1 Third Department of Medicine and 2 Department of Anatomy, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan
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ABSTRACT |
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The development of a variety of enteroendocrine cells of the gut is poorly understood. We tested whether immature intestinal stem cells were switched to multiple enteroendocrine hormone-producing cells by in vitro transfer of a homeobox gene. We transfected the pancreatic-duodenal homeobox 1 gene (Pdx1) into IEC-6 cells, an embryonic intestinal epithelial cell line derived from a normal rat, and selected the cells that overexpressed Pdx1 by 150-fold compared with control. The cells were examined for differentiation into enteroendocrine cells by immunocytochemical and electron microscopic analyses. Transfected cells cultured on micropore filters formed a trabecular network piled up on monolayer cells. These trabecular cells showed nuclear localization of Pdx1 protein and contained well-developed rough endoplasmic reticulum as well as many secretory granules of pleomorphic shape in the cytoplasm. Antibodies against chromogranin A, serotonin, cholecystokinin, gastrin, and somatostatin stained these secretory granules in the cytoplasm. Furthermore, immunofluorescence double staining analysis showed that different hormones were produced within a cell. These results provide the evidence that immature intestinal epithelial cells can differentiate into multiple hormone-producing enteroendocrine cells in response to overexpression of Pdx1.
enteroendocrine hormone; development; transcription factors; IEC-6 cells
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INTRODUCTION |
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ENTEROENDOCRINE CELLS of the digestive tract consist of many subtypes, which are stained with a variety of peptides as well as serotonin. Recent studies in vitro and in vivo suggest that enteroendocrine cells may share a common lineage with epithelial absorptive, goblet, and Paneth cells. All of these cells are derived from primitive intestinal stem cells located in the intestinal crypts (19, 27). However, the process by which the stem cells become enteroendocrine cells is not well known (6). Many transcription factors have been suggested to be involved in this process (1, 15, 29). In particular, BETA2 is critical for the normal development of specialized cell types arising from the gut endoderm, because secretin- and CCK-producing enteroendocrine cells fail to develop in the absence of BETA2 (15). Another homeobox gene, pancreatic/duodenal homeobox 1 (Pdx1) (10, 14, 17), is also expressed in duodenum, stomach, and pancreatic islets, although, compared with basic helix-loop-helix factor (bHLH) NeuroD/Beta2 (15) expression, it is suggested that Pdx1 has a relatively minor role in the normal differentiation of enteroendocrine cells (8, 16). However, it is also true that Pdx1 is required for organogenesis of the pancreas as well as enteroendocrine cells as revealed in Pdx1 knockout mice (5, 8). Thus the overexpression of Pdx1 may affect differentiation of immature intestinal cells into enteroendocrine cells.
To test this hypothesis, we selected an immature intestinal stem cell
line, IEC-6, that is derived from normal rat small intestine (21). These cells have characteristics of immature
intestinal crypt cells. They exhibit undifferentiated morphology and
have limited expression of intestinal cell-specific genes.
Interestingly, Suh and Traber (23) showed that
overexpression of a caudal-related homeodomain protein, Cdx2, in IEC-6
cells enabled them to differentiate into two cell types, a goblet
cell-like cell and an absorptive enterocyte-like cell. Recently, we
showed (9) that exposure of IEC-6 cells to insulin-like
growth factor-1 and insulin leads to differentiation of IEC-6 cells to
some extent through stimulation of the autocrine/paracrine secretion of
transforming growth factor-1. These observations show that IEC-6
cells provide a suitable model for examining enteroendocrine cell formation.
Thus, in this study, we transfected and overexpressed Pdx1 in IEC-6 cells. Fifty positive clones were obtained, and two stable cell lines (IEC-6-YK14 and IEC-6-YK15) showing the highest levels of Pdx1 mRNA expression were examined for enteroendocrine characteristics by immunocytochemical and electron microscopic analyses.
