1 Department of Medical Biochemistry, Box 440, Göteborg University, S-405
30 Göteborg, Sweden
2 Department of Obstetrics and Gynecology, University of Pennsylvania School of
Medicine, 1355 Biomedical Research Building II/III, 421 Curie Boulevard,
Philadelphia, PA 19104, USA
Author for correspondence (e-mail:
henrik.semb{at}endo.mas.lu.se)
Accepted 8 December 2004
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SUMMARY |
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Key words: Pancreas, Morphogenesis, Blood vessel, Mesenchyme, Sphingosine-1-phosphate, Mouse
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Introduction |
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Soon after cell fate determination of the pancreatic endoderm,
morphogenesis begins with folding of both the dorsal and ventral pancreatic
endoderm, processes regulated by soluble factors (members of the FGF and
activin families) secreted from the mesenchyme
(Le Bras et al., 1998;
Miralles et al., 1999
;
Scharfmann, 2000
). Whereas the
lateral and ventral gut mesenchyme is splanchnic-derived and present before
gut closure and pancreas commitment, the dorsal pancreatic mesenchyme is
recruited from a hitherto unknown subpopulation of lateral plate mesenchymal
cells.
Mice deficient for islet 1 (Isl1) and the cell adhesion molecule N-cadherin
lack a dorsal pancreas due to failure to form the dorsal pancreatic mesenchyme
(Ahlgren et al., 1997;
Esni et al., 2001
). Recently,
we showed that N-cadherin is required for formation of the dorsal pancreas, by
mediating dorsal pancreatic mesenchymal cell survival
(Esni et al., 2001
). Notably,
whereas Pdx1 expression is initiated within the N-cadherin null
(Cdh2-/-) dorsal pancreatic endoderm, it later disappears
(at 9.5 days postcoitum, dpc) (Esni et al.,
2001
). Cdh2-/- mice die of cell adhesion
defects in the heart around 9.5 dpc
(Radice et al., 1997
).
However, these mice exhibit additional defects in development of the nervous
system, somites and yolk sac. Importantly, when cadherin function was restored
within the heart of N-cadherin-deficient mice by expressing either N-cadherin
or E-cadherin under the regulation of a cardiac-specific promoter (
myosin heavy chain) (cardiac-rescued Cdh2-/- mice), the
embryos survived until 10.5-11 dpc due to rescued heart and vascular function
(Luo et al., 2001
).
To address whether the pancreatic phenotype in N-cadherin-deficient mice reflects a cell-autonomous function of N-cadherin within the mesenchyme or if it is secondary to other defects, e.g. cardiac and/or vascular function, we analysed pancreas development in cardiac-rescued Cdh2-/- mice. Unexpectedly, morphogenesis of the dorsal pancreatic bud was rescued in 9.5 dpc cardiac-rescued Cdh2-/- mice, suggesting that the pancreatic phenotype of N-cadherin-deficient mice was secondary to the cardiac/vascular defects. Using an in vitro pancreatic explant model, we show that sphingosine-1-phosphate (S1P)-mediated G-protein-coupled signalling rescues formation of the dorsal pancreas in N-cadherin-deficient mice in vitro, by specifically triggering proliferation of the dorsal pancreatic mesenchyme.
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Materials and methods |
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Immunohistochemistry and immunoreagents
For immunohistochemistry, embryos were fixed and sectioned as previously
described (Esni et al., 1999).
Antibodies directed against N-cadherin, Pdx1 and Isl1 were used as previously
described (Esni et al., 1999
;
Esni et al., 2001
). Endothelial
cells, F-actin, and proliferating cells were detected with anti-CD31 (PECAM,
PharMingen, 1:100), endomucin, V.5C7
(Morgan et al., 1999
),
Phalloidin-Alexa 568 (Molecular probes, 1:40) and anti-Ki67 (Novocastra
Laboratories Ltd, 1:200), respectively. Biotin-conjugated anti-rat (1:1000)
and anti-rabbit (1:1000) antibodies were purchased from Jacksons Immuno
Research Laboratory Inc. Alkaline-phosphatase conjugated anti-rat (1:1000) was
purchased from Southern Biotechnology Associates, Inc. The Vectastain ABC and
Dab substrate kit for peroxidase were from Vector Laboratories Inc. and used
according to the manufacturer's instructions. Endomucin was detected with a
TSA kit from Molecular probes and used according to the manufacturer's
instructions with slight modifications. Sections were permeabilized and
blocked in 5% skim milk dissolved in PBST (PBS pH 7.4, 1% Triton X-100) and
subsequently incubated with PBSLEC (PBS pH 6.8, 1% Triton X-100, 0.1 mM
CaCl2, 0.1 mM MgCl2, 0.1 MnCl2) for 60
minutes at room temperature. The endomucin antibody was diluted 1:20 and
incubated overnight at 4°C. Hematoxylin and eosin staining was performed
by standard procedure.
