Development 120, 3451-3462 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
3451
TGF-β1 influences the relative development of the exocrine and endocrine
pancreas in vitro
F. Sanvito, P.-L. Herrera, J. Huarte, A. Nichols, R. Montesano, L. Orci and J.-D. Vassalli
Department of Morphology, University of Geneva Medical School, CH-1211 Geneva 4, Switzerland
SUMMARY
Pancreatic rudiments from E12.5 mouse embryos undergo
extensive development and differentiation when cultured in
three-dimensional gels of extracellular matrix proteins for
up to 12 days. Whereas collagen gels promote the
formation of numerous exocrine acini and relatively small
clusters of endocrine cells, in basement membrane (EHS)
matrices the development of endocrine cells is dramatically
favoured over that of acinar tissue. Buds embedded in a
collagen gel contiguous to an EHS gel also fail to develop
acini, suggesting the involvement of diffusible factor(s).
Addition of cytokines to cultures of pancreatic buds in
collagen gels modifies the relative proportions of the epithelial components of the gland. In the presence of EGF the
proportion of the tissue occupied by ducts overrides that of
acinar structures, whereas the endocrine portion of the
tissue is not significantly modified. TGF-β1 partially
mimicks the effect of EHS matrix in inhibiting the development of acinar tissue without decreasing the amount of
ducts and mesenchyme; TGF-β1 also promotes the development of endocrine cells, in particular of insulin-containing β cells and of cells expressing genes of the PP-fold
family. These results show that cytokines can modulate the
development of the pancreas and suggest a role for TGFβ1 in regulating the balance between the acinar and
endocrine portions of the gland in vivo. More generally,
they are compatible with the notion that, during organogenesis, cytokines act as paracrine factors responsible for
the development and maintenance of appropriate proportions of different tissue constituents.
INTRODUCTION
Schuger et al., 1990b; Nogawa and Takahashi, 1991; Weller et
al., 1991; Warburton et al., 1992), very little is known about
the regulatory factors that control the development of pancreatic acini or islets of Langerhans. A mesenchymal factor that
enhances the proliferation of exocrine and endocrine cells in
pancreatic epithelial rudiments was described more than 20
years ago, but it has been only partially characterized (Ronzio
and Rutter, 1973).
Culture of epithelial cells in reconstituted collagen or
basement membrane matrices has been shown to promote histotypic organization and functional differentiation in a variety
of systems (see for example, Yang et al., 1980; Chambard et
al., 1981; Montesano et al., 1983, 1991; Barcellos-Hoff et al.,
1989). We have therefore embedded pancreatic rudiments in
three-dimensional matrices and examined their fate during in
vitro culture. In contrast to gels of type I collagen, which
allowed the development of all pancreatic constituents,
basement membrane (EHS) matrices selectively stimulated the
development of the endocrine pancreas. Since this effect was
due to a diffusible factor, we explored the effect of adding
exogenous growth factors to pancreatic buds embedded in
collagen gels. We have found that EGF affects the relative
development of the different exocrine constituents of the
pancreas, and that TGF-β1 enhances the formation of
endocrine cells while completely inhibiting the development
of acinar tissue. These results suggest an important modulatory
In mammals the pancreas originates from the primitive gut
through the fusion of two anlagen, which first appear as dorsal
and ventral epithelial evaginations surrounded by caps of
mesenchymal cells (Pictet et al., 1972). The original buds give
rise to branching epithelial ducts from which clusters of
endocrine cells and acini of exocrine cells progressively differentiate (Pictet and Rutter, 1972). The development of the
mammalian pancreas represents an attractive model to study
the molecular signals that direct the differentiation of epithelial cells along different lineages, since the same endodermal
precursor cells are believed to differentiate along two
divergent pathways to form the exocrine and endocrine
portions of the definitive gland (Le Douarin, 1988). In
addition, as yet unidentified mechanisms must operate to
ensure that the different constituents of the tissue develop in
appropriate proportions.
The identification of the molecular signals that trigger and
control the differentiation of the various pancreatic cell types
has been hampered by the lack of suitable model systems
allowing in vitro manipulation and monitoring of these developmental decisions. In particular, despite increasing
knowledge of the mechanisms that regulate the development
of other parenchymal organs such as salivary and mammary
glands, lung and metanephric kidney (Bernfield et al., 1984;
Key words: TGF-β1, EGF, pancreas, embryo, mouse, apoptosis,
extracellular matrix, growth factors
3452 F. Sanvito and others
role for cytokines and in particular TGF-β1 in pancreatic
development and differentiation.
MATERIALS AND METHODS
Culture of pancreatic rudiments in collagen gels
NMRI mice were mated overnight and the morning when the vaginal
plug appeared was defined as day 0.5 of gestation (E 0.5). Pregnant
mice with embryos of gestational age E11.5 or E12.5 were killed by
cervical dislocation. Embryos were removed from the uterus and
placed in Hank’s balanced salt solution (Gibco, Basel, Switzerland).