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MATERIALS AND METHODS |
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Cell lines and culture conditions. We purchased IEC-6 cells, a nontransformed, immature intestinal cell line derived from rat small intestine, from the American Type Culture Collection (Rockville, MD) at passage 11. The IEC-6 stock cells were maintained in T-150 flasks in DMEM supplemented with 5% dialyzed fetal bovine serum (FBS), 1 g/l D-glucose, 3.7 g/l NaHCO3, 0.1 g/l streptomycin, and 105 U/l penicillin G. The flasks were incubated at 37°C under a humidified atmosphere of 95% air-5% CO2. We subcultured these cells once a week and changed the medium three times weekly. IEC-6 cells with or without Pdx1 overexpression were seeded onto a six-well 0.5-µm micropore filter (Falcon cell culture insert, Becton Dickinson) and cultured for 4 days in DMEM containing 5% FBS before studies. The cells were then washed twice with PBS and used for histological and molecular biological analyses.
Stable transfectants. The complete coding sequence of the mouse Pdx1 cDNA was inserted into pcDNA3 plasmid to yield pcDNA3-PDX1 (28). Ten micrograms of pcDNA3-PDX1 or an equal amount of the empty vector, pcDNA3, were transfected into IEC-6 cells by electroporation at 250 V and 975 µF with a Gene Pulser (Bio-Rad, Hercules, CA). Clones resistant to selection medium containing 0.6 mg/ml of G418 (Calbiochem-Novabiochem, La Jolla, CA) were isolated and screened for Pdx1 expression by Northern blot analysis. The Pdx1 signal was compared with that from RNA extracted from neonatal rat duodenal mucosa. We obtained 50 positive clones and examined the two stable cell lines (IEC-6-YK14 and IEC-6-YK15) showing the highest levels of Pdx1 mRNA. In this report, we show the results of IEC-6-YK14 cells with the highest level of Pdx1 mRNA expression at passages 3-10, because we obtained similar results in IEC-6-YK15 cells. There was no difference in growth curve between IEC-6-YK14 cells and IEC-6 empty cells under these culture conditions.
RNA extraction and Northern blot analysis.
Neonatal Sprague-Dawley rats were anesthetized and killed by
intraperitoneal injection of pentobarbital sodium. The duodenal mucosa
and pancreas were dissected, washed with PBS, snap frozen, and then
stored at 70°C. Cultured cells were washed three times with PBS,
harvested by digestion with 10% trypsin before addition of TRIzol
(GIBCO BRL, Rockville, MD), and then stored at
70°C for completion
of RNA extraction at a later date. Total RNA was extracted by using
acid guanidinium isothiocyanate-phenol-chloroform as described
previously (13). Ten-microgram aliquots of the RNAs were
used for Northern blot analysis. The Northern blots were hybridized
with a fragment of the mouse Pdx1 cDNA that was released by
digestion with Sma I subcloned into the pcDNA-PDX1 plasmid
for amplification. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
cDNA was used for normalization to evaluate differences between
different samples.
Immunocytochemical analysis. The cells were fixed for 2 h with 4% paraformaldehyde, 0.2% picric acid, and 0.5% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at 4°C and then incubated for an additional 12 h with 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB at 4°C. After the cells were washed for 24 h with PBS at 4°C, they were incubated for 48 h with the following antibodies diluted 1:5,000 in PBS containing 0.3% Triton X-100 (PBST) at 4°C. Rabbit antisera to a synthetic peptide of Pdx1 (28), rat chromogranin A, CCK, gastrin, or gastric inhibitory polypeptide (GIP) (Yanaihara Shizuoka), mouse monoclonal antibodies to somatostatin (3) and serotonin (5-hydroxytryptamine; Ref. 26), and rabbit antiserum to apolipoprotein A-1 (25) were used in this study. After being washed with PBST, the cells were incubated for 2 h with species-specific biotinylated IgG (Vector Labs, Burlingame, CA) diluted to 1:1,000 in PBST at room temperature and then reacted for 1.5 h with avidin-biotin peroxidase complex (Vector Labs) diluted to 1:1,000 in PBST at room temperature. The immunoreaction was then visualized by developing with 0.05 M Tris · HCl buffer (pH 7.6) containing 0.01% 3,3'-diaminobenzidine, 1% ammonium nickel sulfate, and 0.0003% H2O2 for 30 min at room temperature. The micropore filters with stained cells were mounted on gelatin-coated glass slides, dehydrated by graded ethanol, coverslipped with Entellan (Merck, Darmstadt, Germany), and observed by using light microscopy. For negative-stained cells, counterstaining with 0.1% neutral red solution was performed.
