flk1 mutant
embryos, compared to wild type, which was confirmed by a lack of cells
expressing platelet endothelial cell adhesion molecule (Pecam; CD31), a marker
for embryonic endothelial cells (Vecchi et
al., 1994). RT-PCR products for Pecam and Tal1,
the latter encoding an early endothelial transcription factor
(Visvader et al., 1998
), were
detectable in the dorsal midgut region of control embryos, but not in that of
Flk1 homozygous nulls (Fig.
2J).
|
The expression of Pdx1 is regulated by transcription factors expressed in
the dorsal endoderm, including Foxa2 (formerly Hnf3ß) and Hnf6
(Wu et al., 1997;
Gerrish et al., 2000
;
Jacquemin et al., 2003
). In
situ hybridization showed that at 13-14S, the expression of Foxa2 was
a bit weaker than in wild type, but still positive in
Flk1-/- embryos (Fig.
3A,B). Also, Hnf6 was expressed in the Flk1 null
embryos in nearly the same domain of cells as in the controls
(Fig. 3C,D). These expression
patterns are consistent with the observation that endothelial cells are not
necessary for the initial field of dorsal endoderm-expressing Pdx1. The
notochord was not affected morphologically in Flk1-/-
embryos, and Shh was repressed in the dorsal endoderm as in wild type
(Fig. 3E,F; arrows to dorsal
endoderm) (Kim et al., 1997
;
Apelqvist et al., 1997
;
Hebrok et al., 2000
). Thus,
notochord interactions with the dorsal endoderm physically and functionally
precede those of the dorsal aorta, as originally suggested by Lammert et al.
(Lammert et al., 2001
).
|
|
RT-PCR (Fig. 4A,B) and
immunohistochemistry studies (Fig.
4D,E) showed that insulin and glucagon are also not detectably
induced in Flk1-/- embryos. This contrasts with the
phenotype of mouse embryos containing a null mutation of the Ptf1a
gene, in which some insulin and glucagon-positive cells develop dorsally,
although pancreatic development is disrupted there
(Krapp et al., 1998;
Kawaguchi et al., 2002
). Thus,
endothelial cells and/or the aorta induce or permit the expression of dorsal
Ptf1a, pancreatic bud outgrowth, and critical endocrine genes.
Initial ventral pancreas development is not affected in Flk1-/- embryos
The absence of endothelial cells and the vitelline vein in the ventral
pancreas area of Flk1-/- embryos was confirmed by staining
for Pecam (Fig. 5A,B).
Occasionally we saw a Pecam-positive cell that was distal to the ventral
pancreatic bud (Fig. 5B, blue
arrowhead). Also, while RT-PCR showed a strong decrease in the expression of
Pecam and Tal1 in ventral regions of dissected embryos
(Fig. 5G), at higher PCR cycles
and long exposures we could detect some products
(Fig. 5G, lanes 10, 12),
consistent with the previously reported appearance of some angioblasts in the
umbilicus near the gut in Flk1 embryos
(Shalaby et al., 1995). Thus
while the vitelline veins and other local vasculatures are completely absent,
a few distal angioblasts persist ventrally in Flk1 homozygotes.
|
We note that the absence of endothelial cells did prevent the induction of glucagon ventrally (Fig. 5G). Insulin was not yet expressed ventrally in wild-type embryos during the period (data not shown). Since glucagon was not induced either dorsally or ventrally in Flk1-/- embryos, but Ptfla was only not induced dorsally, endothelial cell interactions appear to control endocrine gene induction at a step later than Ptfla expression.