Dorsal or ventral pancreatic buds were dissected, washed in sterile
culture medium and embedded in three-dimensional collagen gels as
follows: 8 volumes of rat tail collagen stock solution (approximately
1.5 mg/ml) were quickly mixed with 1 volume of 10× concentrated
minimal essential medium (Gibco, Basel, Switzerland) and 1 volume
of sodium bicarbonate (11.76 mg/ml) in a sterile flask kept on ice
(Montesano et al., 1983). 200-300 µl of the solution were dispensed
into 15 mm wells of four-well plates (NUNC) and allowed to gel at
37°C; pancreatic buds were placed on the surface of the collagen gel
and covered with a further 200-300 µl of collagen solution. After the
second layer of collagen had gelled, complete medium (400-600 µl
per well), consisting of RPMI 1640 (Gibco, Basel, Switzerland) supplemented with 4 mM glutamine, 100 U/ml penicillin, 110 µg/ml
streptomycin and 5.5 mM glucose, was added. When indicated, 10%
heat-inactivated fetal calf serum (HI-FCS) was added.
Growth factors were added to both the collagen gels and the
medium. Porcine platelet transforming growth factor-β1 (TGF-β1)
was purchased from R & D Systems Europe (Oxon, UK); recombinant human epidermal growth factor (EGF) and porcine plateletderived growth factor (PDGF) were from Boehringer Mannheim
(Rotkreuz, Switzerland); nerve growth factor (NGF) was a generous
gift from Dr L. Aloe (Istituto di Neurobiologia, CNR, Italy); rat
insulin-like growth factor-II (IGF-II), human recombinant basic
fibroblast growth factor (bFGF) and recombinant human hepatocyte
growth factor (HGF) were kindly provided by Dr N. Yanaihara (Lab.
of Bioorganic Chemistry, Shizuoka-shi, Japan), Dr P. Sarmientos
(Farmitalia Carlo Erba, Milano, Italy) and Dr T. Nakamura (Biomedical Research Center, Osaka, Japan), respectively.
Medium and growth factors were replaced every 48 hours and the
buds were fixed, at the indicated times, in 2% glutaraldehyde in 0.1
M phosphate buffer or in Bouin’s solution, postfixed in osmium
tetroxide, dehydrated and embedded in Epon 812, as described previously (Bendayan et al., 1980).
Preparation of basement membrane-like matrix
The basement membrane-like matrix was obtained from the Engelbreth-Holm-Swarm (EHS) tumor, that was initially provided by Dr J.M. Foidart (Liège, Belgium) and subsequently maintained in our laboratory by subcutaneous or intramuscular inoculation in C57 black
mice. The procedures described by Kleinman et al. (1986) and Kramer
et al. (1986) were followed with slight modifications. Briefly, 3-4
weeks after inoculation, the tumors were harvested and homogenized
in 3.4 M NaCl, 50 mM Tris-HCl, pH 7.4, containing protease
inhibitors (10 mM EDTA, 2 mM N-ethylmaleimide, and 1 mM
phenylmethylsulfonylfluoride). After centrifugation at 500 g for 10
minutes, the supernatant was discarded, the pellet was resuspended in
Tris-HCl buffer with protease inhibitors (see above), and the washing
procedure was repeated three more times. The homogenate was then
centrifuged at 30,000 g for 30 minutes, the supernatant discarded and
the pellet resuspended in an equal volume (1 ml/g) of freshly prepared
2 M urea in Tris-HCl buffer with protease inhibitors. The suspension
was stirred overnight at 4°C, and the insoluble material was removed
by centrifugation at 30,000 g for 1 hour. The supernatant was dialyzed
for 24-48 hours at 4°C under sterile conditions, first against 0.15 M
NaCl in 50 mM Tris-HCl, and then overnight against a 1:10 dilution
of MEM with Earle’s salts (Gibco). The dialyzed extract was centrifuged at 30,000 g at 4°C for 30 minutes to remove small amounts
of insoluble material and the supernatant frozen at −20°C in small
aliquots. For gel formation, 8:1:1 volumes of EHS extract, 10× MEM
and sodium bicarbonate (11.76 mg/ml) were mixed on ice, dispensed
into culture wells and allowed to gel for about 30 minutes at 37°C
before adding complete culture medium. Commercially available
EHS matrix (MatrigelTM) was purchased from Collaborative Research
Incorporated (Bedford, MA) and gelled by incubation at 37°C for 30
minutes. Pancreatic rudiments were suspended in EHS matrix or
Matrigel before gelation occurred.
Immunocytochemistry and electron microscopy
Consecutive semithin sections (1 µm thick) of non-osmified pancreatic buds were collected on glass slides. The Epon was removed
(Maxwell, 1978) and the sections incubated with antibodies (see
below) for 2 hours, washed 5 minutes, incubated with FITC-conjugated anti-IgG antibodies for 1 hour at room temperature and counterstained with 0.003% (w/v) Evans blue. Sections were examined
and photographed with a Zeiss Axiophot epifluorescence microscope.
Rabbit anti-porcine glucagon serum directed against the C-terminal
region of glucagon was provided by Dr R. H. Unger (Dallas); guinea
pig anti-porcine insulin serum by Dr P. Wright (Indianapolis); rabbit
anti-bovine pancreatic polypeptide (anti-bPP) serum by Dr R. E.
Chance (Indianapolis); rabbit anti-synthetic somatostatin sera by Dr
R. Guillemin (San Diego, CA) and by Dr Y. Patel (Montreal, Canada).
Rabbit anti-human pancreatic polypeptide (anti-hPP) and rabbit antisynthetic porcine peptideYY (anti-PYY) were purchased respectively
from Peninsula Laboratories (Belmont; CA) and Euro-Diagnostica
(Malmö, Sweden); mouse monoclonal anti-BrdU IgGs were from
Becton Dickinson & Co. (San José, CA). FITC-conjugated sheep IgG
against guinea pig IgG, FITC-conjugated goat IgG against rabbit IgG,
and FITC-conjugated rabbit IgG against mouse IgG were purchased
from Biosys (Switzerland).