For the double immunofluorescence staining, the fixed cells were incubated for 48 h in either a mixture of the specific antibodies against chromogranin A and serotonin or antibodies against serotonin and somatostatin, which were diluted to 1:5,000 in PBST at 4°C. After being washed with PBST, the cells were incubated for 2 h at room temperature with either a mixture of FITC-labeled anti-mouse IgG (Vector Labs) and Texas red-labeled anti-rabbit IgG (Vector Labs) for chromogranin A-serotonin or serotonin-somatostatin double staining. After rinsing cells with PBST, we mounted micropore filters with attached cells on the glass slides, and cells were dried, coverslipped with liquid paraffin, and observed under a confocal laser scanning image system (MRC-600; Bio-Rad). We also performed immunofluorescence staining for Pdx1, CCK, gastrin, GIP, and apolipoprotein A-1, in which Texas red-labeled anti-rabbit IgG (Vector) was used as a second antibody and was observed by using the confocal laser scanning image system. The specificity of the positive staining was examined by an immunocytochemical absorption study. The primary antibodies were replaced with an antigen-antibody mixture in which we used recombinant rat chromogranin A, serotonin, synthetic somatostatin 14, and recombinant Pdx1 peptide at a concentration of 10 mM each.Electron microscopy. The cultured cells were fixed as described in Immunocytochemical analysis and incubated for 1 h with 1% OsO4 in 0.1 M PBS at 4°C. The samples were then dehydrated with a graded series of ethanol and propylene oxide and embedded in epoxy resin. Ultrathin sections were made in an ultramicrotome (Ultracut E; Reichert-Jung, Vienna, Austria) vertically or horizontally along the filter and mounted on 200-mesh copper grids. Cells were stained for 20 min with 2% uranyl acetate and for an additional 5 min with Reynolds' solution and then observed under an electron microscope (H-7100; Hitachi, Tokyo, Japan).
For the immunoelectron microscopic study, fixed cells were incubated overnight with LR Gold resin (London Resin, Basingstoke, UK) atRT-PCR analysis.
Using 1 µg of the total RNA as a template and a set of antisense
oligonucleotides complementary to each mRNA, we reverse transcribed the
first cDNA strands in 30 µl of a reaction mixture containing reagents
(Takara, Kyoto, Japan). For the subsequent PCR, 1 µl of the reaction
mixture was used each time. The specific oligonucleotide primers (5'
and 3') were used for the amplification of each transcription factor.
The thermal cycle profile was as follows. A single 1-min denaturing
step at 94°C was followed by 30 cycles of 30 s at 94°C, 45 s at 54°C, and 1 min at 72°C. The primer pairs were
as follows (forward and reverse): NeuroD/Beta2,
5'-GCAAAGGTTTGTCCCAGC-3' and 5'-ACGTGGAAGACGTGGGAG-3'; hepatocyte
nuclear factor (HNF)-1, 5'-ATGAGCCGTCGTCTCCTC-3' and
5'-GTTGGATGGCAGCAGGTG-3'; HNF-3
, 5'-AACATGAACTCCGGCCTGGG-3' and
5'-AGCTGGCTAGCCTTTCCGTG-3'; HNF-3
, 5'-GGCTCCTTCTGGACCCTG-3' and
5'-ACCTCGCTTGTGCTCCTG-3'; HNF-4
, 5'-TACATCAACGACCGGCAG-3' and
5'-GTGGAGTCTCAGGGGTGG-3'; glucose-6-phosphate dehydrogenase,
5'-GACCTGCAGAGCTCCAATCAAC-3' and
5'-CACGACCCTCAGTACCAAAGGG-3'. Each PCR product was
confirmed by its respective size based on the expected size of each
transcription factor.