Aorta fragments induce Ptf1a in dorsal endoderm explants of Flk1-/- embryos
To test the hypothesis that the aorta itself, rather than factors in the
bloodstream or other secondary effects, regulates the induction of
Ptf1a in the dorsal endoderm, we performed tissue recombination
studies. Dorsal endoderm fragments were dissected from
Flk1-/- embryos at 7-10S
(Fig. 6A-C) and cultured for 24
hours with or without aortae dissected from control embryos
(Fig. 6E), which is comparable
to the E8.5-9.5 transition in vivo. By this strategy, the isolated dorsal
endoderm is genetically depleted of endothelial cells, which was confirmed by
RT-PCR analysis showing the absence of Pecam expression
(Fig. 6F, lanes 3, 4; `no
aorta' explants). RT-PCR analysis also showed that the dissected aorta from
wild-type embryos was Hnf6 negative, and thus was not contaminated
with endoderm, and that the isolated Flk1-/- dorsal
endoderm expressed Hnf6, as expected
(Fig. 6D). Owing to the smaller
size of the Flk1-/- embryos, the endoderm was difficult to
dissect. To be sure that the dissected dorsal endoderm, underlying the
somites, lacked contaminating ventral-lateral endoderm, which does not require
endothelial cells for Ptf1a induction
(Fig. 5H), we selected only
those Hnf6-positive, endoderm-only explants which, upon culture, were
negative for the expression of Hex
(Fig. 6F), as Hex
marks both hepatic and ventral pancreatic progenitor cells and not the dorsal
pancreatic progenitors (Martinez-Barbera
et al., 2000; Bort et al.,
2004
). We also assessed the presence of mesenchyme cells by RT-PCR
for Wt1, which in situ hybridization has shown to be expressed
specifically in prospective dorsal and lateral mesenchyme cells
(Armstrong et al., 1992
). We
found that Wt1 levels in the dorsal pancreatic region normally
increase during E8.5-9.5 (Fig.
6G), consistent with the appearance of mesenchymal cells around
the dorsal pancreatic endoderm (Fig.
1B,C).
|
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Discussion |
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We find that the aortal endothelium induces Ptf1a in a field of
dorsal endoderm cells that, by the 13-15S stage, already have Shh
expression repressed and Hnf6 and Pdx1 expression induced
(Fig. 3B,D,F). Genetic lineage
tracing studies have shown that early endoderm cells expressing Ptf1a
contribute to all pancreatic cell lineages and thereby constitute the
definitive pancreatic progenitor population
(Krapp et al., 1998;
Kawaguchi et al., 2002
). These
and other studies (Chiang and Melton,
2003
) also showed that Ptf1a expression normally
initiates in the Pdx1-positive cell population. Taken together, these points
show that endothelial cell interactions in vivo
(Fig. 4A,B) and in vitro
(Fig. 6E,F), contribute to the
initial differentiation of dorsal pancreatic progenitor cells from the
endoderm by inducing the transcription factor gene Ptf1a.
From our embryonic tissue recombination studies, it appears that the aortal
endothelium induces Ptf1a directly in the dorsal endoderm, rather
than through an intermediary cell such as lateral mesenchyme cells, which are
necessary for ventral pancreatic development
(Kumar et al., 2003). Also,
the aorta exhibits an extensive and apparently direct interaction with the
Pdx1-positive dorsal endoderm during the 12-15S stage
(Fig. 1B), when Ptf1a
is first induced (Fig. 4C, lane
6), apparently prior to mesenchyme cells interposing extensively between the
tissues (Fig. 1C). By contrast,
although the vitelline veins are near the ventral Pdx1-positive endoderm
cells, we can discern mesenchyme cells interposing between the tissues at all
stages (Fig. 1E-I) and our data
show that the vitelline veins and other local vasculatures are not required
for ventral Ptf1a induction (Fig.
5G). It remains possible that rare, distal angioblasts that
persist in Flk1-/- embryos could promote ventral
pancreatic development, perhaps in an analogous manner to that described
recently in normal early pancreatic bud development in cloche mutant
zebrafish embryos that have a greatly diminished vasculature
(Field et al., 2003
).
Alternatively, initial ventral pancreatic bud development in the mouse, and
pancreatic bud development in general in zebrafish, may be independent of
vascular cell signaling. Regardless, it thus appears that, as for other
pancreatic transcription factors described in the Introduction, the induction
of Ptf1a, which is necessary for pancreatic differentiation in
general (Krapp et al., 1998
;
Kawaguchi et al., 2002
), is
differentially dependent upon vascular cells for the mouse dorsal and ventral
pancreatic progenitor cell populations.