Specificity of anti-bPP, anti-hPP and anti-PYY antibodies was
assessed with an ELISA assay (data not shown), using bovine PP, rat
neuropeptide Y (NPY), porcine synthetic peptide YY (PYY) and
porcine glucagon as antigens. The results indicate that anti-bPP recognizes PP, NPY and PYY, but not glugagon; anti-bPP thus reveals
all three members of the PP-fold family (Hazelwood, 1993). Anti-hPP
and anti-PYY are specific for PP and PYY, respectively.
Electron microscopy was performed on ultrathin sections of Eponembedded osmified samples, collected on 150-mesh copper grids and
stained with uranyl acetate and Reynold’s lead citrate. Ultrathin
sections of Epon-embedded non-osmified samples were mounted on
nickel grids, and immunolabelled by the protein A-gold method (Roth
et al., 1978). Sections were incubated for 2 hours at room temperature with a 1:100 dilution of guinea pig anti-porcine amylase serum
(Bendayan et al., 1980); the grids were then rinsed in distilled water
and incubated for 1 hour at room temperature on a drop of a solution
of protein A coupled to 10 nm gold particles and double stained with
uranyl acetate (20 minutes) and Reynold’s lead citrate (10 minutes).
All samples were examined using a Philips EM 300 transmission
electron microscope.
Morphometry
The volume density (relative volume) of the different tissue components and of pancreatic hormone-containing cell types was determined
using the point-counting method of morphometric analysis (Weibel,
1979) on positive prints. Groups, corresponding to different conditions tested, were compared by a Krushal-Wallis test; pairwise comparisons were performed with the Bonferroni correction (Wallenstein
et al., 1980).
To estimate the real volume of cultured buds, 20 rudiments in
collagen gels, 26 rudiments in EHS matrix and 10 rudiments in
Cytokines and pancreatic differentiation 3453
collagen gels in the presence of TGF-β1 were measured after 8-10
days in culture and the following formula was used:
V = (4/3)π.r1.r2.r3 ,
assuming that r2=r3. The absolute volumes of the different components (acinar cells, ducts, endocrine cells, etc.) were estimated using
the following formula:
absolute volume = (total pancreatic volume × volume density)/100 .
To determine the number of cells undergoing DNA synthesis in
cultured buds, a final concentration of 10−4 M bromo-deoxyuridine
(BrdU; Sigma Chemical Co., St.Louis, MO) was added 12 hours
before fixation in Bouin’s. The volume density of proliferating cells,
demonstrated by anti-BrdU staining, was calculated by using the
point-counting method of morphometric analysis (Weibel, 1979) on
3 randomly selected positive prints of at least 3 pancreatic rudiments
from each group analyzed. For these two studies, groups were
compared by an analysis of variance (ANOVA).
Fig. 1. Dorsal pancreatic buds from E12.5 mouse embryos. (A) Semithin section of a freshly dissected bud showing a central epithelial mass
surrounded by a mesenchymal cap. The epithelial mass contains narrow lumen-like intercellular spaces (arrowheads). (B,C,D,E,F) Semithin
sections of buds cultured in collagen gels for 3 (B), 4 (C,D), 9 (E) and 15 (F) days. During the first 3 days in culture epithelial cells actively
proliferate (see Fig. 2A) and give rise to branching ducts (B). Exocrine cells, containing numerous zymogen granules and forming acinar
structures, are first detected after 4 days in culture (C,D); the arrowhead in D (phase-contrast microscopy) points to a mitotic exocrine cell.
Between 8 and 10 days, pancreatic buds cultured in a collagen gel show ducts, acini and clusters of endocrine cells (see Fig. 3A,B) in
paraductal region (E). After about 15 days in culture acinar cells are undetectable (F). Bars, 30 µm (A,B) and 10 µm (D); magnification is the
same in A, C, E and F.
3454 F. Sanvito and others
Quantification of apoptosis was performed on pancreatic rudiments
cultured for 7 days in collagen gels and further incubated for 24 hours
in the presence or absence of 1 ng/ml TGF-β1 (22 buds for each
condition). The number of apoptotic bodies immediately adjacent to
a measured length of basal acinar contour was counted, and the ratio
between the number of apoptotic bodies and the length of acinar
contour referred to as the frequency of apoptotic bodies. This
parameter was determined on 10 randomly selected electron micrographs for each bud. Statistical evaluation was performed on the logarithms of the frequencies by a nested ANOVA.
RNA extraction, cDNA synthesis and PCR
Total RNA was extracted from pancreata of E11.5 to newborn mice
according to the method of Chomczynski (Chomczynski and Sacchi,
1987). cDNAs were synthesized from 1 µg of total RNA using
oligo(dT) as primer for M-MLV reverse transcriptase (Promega).
cDNAs were amplified by PCR with the following mouse TGF-β1
primers: 5′ primer, 5′-TCCCGTGGCTTCTAGTGCTG-3′, where 5′ =
residue 396; 3′ primer (antisense), 5′-ATTTTAATCTCTGCAAGCGCA-3′, where 5′ = residue 836 (numbering according to mouse TGFβ1 cDNA sequence, as described by Derynck et al., 1986); the
expected size of the amplified cDNA is 440 bp. PCR was performed
essentially as described (Saiki et al., 1988), in a 30 times repeated
temperature cycle: 1 minute at 94°C, 1 minute at 56°C and 1 minute
at 72°C. 10 µl of the PCR mixtures were loaded on 5% acrylamide
gels that were stained with ethidium bromide after electrophoresis.