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RESULTS |
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Morphological changes in IEC-6 cells that overexpress Pdx1.
We obtained 50 G418-resistant clones after transfection of IEC-6 cells
with pcDNA3-Pdx1 and then used two stable clones, IEC-6-YK14 and
IEC-6-YK15, exhibiting the highest level of Pdx1 mRNA
expression in the following experiments (Fig.
1). The level of Pdx1 mRNA in
IEC-6-YK14 cells was 150-fold higher than that in IEC-6 cells containing empty vector. This increase was specific for
Pdx1, because the level of Cdx2 mRNA in the
IEC-6-YK14 cells was not increased (data not shown).
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Immunocytochemical analyses of production of multiple hormones in
IEC-6-YK14 cells.
To characterize the trabecular cells, we stained them with an antibody
against chromogranin A, an acidic protein present in the secretory
granules of a wide variety of endocrine cells, and stained them with an
antibody against apolipoprotein A-1 as a marker of absorptive
enterocytes. IEC-6 cells containing empty vector cultured on micropore
filters showed no staining for chromogranin A (Fig.
4a) but positive staining with
apolipoprotein A-1 (Fig. 4c). In contrast, IEC-6-YK14
cells showed positive staining for chromogranin A only in
trabecular network-forming cells (Fig. 4b) but negative
staining with apolipoprotein A-1 (Fig. 4d). Furthermore, those trabecular cells also positively stained with antisera against serotonin (Fig. 5, a,
d, and g), somatostatin (Fig.
5h), CCK (Fig. 5j), and gastrin (Fig.
5k). However, immunoreactive insulin, glucagon, glucagon-like peptide-1 (GLP-1), and GIP were not detected (data not shown).
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Electron microscopic features of IEC-6-YK14 cells.
IEC-6-YK14 cells cultured on micropore filters for 4 days were
examined by electron microscopy. Two different types of cells were
seen, i.e., a monolayer of thin-body cells that were in contact with
the micropore filter and thick-body cells piled up on the monolayer
cells (Fig. 6a). These
findings and those obtained by light microscopy indicated that the
trabecular network-forming cells were thick-body cells. In these cells,
the rough endoplasmic reticulum was well developed, and, most
characteristically, the cells had many cytoplasmic granules of
pleomorphic shapes (Fig. 6b). Moreover, immunoelectron
microscopic analysis for localization of chromogranin A showed that the
cores of some granules were colocalized with immunogold particles, but
other granules were not (Fig. 6c). In IEC-6 empty
cells, on the other hand, the endoplasmic reticulum was poorly
developed and granules were not observed in the cytoplasm (Fig.
6d).
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mRNA expression of transcription factors in IEC-6-YK14 cells.
We examined the expression of some important nuclear
transcription factors in IEC-6 cells; expressions in IEC-6-YK14 cells were compared with those of either IEC-6 empty cells or neonatal rat
small intestine by RT-PCR (Fig. 7). IEC-6
cells, IEC-6 empty cells, and IEC-6-YK14 cells were expressed with
NeuroD/Beta2, HNF-1, HNF-3
, HNF-3
, and HNF-4
, which
were similar to those of neonatal rat small intestine.