Whole-mount Immunohistochemistry
Whole-mount immunohistochemistry was performed as previously described
(Ohlsson et al., 1993).
Preparation of agarose-beads
Agarose-beads from Bio-Rad were rinsed three times in PBS and added onto a
bacterial plate, where they were dried prior to being soaked in different
factors. Beads were incubated in plasma (prepared from 15.5 dpc wild-type
embryos), S1P (Sigma, final concentration 0.1 µM) dissolved in PBS
supplemented with 100 µg/ml BSA and in pertussis toxin (Sigma, final
concentration 0.2 µg/ml) dissolved in PBS supplemented with 100 µg/ml
bovine serum albumin (BSA) at 37°C for one hour and stored at 4°C.
Beads were prepared weekly.
Culture of pancreatic rudiments
Isolation, recombination and culture of pancreatic rudiments were carried
out essentially as previously described
(Ahlgren et al., 1996;
Esni et al., 2001
;
Gittes and Galante, 1993
).
Pre-soaked agarose-beads (see above) were added to the rudiments. The explants
were cultured for 48 hours in a humidified incubator at 37°C with 5%
CO2 and subsequently whole-mount stained with Pdx1 and CD31
antibodies.
For cultures of dorsal pancreatic mesenchyme, the same mesenchyme was removed from 10.5 dpc wild-type endoderm in ice-cold PBS without using trypsin. Mesenchyme pooled from several embryos was plated onto 0.1%-gelatine-coated coverslips positioned in a 24-well tissue-culture plate and cultured in `199' medium (Gibco), supplemented with 10% inactivated fetal calf serum (FCS), 50 U/ml penicillin/streptomycin (Gibco), and 1.25 µg/ml fungizone (Gibco), for 24 hours in a humidified incubator at 37°C prior to the addition of different factors. The factors were added for another 24 hours before the samples were processed for further analysis.
Proliferation assay
BrdU-labelling was used to detect proliferating cells in two-day-old
cultures of pancreatic mesenchyme. Cells were incubated with BrdU (Roche) for
1 hour, fixed and stained for proliferating cells according to the
manufacturer's recommendations. To stain the nuclei, DAPI (1:1000) was added
in the last wash. The cells were photographed, counted, and the proliferation
index was determined as the relative number of BrdU-labelled cells in the
cultures. The assay was repeated three times for each factor,
n=3.
RNA preparation and RT-PCR
RNA was prepared from dorsal pancreatic mesenchyme of 10.5 dpc wild-type
embryos by using the Qiagene RNA isolation kit (Qiagene). cDNA synthesis was
performed by using Superscript II from Gibco. PCR was run with primers
specific for SIP1-5, Pdx1, Isl1, and ß-actin.
5'-TAGCAGCTATGGTGTCCACTAG-3' (sense) and
5'-GATCCTGCAGTAGAGGATGGC-3' (anti-sense) for SIP1,
5'-ATGGCAACCACGCATGCGCAG-3' (sense) and 5'-GGCGGTGAAGATACTGATGAG-3' (anti-sense) for SIP3,
5'-ATGGGCGGCTTATACTCAGAG-3' (sense) and 5'-GACGGAGAAGATGGTGACCAC-3' (anti-sense) for SIP2, 5'-CTCCTCATTGTCCTGCACTA-3' (sense) and 5'-GATCATCAGCACGGTGTTGA-3' (anti-sense) for SIP4, 5'-TGTTTCTGCTCCTTGGCAGC-3' (sense) and 5'-ATCTCGGTTGGTGAAGGTGT-3' (anti-sense) for SIP5, 5'-CCACCCCAGTTTACAAGCTC-3' (sense) and 5'-TGTAGGCAGTACGGGTCCTC-3' (anti-sense) for Pdx1,
5'-ATGGTGGTTTACAGGCGAACC-3' (sense) and
5'-GGGCAGAAACAACATCAGAACCTG-3' (anti-sense) for Isl1,
5'-GTGGGCCGCTCTAGGCACCAA-3' (sense) and
5'-CTCTTTGATGTCACGCACGATTTC-3' (anti-sense) for ß-actin.