RESULTS
Development of pancreatic buds in collagen gels
When they first arise from the primitive gut, pancreatic buds
consist of a central mass of epithelial cells containing narrow
lumen-like intercellular spaces and surrounded by a mesenchymal cap (Fig. 1A). Immunofluorescence on mouse E12.5
pancreatic buds reveals a small proportion of epithelial cells
containing glucagon and/or one or another member of the PPfold family (i.e. PP, NPY or PYY; Herrera et al., 1991;
Hazelwood, 1993; Teitelman et al., 1993; Upchurch et al.,
1994).
When embedded in three-dimensional collagen gels, mouse
E12.5 pancreatic buds underwent morphological changes reminiscent of normal development in vivo. After 3 days in culture,
the epithelial portion of the tissue had developed into numerous
branching ducts delimited by a single layer of epithelial cells
(Fig. 1B), many of which were actively proliferating, as
revealed by incorporation of BrdU (Fig. 2A). Clusters of
epithelial cells apparently budding from the developing ducts
were also observed; some of these cells could be revealed by
anti-glucagon- and/or anti-bPP immunofluorescent staining.
Rare epithelial cells containing a few zymogen granules could
Fig. 2. Incorporation of BrdU in E12.5 pancreatic buds cultured in collagen gels either in the absence (A,C) or in the presence of TGF-β1 (1
ng/ml; B,D). At 3 days (A,B) the proliferation rate in pancreata in the presence or absence of TGF-β1 is similar, whereas after 6 days (C,D)
TGF-β1-treated buds contain less proliferating cells than the parallel control buds. Proliferating epithelial cells decrease between 3 and 6 days
in both conditions. Bar, 30 µm.
Cytokines and pancreatic differentiation 3455
Fig. 3. Development of pancreatic buds cultured in different conditions. Pairs of consecutive semithin sections from cultured E12.5 dorsal buds
were stained with different anti-hormone antibodies. (A,B) Pancreatic bud grown for 9 days in a collagen gel and stained with anti-glucagon
(A) or anti-bPP (B) antibodies. The epithelial portion of the bud consists of ducts, acini (arrowheads) and clusters of endocrine cells adjacent to
the ducts. (C,D) Pancreatic bud grown for 8 days in basement membrane-like extracellular matrix (EHS) and stained with anti-bPP (C) or antisomatostatin (D) antibodies. The bud is characterized by an increased number of endocrine cells, which form clusters budding from the ducts,
and by the absence of acini. (E,F) Pancreatic bud grown for 10 days in a collagen gel in the presence of TGF-β1 (1 ng/ml) and stained with
anti-bPP (E) or anti-insulin (F) antibodies. TGF-β1 partly mimics the effects of the EHS matrix in increasing the number of endocrine cells
(particularly of those containing the PP-fold family peptides and insulin) and in drastically reducing the development of acini. (G,H) Pancreatic
bud grown for 9 days in a collagen gel in the absence of serum and stained with anti-glucagon (G) or anti-insulin (H) antibodies. The
development of the endocrine component (that presents here an islet-like organization) is not affected by the absence of serum, whereas the
amount of acinar cells (arrowhead), is increased (for quantification, see Fig. 4). Bar, 30 µm.
3456 F. Sanvito and others
be detected by electron microscopy. Cells containing numerous
zymogen granules and organized in acinar structures were first
observed after 4 days in culture; the acini were embedded in a
loose stroma containing mesenchymal cells (Fig. 1C,D). The
presence of amylase, in zymogen granules, was confirmed by
protein A-gold immunocytochemistry (data not shown). After
6 days, the relative volume occupied by proliferating epithelial cells was 4-fold lower than at 3 days (Fig. 2C; P<0.0001);
this reduced rate of proliferation was maintained at least up to
10 days. After 8 days in culture in collagen gels, the amount
of acini was increased and endocrine cells containing one or
more islet hormone were present either as single cells in paraductal regions or as clusters of variable size that sometimes
acquired an islet-like organization (Figs 1E, 3). Surprisingly,
the fraction of endocrine cells containing glucagon was quite
low, even in cultured dorsal buds, in which at a corresponding
stage in vivo, as well as in the adult, A cells are abundant (Orci
et al., 1976; Orci, 1982; Herrera et al., 1991). During subsequent culture in collagen gels, acinar cells progressively
decreased in number and were undetectable after about 15 days
in culture (Fig. 1F). In contrast, no obvious changes in the composition and organization of the endocrine portion of the tissue
were observed.
Similar results were obtained when E11.5 or E12.5 dorsal or
ventral pancreatic buds were cultured in the same conditions.
Taken together, these observations indicate that pancreatic
primordia continue to develop when cultured in collagen gels,
albeit somewhat more slowly
than in vivo, yielding, after 8
days in culture, a histological
pattern resembling that of an
E16.5 pancreas. However, two
differences are noteworthy in
buds developing in culture: the
relative paucity of glucagoncontaining cells and the limited
viability of the acinar tissue.
Development of
pancreatic buds in
basement membrane gels
There is considerable evidence
that basement membranes play
important roles in the development of epithelial tissues
(Golosow and Grobstein, 1962;
Wessells and Cohen, 1967;
Bernfield et al., 1984; Schuger
et al., 1990b; Takahashi and
Nogawa, 1991), and numerous
studies have shown that
basement
membrane-like
matrices obtained from the EHS
tumor promote tissue-specific
cell organization and cytodifferentiation in various epithelial
systems (Kleinman et al., 1986;
Barcellos-Hoff et al., 1989;
Schuger et al., 1990a). We
therefore examined the development of pancreatic buds
cultured in an EHS-derived matrix and compared it with that
achieved in collagen gels.