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DISCUSSION |
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In this report, we show that an immature intestinal crypt cell line undergoes differentiation into multiple hormone-producing enteroendocrine cells after being overexpressed with Pdx1. Compared with wild-type IEC-6 cells cultured on micropore, IEC-6-YK14 cells overexpressing Pdx1 consisted of two cell types: one cell type formed a trabecular cell network, and the other formed a round cell mass surrounded by trabecular cells. The trabecular cells showed strong Pdx1-positive immunoreactivity in the nucleus compared with nontrabeculated cells. Only trabecular cells acquired enteroendocrine cell-like characteristics, leading to the synthesis of chromogranin A and hormones including serotonin, somatostatin, CCK, and gastrin. However, we could not show synthesis of insulin, glucagon, GLP-1, or GIP in those cells under these culture conditions. Consistently, an expression of apolipoprotein A-1, a typical enterocyte marker (30), was lost in IEC-6-YK14 cells, although IEC-6 cells expressed the marker. These data suggest that overexpression of Pdx1 induces the differentiation into endocrine cells specific for the upper small intestine but not the pancreas. Immunofluorescence double staining for these products showed that multiple hormones were colocalized in the same cells. These data are intriguing in view of the evidence that colocalization of multiple hormones in one cell is a characteristic feature of an early stage of endocrine cell differentiation (22).
Our results directly indicate that overexpression of Pdx1 can play some role in the differentiation of enteroendocrine cells from immature epithelial cells. Although Pdx1 seems to be important for enteroendocrine cell formation, the differentiation of intestinal stem cells into nonendocrine cells requires the factor Cdx2 (12, 23). This factor was not normally expressed in IEC-6 cells in the presence or absence of Pdx1. Wild-type IEC-6 cells form a simple monolayer of flat epithelial cells in culture. Overexpression of Cdx2 in IEC-6 cells enables them to differentiate into absorptive enterocytes or goblet cells. These cells grown in culture tend to form clusters of round cells and a surrounding trabecular cell network (latticelike structure) (23). Electron microscopic analysis also reveals that the cells overexpressing Cdx2 grow in piles. The piles consist of a lower layer of flat cells and an upper layer of trabecular cells, which are differentiated cells. These morphological characteristics were also seen in the cells overexpressing Pdx1 in the present study. We found that the upper cells had strong immunostaining for various hormones but the lower monolayer of nontrabeculated flat cells did not. On the basis of these observations, it appears that a divergent mature intestinal cell lineage can arise from IEC-6 cells in response to overexpression of either Pdx1 or Cdx2. Cdx2 expression stimulates immature cells to the formation of more differentiated absorptive enterocytes and goblet cells, and Pdx1 overexpression leads to the formation of enteroendocrine cells.
How do our results provide a better understanding of intestinal cell
differentiation into enteroendocrine-like cells? According to published
studies, enteroendocrine cell differentiation requires other more
important transcription factors including bHLH NeuroD/Beta2 (15), neurogenin-3 (4), and members of the
HNF family (2, 11, 20, 22, 24). Furthermore, it has also
been reported that Hes1, which is activated by Notch signaling, is
shown to inhibit neurogenin 3 and NeuroD cascade, resulting in
impairment of terminal differentiation of endodermal endocrine cells
(7). Mice homozygous for a null mutation in some of these
transcription factors showed marked morphological changes, with defects
in endocrine function not only in the small intestine but also in the
pancreas. For example, secretin- and CCK-producing enteroendocrine
cells failed to develop in the absence of NeuroD/Beta2
(15). Furthermore, foregut morphogenesis is severely
affected and tube formation is defective in HNF-3/
embryos (1, 29). In the present study, we found that
HNF-1
, HNF-3
, HNF-3
, HNF-4
, and NeuroD/Beta2 were expressed
in IEC-6 cells at the basal culture conditions (Fig. 7). However,
analysis of complex interactions among many transcription factors for
the control of the differentiation of intestinal cells into
enteroendocrine cells awaits future study, because the levels of these
transcription factors in IEC-6 cells with or without Pdx1
overexpression were too low to analyze further. Thus the endocrine
differentiation seen by Pdx1 overexpression may indicate
that the relatively immature IEC-6 cells have retained some of the
multipotential properties of the intestinal stem cell that permit
forced differentiation into enteroendocrine cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Yoshitaka Kajimoto (Dept. of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine) for providing us with Pdx1 cDNA and antiserum. We also thank Dr. P. G. Traber (University of Pennsylvania) for providing the Cdx2 cDNA and Dr. C. V. E. Write (Vanderbilt University School of Medicine) for kind advice on Pdx1.