In situ hybridization
In situ RNA hybridization was performed with slight modifications of
previously described protocols (Bostrom et
al., 1996). Hybridization solution consisted of 55% formamide, 20%
dextran sulphate, 1 mg/ml yeast tRNA, 1 x Denhardt's solution, 5 mM
EDTA, 0.2 M NaCl, 0.013 M Tris-HCl, 5 mM NaH2PO4, 5 mM
Na2HPO4, and probes were denaturized at 80°C for 5
minutes. Digoxigenin-labelled probes of SIP1 and SIP3
were generated from cDNA fragments in the pBluescript KS+ vector. The
anti-sense probes were generated by T3 RNA polymerase-mediated transcription
after NotI digestion, whereas the sense probes were generated by
T7 RNA polymerase-mediated transcription after EcoRI
digestion. SIP2 probes were generated from S1P2
cDNA in the pCMV SPORT 6 vector. The anti-sense probe was generated by
T7 RNA polymerase-mediated transcription after SalI
digestion and the sense probe was generated by Sp6 RNA
polymerase-mediated transcription after NotI digestion.
SIP1 and SIP3 cDNAs were gifts from M.
Takemoto, and SIP2 cDNA was purchased from Invitrogen life
technologies. Hybridization temperatures were: SIP1, 57°C;
SIP2 and SIP3, 61°C.
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Results |
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Members of the S1P receptor subfamily that bind S1P with high affinity
include S1P1, S1P2, S1P3, S1P4,
and S1P5 (Pyne and Pyne,
2000; Spiegel and Milstien,
2003
). RT-PCR and in situ hybridization analyses of the 10.5 dpc
wild-type dorsal pancreatic bud revealed that S1P1,
S1P2, and S1P3 mRNAs were all expressed
within the mesenchyme (Fig. 4).
Whereas S1P1 mRNA was specifically expressed in
endothelial cells, both S1P2, and S1P3
mRNAs were preferentially expressed in the mesenchyme
(Fig. 4B-J). The fact that
pre-incubation with pertussis toxin (PTX), which ADP-ribosylates and
inactivates Gi, blocked the S1P-mediated rescue of the dorsal
pancreas in Cdh2-/- explants (% ventral and dorsal
pancreas: Cdh2-/-+S1P (64%) vs
Cdh2-/-+PTX and S1P (33%);
Table 1,
Fig. 2F), and that PTX blocked
early dorsal bud formation in wild-type explants (30%,
Table 1,
Fig. 2G), suggest that
G-protein-coupled S1P receptors play a role in early pancreas development.
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S1P-induced rescue of the Cdh2-/- dorsal pancreatic bud outgrowth does not correlate with apparent changes in vascular density
To begin to explore whether S1P may affect endoderm-endothelial cell
interactions, we examined the distribution of blood vessels in our in vitro
system. To visualize blood vessels around the pancreatic buds, we colabelled
explants with antibodies against Pdx1 and CD31. In addition to rescuing
formation of the Cdh2-/- dorsal pancreatic endoderm, S1P
rescued growth of Cdh2-/- mesenchyme and endothelium
(Fig. 6). Importantly, the
distribution of endothelial cells and blood vessels around the presumptive
dorsal and ventral pancreas was apparently unaffected by N-cadherin ablation
and addition of S1P (Figs 1,
6), suggesting that N-cadherin
and S1P are not primarily involved in the localization of endothelial
cells/blood vessels near the pancreatic endoderm.
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Discussion |
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Soon after specification of the dorsal pancreatic endoderm, the dorsal
pancreatic mesenchyme is recruited around the endoderm. Intimate reciprocal
interactions between the epithelium (endoderm) and the mesenchyme mediate
further growth and differentiation of the dorsal pancreas. Previous studies
have demonstrated that in the absence of the mesenchyme, the dorsal pancreas
does not emerge (Ahlgren et al.,
1997; Esni et al.,
2001
; Scharfmann,
2000
). Consequently, it was necessary to address whether the
S1P-mediated rescue of the dorsal pancreatic bud formation in
Cdh2-/- explants was specifically mediated by an effect on
the endoderm or mesenchyme. Indeed, using whole pancreatic and mesenchyme
explants, we show that S1P exerted its effect specifically on the mesenchyme
by stimulating mesenchymal cell proliferation.