After 8 days in culture in EHS-derived matrix, prominent
clumps of endocrine cells were seen to bud from ductal epithelium. The majority of these cells could be stained using an antibPP antiserum, which reveals all three members of the PP-fold
family (Fig. 3C; Teitelman et al., 1993; Upchurch et al., 1994;
unpublished observations). Staining using specific anti-hPP
and anti-PYY antibodies demonstrated the presence of both
peptides, with PYY-containing cells being most abundant.
Glucagon-, insulin- and somatostatin- (Fig. 3D) containing
cells were frequently observed. A quantitative evaluation of the
volume density of the different types of endocrine cells in pancreatic buds cultured in collagen or in EHS-derived gels
revealed that the development of the epithelial buds and of the
endocrine portion of the tissue was significantly enhanced
(Figs 4, 5; P<<0.0001) in the basement membrane-like extracellular matrix. A strikingly different pattern of development
was observed in the case of the exocrine tissue: while the
ductular portion of the tissue was similar in collagen and in
EHS-derived gels, the acinar component was completely
absent in buds cultured in the EHS matrix (Figs 3C,D, 4). In
experiments in which pancreatic buds were grown in gels
composed of a 1:1 mixture of EHS matrix and type I collagen,
the pattern of development was similar to that achieved in EHS
matrix alone.
Surprisingly, although the results described above were con-
Fig. 4. Volume densities (expressed as
percentages) of the different tissue
components in E12.5 buds cultured for 810 days in collagen gels (CTR), in EHS
matrix (EHS), in collagen gels in the
presence of either EGF (5 ng/ml) (EGF)
or TGF-β1 (1 ng/ml) (TGF), or in
collagen gels in the absence of serum
(CTRSF). Results are presented as boxplots (Williamson, 1989) in which boxes
symbolize the interquartile space of each
distribution, the median (d) and the mean (×) being indicated within the box; triangles (m) are the
extreme values of each distribution. Numbers indicate the size of the sampling.
Cytokines and pancreatic differentiation 3457
sistently reproducible with several distinct batches of EHS
tumor extract prepared in our laboratory, commercially
available EHS matrix (MatrigelTM) did not produce the same
effect. Buds embedded in Matrigel gave rise to large cysts,
which in semithin sections appeared as enlarged (ectatic) ducts
delimited by flattened epithelial cells. Acinar and endocrine
cells were rare and often poorly preserved (data not shown).
The striking effects of EHS-derived matrix on the development in cultured pancreatic buds might have been due to
insoluble basement membrane components (such as laminin,
type IV collagen or heparan sulfate proteoglycans) or to diffusible factors present in the EHS matrix, which is known to
contain a number of cytokines (Vukicevic et al., 1992). To distinguish between these possibilities, pancreatic buds were
embedded in a collagen gel, which was subsequently covered
with a layer of EHS matrix. This resulted in a pattern of pancreatic development similar to that observed by culturing pancreatic rudiments directly within the EHS matrix, suggesting
that soluble mediators diffusing from the EHS matrix into the
adjacent collagen gel were responsible for the increased development of endocrine tissue and the disappearence of acinar
cells.
Effect of exogenous growth factors on the
development of pancreatic buds in collagen gels
In view of the results described above, we investigated whether
the addition of well characterized cytokines to pancreatic
buds in collagen gels might
affect their development, as
determined after 10 days in
culture. Addition of either
bFGF (30 ng/ml), rhHGF (20
ng/ml), IGF-II (50 ng/ml),
PDGF (50 ng/ml) or NGF (1, 10
or 100 ng/ml) did not detectably
influence pancreatic differentiation (data not shown). rhEGF
(5 ng/ml) stimulated the
formation of duct-like structures, while drastically decreasing the development of acini
(Fig. 4); the formation of the
endocrine part of the tissue was
not affected (Fig. 5).
Pancreatic buds cultured for
8-10 days in the presence of
TGF-β1 (1 ng/ml) were characterized by the absence of acinar
cells and an abundance of
epithelial buds neighbouring
ductular structures; all types of
endocrine cells were present in
these epithelial buds, with a preponderance of cells containing
insulin and/or a member of the
PP-fold family (Fig. 3E,F). The
shape and arrangement of
stromal cells was also modified
by TGF-β1: as compared to
stromal cells in control cultures,
they appeared more elongated and oriented in parallel arrays.
Similar effects were observed with TGF-β1 at concentrations
of 0.1, 0.3 and 3 ng/ml; at the lower concentrations a few acinar
cells were present. Specimens were also fixed at earlier time
points (data not shown); this revealed that after the first 5 days
in culture the buds cultured in the presence of TGF-β1 were
not detectably different from those cultured in the absence of
the growth factor, i.e. they contained well-developed acini
formed by zymogen granule-containing cells. Incorporation of
BrdU showed that the relative volume occupied by the proliferating epithelial cells after 3 days in culture was similar in the
presence or absence of TGF-β1 (Fig. 2A,B); at 6 days, TGFβ1-treated cultures contained 2-fold less proliferating cells
than parallel control cultures (Fig. 2D; P<0.001).