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FOOTNOTES |
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This work was supported, in part, by a grant for scientific research from the Ministry of Education, Science and Culture of Japan (no. 10671064).
Address for reprint requests and other correspondence: A. Kashiwagi, Third Dept. of Medicine, Shiga Univ. of Medical Science, Otsu, Shiga 520-2192 Japan (E-mail: kasiwagi{at}belle.shiga-med.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 June 2000; accepted in final form 19 March 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ang, SL,
and
Rossant J.
HNF-3 is essential for node and notochord formation in mouse development.
Cell
78:
561-574,
1994[ISI][Medline].
2.
Erickson, RH,
Lai RS,
and
Kim YS.
Role of hepatocyte nuclear factor 1 and 1
in the transcriptional regulation of human dipeptidyl peptidase IV during differentiation of Caco-2 cells.
Biochem Biophys Res Commun
270:
235-239,
2000[ISI][Medline].
3.
Fujimiya, M,
Christopher HS,
McIntosh CH,
Kimura H,
and
Kwok YN.
Effect of carbachol on luminal release of somatostatin from isolated perfused rat duodenum.
Neurosci Lett
145:
229-233,
1992[ISI][Medline].
4.
Gradwohl, G,
Dierich A,
LeMeur M,
and
Guillemot F.
Neurogenin 3 is required for the development of the four endocrine cell lineages of the pancreas.
Proc Natl Acad Sci USA
97:
1607-1611,
2000
5.
Habener, JF,
and
Stoffers DA.
A newly discovered role of transcription factors involved in pancreas development and the pathogenesis of diabetes mellitus.
Proc Assoc Am Physicians
110:
12-21,
1998[ISI][Medline].
6.
Höcker, M,
and
Wiedenmann B.
Molecular mechanisms of enteroendocrine differentiation.
Ann NY Acad Sci
859:
160-174,
1998
7.
Jensen, J,
Pedersen EE,
Galante P,
Hald J,
Heller RS,
Ishibashi M,
Kageyama R,
Guillemot F,
Serup P,
and
Madsen OD.
Control of endodermal endocrine development by Hes-1.
Nat Genet
24:
36-44,
2000[ISI][Medline].
8.
Jonsson, J,
Carlsson L,
Thomas E,
and
Edlund H.
Insulin-promoter-factor 1 is required for pancreas development in mice.
Nature
371:
606-609,
1994[ISI][Medline].
9.
Kojima, H,
Hidaka H,
Matsumura K,
Fujita Y,
Nishio Y,
Maegawa H,
Haneda M,
Yasuda H,
Fujimiya M,
Kikkawa R,
and
Kashiwagi A.
Concerted regulation of early enterocyte differentiation by insulin-like growth factor I, insulin, and transforming growth factor-1.
Proc Assoc Am Physicians
110:
197-206,
1998[ISI][Medline].
10.
Larsson, LI,
Madsen OD,
Serup P,
Jonsson J,
and
Edlund H.
Pancreatic-duodenal homeobox 1role in gastric endocrine patterning.
Mech Dev
60:
175-184,
1996[ISI][Medline].
11.
Levinson-Dushnik, M,
and
Benvenisty N.
Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells.
Mol Cell Biol
7:
3817-3822,
1997.
12.
Lorentz, O,
Duluc I,
Arcangelis AD,
Simon-Assmann P,
Kedinger M,
and
Freund JN.
Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation.
J Cell Biol
139:
1553-1565,
1997
13.
Mashima, H,
Ohnishi H,
Wakabayashi K,
Mine T,
Miyagawa J,
Hanafusa T,
Seno M,
Yamada H,
and
Kojima I.
Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells.
J Clin Invest
97:
1647-1654,
1996
14.
Miller, CP,
McGehee RE, Jr,
and
Habener JF.
IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.