Although we have not identified the cellular source of the S1P involved in
pancreas development, the following observations indicate that it is blood
vessel-derived. First, S1P is produced and secreted by cells of haematopoietic
origin, such as activated platelets, monocytic and mast cells
(Yatomi et al., 1995). Second,
at the time of dorsal pancreatic mesenchyme recruitment (20-25 somites),
immature endothelial cell-cell contacts and holes in endothelial cells (P.
Wastesson, personal communication) result in leakage of smaller molecules,
such as S1P, from the circulation (dorsal aorta)
(Fig. 7). Third, rescue of the
dorsal pancreatic bud in cardiac-rescued Cdh2-/- embryos
correlates with rescue of an intact circulatory system
(Luo et al., 2001
). Finally,
the dorsal pancreatic bud in Cdh2-/- embryos was rescued
with similar efficiencies in vitro by plasma from wild-type embryos and
S1P.
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A functional role of blood vessels in early pancreatic development has
previously been reported to involve cell interactions between the endoderm and
endothelial cells, and to be independent of the supply of oxygen and nutrients
(Lammert et al., 2001;
Yoshitomi and Zaret, 2004
).
These studies showed that endothelial cell interactions are required for
induction and maintenance of transcription factors known to regulate
pancreatic endoderm specification, such as Pdx1 and Ptf1a, and endocrine cell
differentiation. Moreover, it was demonstrated that specification of the
dorsal, but not the ventral, pancreatic endoderm requires signals from the
endothelial cells (Yoshitomi and Zaret,
2004
). Thus, these studies demonstrate that independent of
vascular function, endothelial cells directly regulate the specification and
differentiation of the early pancreatic endoderm.
These studies did not, however, address the functional role of blood
vessels on subsequent steps in pancreas development, such as formation of the
dorsal pancreatic mesenchyme and emergence of the dorsal pancreas. Concomitant
with the recruitment of the dorsal pancreatic mesenchyme, the aorta becomes
displaced by the invading mesenchymal cells, which through reciprocal
interactions with the endoderm stimulates dorsal pancreatic bud emergence
(Scharfmann, 2000). Based on
our findings the following model for how blood vessel function contributes to
pancreas development subsequent to endoderm specification is proposed
(Fig. 7). Blood vessel
(aorta)-derived S1P escapes from the circulation through immature endothelial
cell-cell contacts and holes in endothelial cells, and binds S1P receptors
(S1PRs) on dorsal pancreatic mesenchymal cells or endothelial cells.
Activation of heterotrimeric G-protein-coupled S1PR-mediated intracellular
signalling in mesenchymal and/or endothelial cells results in enhanced
mesenchymal cell proliferation/survival in a cell-autonomous or indirect
manner, respectively. In analogy with many epithelial organs, such as the
lung, kidney, hair and gut, the accumulation/condensation of mesenchymal cells
is necessary for proper signalling, i.e. the release of soluble growth
factors, to stimulate growth and differentiation of the epithelium, in this
particular case the dorsal pancreatic endoderm. Whether S1P, in addition to
its role in mesenchymal cell proliferation/survival, participates in the
recruitment of the lateral plate mesenchyme-derived dorsal pancreatic
mesenchyme by stimulating cell migration remains to be shown.
In conclusion, we provide evidence that adds new dimensions to how blood vessels control pancreatic development. Our findings suggest that subsequent to dorsal pancreatic endoderm specification by endothelial cell interactions, blood vessel-derived S1P regulates dorsal pancreatic mesenchyme formation and thereby emergence of the dorsal pancreatic bud. Taken together, all available data on the functional role of blood vessels in pancreatic development suggest that the patterning and function of blood vessels concomitant with organogenesis may be of general importance for guiding differentiation and morphogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Section of Endocrinology, Stem Cell Center, University of Lund, BMC, B10,
Klinikgatan 26, SE-221 84 Lund, Sweden
Present address: Department of Surgery, Johns Hopkins University, 720
Rutland Avenue, Ross 743, Baltimore, MD 21205, USA
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