A quantitative analysis of the frequency of each cell type
present in pancreatic buds after 10 days in culture confirmed
that acinar cells were virtually absent in specimens cultured in
the presence of TGF-β1 (Fig. 4), whereas the endocrine
component was increased (Fig. 5). All endocrine cell types
were significantly increased; the increase in abundance of A
and D cells was less pronounced in TGF-β1-supplemented
collagen gels than in EHS matrices (Fig. 5). The total volumes
of the pancreatic rudiments cultured in the various experimental conditions were also determined, and an estimation of
volumes occupied by the different components was computed.
In the presence of either EHS matrix or TGF-β1, the total
Fig. 5. Volume densities of the different
endocrine cell types, as revealed by antihormone staining, in E12.5 pancreatic buds
cultured as in Fig. 4. The volume densities
of total endocrine cells were calculated by
adding the volume densities of each of the
four endocrine cell types.
3458 F. Sanvito and others
volume of the endocrine compartment was increased by 4.5- and 3fold (P<0.0001), respectively, as
compared to control conditions.
Taken together, these observations suggest that TGF-β1 induces
the regression of the acinar compartment of the developing
pancreas and promotes differentiation of the endocrine tissue. In
this respect, the effect of TGF-β1
mimics, at least in part, that of
EHS matrix.
Development of pancreatic
buds in serum-free medium
Besides extracellular matrices,
another rich source of cytokines
affecting growth and differentiation in tissue culture is serum.
The experiments described thus
far were all performed in medium
containing 10% HI-FCS. Pancreatic rudiments cultured for 10
days in collagen gels in serumfree medium showed a different
pattern of development: while
mesenchyme and ductular cells
were less abundant (Fig. 4;
P<0.0001), acinar cells organized
in densely packed structures were
particularly conspicuous (Figs
3G,H, 4; P<0.0001). The total
volume density of endocrine cells
was not significantly different in
the presence or absence of FCS;
however, A cells were more
abundant (P<0.0001) in serumfree cultures (Fig. 5). The addition
of TGF-β1 to serum-free cultures
did not elicit effects comparable to
those observed in the presence of
serum (data not shown). These
results suggest that components
present in serum affect the differentiation of cultured pancreatic
buds; they also show that the
presence of serum is necessary for
the effect of TGF-β1 on differentiation.
TGF-β1 enhances apoptosis
of acinar cells
The striking disappearance of
acinar cells during the incubation
of pancreatic buds in the presence
of TGF-β1 suggested that the
cytokine might selectively prevent
their survival. Thin sections of
pancreatic buds cultured under
different conditions were thus
Fig. 6. Apoptosis of acinar cells. Dorsal pancreatic buds at E12.5 were cultured in collagen gels for 6
days. 24 hours before fixation, TGF-β1 was added to a final concentration of 1 ng/ml. Fragments of
cells containing condensed chromatin and zymogen granules can be seen in close proximity to acini
(A). Apoptotic bodies appear to be engulfed by mesenchymal cells (B,C). Bars, 1 µm.
Cytokines and pancreatic differentiation 3459
examined for signs of apoptosis. Analysis of cultures in
collagen gels with serum, maintained for 10 days revealed the
presence of apoptotic bodies containing fragmented nuclei
with condensed peripheral chromatin and cytoplasmic
organelles, including abundant endoplasmic reticulum and
zymogen granules, which indicates that they were derived from
acinar cells. These apoptotic bodies were either localized in the
interstitium immediately outside the acinar basement
membrane or phagocytosed by adjacent cells (Fig. 6), but they
were not observed within the acinar epithelium itself. Morphological signs of apoptosis were not seen when buds were
cultured for up to 15 days in the absence of serum. In serumcontaining cultures, apoptotic bodies were more abundant in
the presence of TGF-β1. Increased numbers of apoptotic
bodies were also seen when TGF-β1 was added after 6 days in
culture, i.e. at a time when acini are already conspicuous, and
the cultures examined 24 hours later: a quantitative analysis
revealed a 1.7-fold higher frequency of acinar apoptotic bodies
in TGF-β1-treated cultures as compared to control cultures
(P<0.0001; 95% confidence interval between 1,5 and 2.0). No
obvious signs of apoptosis were noted, either in the absence or
presence of TGF-β1, for the other cell types present in the
cultures.
These results indicate that TGF-β1 selectively increases the
level of apoptosis of acinar cells in serum-containing cultures,
and that apoptosis may thus account for the TGF-β1-induced
disappearance of acinar cells.
TGF-β1 mRNA is present in pancreatic buds
throughout development
Previous studies have demonstrated the presence of TGF-β1
mRNA and protein in the adult human pancreas (Yamanaka et
al., 1993). Since this cytokine affects development of cultured
pancreatic buds, it was of interest to determine whether it is
produced during embryogenesis. Total RNA extracted from
mouse pancreatic buds from E11.5 to birth was used to
generate oligo(dT)-primed cDNAs. PCR amplification of the
cDNAs with specific oligonucleotide primers revealed the
presence of TGF-β1 mRNA at all embryonic stages studied.