We found that the aorta, endothelial cells, and/or Ptf1a
expression were necessary to promote the outgrowth of dorsal endoderm cells
into a pancreatic bud (Fig.
2F,H). This effect was only marginal in the ventral endoderm,
where the Pdx1 cell population appeared to be about half the normal size by
30S, but the cells were within a morphologically distinct pancreatic bud
(Fig. 5F). The ability of
Pdx1-positive cells to be maintained ventrally and generate a pancreatic
bud-like structure shows that the embryonic lethality of the
Flk1-/- mutation, and the lack of endothelial cells or a
blood supply, is not generally deleterious to the initiation of pancreatic
morphogenesis. Ventral Ptf1a homozygous null cells in the previous
lineage studies (Kawaguchi et al.,
2002) exhibited a fate change to an intestinal cell type, and
therefore formed part of the gut tube instead of budding into a pancreas.
While most of the dorsal Ptf1a homozygous null cells that expressed a
Ptf1a-cre lineage marker also formed gut cells, a subpopulation
eventually grew out and exclusively expressed endocrine genes, as previously
observed (Krapp et al., 1998
;
Kawaguchi et al., 2002
).
However, the endocrine gene-expressing cells in the Ptf1a null
embryos did not form a normal dorsal pancreas bud or tissue; instead, by
E14.5-16.5 the cells grew into an extended rudiment and eventually the cells
became incorporated into the spleen. Consistent with these observations, we
found that the residual dorsal Ptf1a-negative cells in
Flk1-/- embryos expressed the endocrine progenitor genes
Pdx1, Ngn3 and Neurod1
(Fig. 2D; Fig. 4A,B). The failure to
generate a bud in the dorsal Pdx1-positive, Ptf1a-negative cell population in
such embryos could explain the mouse tissue recombination and Xenopus
aorta excision results of Lammert et al.
(Lammert et al., 2001
), which
resulted in diminished Pdx1-positive endocrine cells. We suggest that the
aorta did not induce a field of cells capable of initiating Pdx1 expression,
but rather specifically induced Ptf1a resulting in the subsequent ability of
those cells to maintain normal amounts of dorsal pancreatic tissue.
Although the initial development of the ventral pancreas is not strongly
affected by the absence of a vasculature, glucagon, an early endocrine cell
marker, was not induced in the ventral pancreatic bud of
Flk1-/- embryos, and both glucagon and insulin were not
induce dorsally. The data indicate that endothelial cells serve a distinct
function in promoting endocrine gene activation, in agreement with the studies
of Lammert et al. (Lammert et al.,
2001), particularly as we observed a capillary network beginning
to form in the early, emerging pancreatic buds at 26S
(Fig. 1D,J).
We note that various earlier studies of mesenchymal cell induction of pancreatic development employed mesenchyme tissue explants isolated from embryos at E9.5 and onwards, which we have shown to contain endothelial cells (Fig. 1D,J). Thus it is possible that at least some of the inductive effects previously attributed to mesenchyme cells could be due to the presence of endothelial cells within the explants.
In summary, we provide evidence that during dorsal pancreatic development, the vascular endothelium plays a central role in activating Ptf1a, one of the initial genes required to specify the pancreatic lineage, yet it does so only for the dorsal endoderm. Specific questions for the future include: What leads to the recruitment of endothelial cells and vessels by endodermal domains that give rise to vascularized tissues? What explains our observations of differences in a requirement for the aorta, but not the vitelline veins, for the initial expression of Ptf1a? What are the signaling molecules involved? In addition, it will be interesting to identify mesodermal inducers of other transcription factors required for the initiation of pancreatic differentiation from the endoderm, and thus understand how distinct mesodermal interactions lead to the activation of the transcription factor network that promotes pancreatic development.
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ACKNOWLEDGMENTS |
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