EMBO J
13:
1145-1156,
1994[Abstract].
15.
Naya, FJ,
Huang HP,
Qiu Y,
Mutoh H,
DeMayo FJ,
Leiter AB,
and
Tsai MJ.
Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice.
Genes Dev
11:
2323-2334,
1997
16.
Offield, MF,
Jetton TL,
Labosky PA,
Ray M,
Stein RW,
Magnuson MA,
Hogan BLM,
and
Wright CVE
PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum.
Development
122:
983-995,
1996
17.
Ohlsson, H,
Karlsson K,
and
Edlund T.
IPF1, a homeodomain-containing transactivator of the insulin gene.
EMBO J
12:
4251-4259,
1993[Abstract].
18.
Okumiya, K,
Matsubayashi K,
Maeda T,
and
Fujimiya M.
Change in subcellular localization of gastrin-like immunoreactivity in epithelial cells of rat duodenum induced by carbachol.
Peptides
17:
225-232,
1996[ISI][Medline].
19.
Podolsky, DK.
Regulation of intestinal epithelial proliferation: a few answers, many questions.
Am J Physiol Gastrointest Liver Physiol
264:
G179-G186,
1993
20.
Quaroni, A,
and
May RJ.
Establishment and characterization of intestinal epithelial cell cultures.
Methods Cell Biol
21B:
404-427,
1980.
21.
Rausa, FM,
Galarneau L,
Belanger L,
and
Costa RH.
The nuclear receptor fetoprotein transcription factor is coexpressed with its target gene HNF-3 in the developing murine liver, intestine and pancreas.
Mech Dev
89:
185-188,
1999[ISI][Medline].
22.
Slack, JM.
Developmental biology of the pancreas.
Development
121:
1569-1580,
1995
23.
Suh, E,
and
Traber PG.
An intestine-specific homeobox gene regulates proliferation and differentiation.
Mol Cell Biol
16:
619-625,
1996[Abstract].
24.
Swenson, ES,
Mann EA,
Jump ML,
and
Giannella RA.
Hepatocyte nuclear factor-4 regulates intestinal expression of the guanylin/heat-stable toxin receptor.
Am J Physiol Gastrointest Liver Physiol
276:
G728-G736,
1999
25.
Taylor, AH,
Raymond J,
Dionne JM,
Romney J,
Lawless DE,
Chan J,
Wanke IE,
and
Wong NCW
Glucocorticoid increases rat apolipoprotein A-1 promoter activity.
J Lipid Res
37:
2232-2243,
1996[Abstract].
26.
Tohyama, I,
Kameyama M,
and
Kimura H.
Quantitative morphometric analysis of two types of serotonin-immunoreactive nerve fibres differentially responding to p-chlorophenylalanine treatment in the rat brain.
Neuroscience
26:
971-991,
1988[ISI][Medline].
27.
Traber, PG,
and
Silberg DG.
Intestine-specific gene transcription.
Annu Rev Physiol
58:
275-279,
1996[ISI][Medline].
28.
Watada, H,
Kajimoto Y,
Miyagawa J,
Hanafusa T,
Hamaguchi K,
Matsuoka T,
Yamamoto K,
Matsuzawa Y,
Kawamori R,
and
Yamasaki Y.
PDX-1 induces insulin and glucokinase gene expressions in aTC1 clone 6 cells in the presence of betacellulin.
Diabetes
4:
1826-1831,
1996.
29.
Weinstein, DC,
Altaba AR,
Chen WS,
Hoodless P,
Prezioso VR,
Jessell TM,
and
Darnell JE, Jr.
The winged-helix transcription factor HNF-3 is required for notochord development in the mouse embryo.
Cell
78:
575-588,
1994[ISI][Medline].
30.
Zannis, VI,
Kurnit DM,
and
Breslow JL.
Hepatic apo A-1 and apo-E and intestinal apo A-1 are synthesized in precursor isoprotein forms by organ cultures of human fetal tissues.
J Biol Chem
257:
536-544,
1982