DISCUSSION
Among the most challenging issues in developmental biology
is the understanding of how, within a given organ, a quantitatively adequate contribution of its different constituents is
achieved and maintained. In exocrine glands, for instance, the
development of excretory ducts must be limited so as to leave
space for the secretory units. The development of the pancreas
is, in this respect, particularly intriguing since, in addition to
the excretory ducts and secretory acini, a relatively fixed proportion (approximately 1%) of the tissue differentiates into the
endocrine islets of Langerhans. Although growth factors or
cytokines are reasonable candidates for the regulation of the
relative proportions of an organ’s tissue constituents, the investigation of these issues is difficult: in vitro studies allow the
environment of the developing organ to be manipulated, but
the conditions must be chosen so as to allow development to
proceed in a manner that mimics in vivo ontogeny as closely
as possible. Three-dimensional cultures of embryonic buds in
extracellular matrix (ECM) gels provide a suitable experimen-
tal system to identify factors that control organogenesis
(Takahashi and Nogawa, 1991). We have therefore explored
the use of collagen and basement membrane-like gels to study
the ontogeny of the murine pancreas in vitro. Gels formed from
type I collagen were permissive for the development of both
the exocrine and endocrine parts of the gland, albeit at a
somewhat slower schedule than that observed in vivo; under
these conditions a pattern compatible with that which occurs
physiologically was observed. Cultures in collagen gels can
thus be used to investigate the putative factors controlling pancreatic development. Interestingly, the relative proportions of
the different endocrine cell types formed during in vitro development were similar, irrespective of whether the cultures were
initiated from dorsal or from ventral buds; this suggests that
the differences in cell composition between juxta-duodenal and
splenic islets of Langerhans in vivo (Orci et al., 1976; Orci,
1982) may be due to different environmental cues rather than
to differences in intrinsic developmental potential of the buds
themselves.
Earlier in vitro studies have pointed to the role of epithelialmesenchymal interactions in pancreatic development and
suggested the importance of diffusible mesenchyme-derived
factors in determining the fate of the epithelial components
(Golosow and Grobstein, 1962; Wessells and Cohen, 1967). Of
particular interest was the demonstration that a ‘mesenchymal
factor’ (MF) could induce isolated rat pancreatic epithelium to
preferentially differentiate into acinar structures and B cells
(Ronzio and Rutter, 1973). These observations set the stage for
the present studies, in that they suggested that epigenetic
phenomena might be involved in determining the relative proportions of tissues and cell types in developing organs. Under
our own experimental conditions, we have found that buds
(containing both epithelial and mesenchymal structures)
cultured in a matrix composed of type I collagen develop all
endoderm-derived pancreatic constituents, in approximately
physiological proportions. In contrast, similar buds cultured in
EHS matrix acquired a very different phenotype: islands of
endocrine cells developed more extensively, while acini were
almost completely absent. Mixing experiments suggested that
the EHS matrix plays a dominant role in the acquisition of this
abnormal phenotype, apparently through the release of diffusible substances. Surprisingly, whereas several batches of
EHS matrix prepared in our laboratory yielded similar results,
commercially available EHS matrix (MatrigelTM) did not
support the development of either acinar or endocrine cells and
the only epithelial structures to form were enlarged, cyst-like
ducts. Since minor components, such as growth factors contaminating the major ECM proteins, are most likely to vary
between preparations of EHS matrix, this observation is also
in accord with the notion that cytokines may influence the
relative development of different pancreatic tissues. These
results therefore led us to explore the possible effects of
cytokines on embryonic buds in collagen gels.
In accord with our hypothesis, we have found that two
cytokines, EGF and TGF-β1, cause profound changes in the
fate of cultured buds. EGF increased the proportion of tissue
occupied by ducts, at the expense of acinar structures; the
endocrine portion of the tissue did not appear to be affected.
TGF-β1 induced a complete involution of acini with morphological signs of apoptosis, and also induced a marked increase
in development of the endocrine tissue. It may be relevant that
3460 F. Sanvito and others
EGF and TGF-β1 are among the cytokines that have been identified in certain preparations of EHS matrix (Vukicevic et al.,
1992): the different patterns of development that we have
observed in cultures embedded in EHS gels or MatrigelTM
could conceivably be due to differences in the concentrations
of these cytokines in the different batches of ECM used. In any
event, our experiments indicate that EGF and TGF-β1 are modulators of pancreatic organogenesis in vitro and that cytokines
can play a part in controlling the relative development of the
different tissues that compose the mature gland.
Previous studies on the effects of EGF (or TGF-α, which is
structurally related to EGF and binds to the same receptor) and
TGF-β on pancreatic and other tissues have yielded results
relevant to our observations. Transgenic mice overexpressing
TGF-α in their pancreas exhibit extensive acinoductular metaplasia, resulting in an increased proportion of duct-like structures, similar to what we have observed in EGF-treated buds
(Jhappan et al., 1990; Sandgren et al., 1990). In cell culture,
TGF-β1 has been shown to inhibit the growth of adult mouse
pancreatic acinar cells and also to induce apoptosis of various
epithelial cell types, including rabbit uterine epithelial cells and
rat hepatocytes (Rotello et al., 1991; Logsdon et al., 1992;
Oberhammer et al., 1992). The effect of TGF-β1 on embryonic
acinar cells in buds developing in vitro is thus compatible with
these earlier findings. It is however puzzling that acinar cells
are only eliminated after an initial few days in culture during
which time they form at an apparently normal rate; this could
be due to a relatively late appearance of TGF-β receptors
during development of the buds. An alternate possibility, i.e.
that the TGF-β effect on acinar structures takes several days to
become manifest because it induces progressive changes in the
composition of the ECM, is less likely: addition of TGF-β1 to
cultures containing well-developed acini leads to their very
rapid involution. Another intriguing aspect of acinar apoptosis
in our organotypic cultures is the localization of the apoptotic
bodies: essentially all were present immediately adjacent to but
clearly separated from the epithelium itself. Apoptotic bodies
could be unambiguously identified as originating from acinar
cells by the presence of well-preserved typical organelles such
as zymogen granules. This suggests either that cells undergoing apoptosis are very rapidly ‘rejected’ from the epithelium,
or that the primary effect of TGF-β1 is to induce epithelial cells
to leave the epithelium. In the latter situation, ‘misplaced’
epithelial cells would subsequently eliminate themselves
through apoptosis; this would resemble anoikis, a form of
apoptosis induced by denied anchorage (Frisch and Francis,
1994; Ruoslahti and Reed, 1994).
Besides inducing involution of acinar cells, the EHS matrix
and TGF-β1 had a striking effect on the development of the
endocrine portion of the pancreatic buds. Both the relative proportion and the total volume of tissue occupied by endocrine
cells were increased as compared to control cultures in
collagen gels; hence this increase does not simply reflect the
loss of part of the exocrine compartment, but must be due to
increased proliferation and/or differentiation of endocrine cells
or their precursors. To our knowledege this positive influence
of TGF-β1 on the endocrine pancreas has not been previously
reported; on most epithelial cell types TGF-β1 exerts an
inhibitory effect on proliferation (Moses, 1992). No dramatic
changes in the relative proportions of the different endocrine
cell types were noticed in response to TGF-β1, although the
increase in B and PP gene family expressing-cells tended to be
more pronounced than the increase in A cells. Whether the
effect of TGF-β1 on the developing islets of Langerhans is
mediated by a direct effect of the cytokine on endocrine cells
or their precursors, whether it is due to the induction of another
growth-stimulating cytokine or whether it is secondary to
changes in the ECM through effects on stromal cells, cannot
be decided at this time.
Our observations demonstrate that exogenous cytokines can
modify pancreatic development. Could the same cytokines be
endogenous physiological modulators of this process? TGF-α
and the EGF receptor have been identified in fetal pancreas
(Miettinen and Heikinheimo, 1992); TGF-βs are present in
adult pancreatic tissue (Yamanaka et al., 1993), and by RTPCR we have demonstrated the presence of TGF-β1 mRNA in
pancreatic buds at all stages of development. These cytokines
could thus play a paracrine role in organogenesis of the
pancreas. The lack of reported effects of TGF-β1 gene inactivation on pancreatic development does not invalidate this
hypothesis, since other members of the TGF-β family may also
be physiologically relevant factors or may substitute for the
missing cytokine (Shull et al., 1992). It is also interesting to
note that endogenous cytokines, in particular TGF-βs, may
play a part in controlling the in vitro development of cultured
buds. The evidence in favor of this is indirect: in long-term
cultures maintained in presence of serum, acinar structures
undergo slow involution, similar to that which occurs, albeit
with much more rapid kinetics, in the presence of exogenous
TGF-β1. As in other systems (Rotello et al., 1991), the effect
of exogenous TGF-β1 on pancreatic buds requires the presence
of serum, and cultures performed in the absence of both TGFβ1 and serum show increased development of acini, which
persist even in long term cultures. The use of antisense
oligonucleotides or antibodies capable of preventing TGF-β
production or activity may help to decide whether endogenous
TGF-β, either synthesized in situ or present in serum, influences the development of cultured buds.
Given that EGF and TGF-β1 affect the development of
epithelial derivatives in the developing pancreas, is one of
these the long sought MF? It has been proposed that MF plays
a role in pancreatic ontogeny by determining the proportion of
endocrine and acinar cells; it increases the development of B
cells and decreases the yield of A cells, but it also enhances
the differentiation of acinar cells (Ronzio and Rutter, 1973).
Thus, in view of our results, neither EGF nor TGF-β1 alone
can account for the effects of MF. However, the data available
on MF suggest that it probably encompasses more than just one
active principle (Filosa et al., 1975); it is possible that some of
its effects are due to the presence of EGF and/or TGF-β, in
combination with other modulatory factors.
In conclusion, we have shown that two cytokines, EGF and
TGF-β1, influence the in vitro differentiation of pancreatic
buds, in particular with respect to the relative development of
the endodermal derivatives. The notion that cytokines can act
as paracrine mediators responsible for achieving the appropriate balance of different tissue constituents during organogenesis is compatible with the observed effects of transgeneencoded TGF-β1 in the mammary gland: overexpression of the
cytokine leads to alterations in the relative development of lobuloalveolar versus ductal tissue (Pierce et al., 1993). It will be
of interest to determine whether molecular ablation of
Cytokines and pancreatic differentiation 3461
members of TGF-β family in knock out mice, or targetted overexpression of these cytokines, alters the structure of the
pancreas, and whether certain developmental pathologies, such
as nesidioblastosis (Heitz et al., 1977; Creutzfeldt, 1985), or
species-related characteristics, such as the relative abundance
of endocrine tissue in the acomys mouse (Gonet et al., 1965),
are due to differences in the cytokine-mediated regulation of
pancreatic ontogeny.
We thank Mrs D. Ben-Asr and Mrs G. Moussard for their skillful
technical assistance, Mr J.-P. Gerber, B. Favri and Mr G. Negro for
photographic work and Mr G. Andrey for computer work. We are
grateful to Dr M. Castellucci for preparing extracts of EHS tumor, to
Ms B. Mermillod for help and advice for all statistical analyses and
to Dr M. Pepper for valuable suggestions. This work was supported
by a grant from the Fonds National Suisse de la Recherche Scientifique (31-34088.92).
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(Accepted 31 August 1994)