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Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute (J.F.H.), Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Joel F. Habener, M.D., Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street, WEL320, Boston, Massachusetts 02114-2696. E-mail: habenerj{at}a1.mgh.harvard.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The early anlages of the pancreatic epithelium form by evagination of a narrow band of foregut endoderm destined to become the dorsal and ventral pancreata which later in development fuse into a single pancreas (2, 3). Pancreatic morphogenesis requires factors derived from the mesenchyme (4, 5). The appearance of cells expressing endocrine-specific (islets of Langerhans) and exocrine-specific (acinar) gene products proceeds in a spatially and temporally ordered manner. Insulin/glucagon-positive cells are detectable on embryonic day 9.5 (e9.5) before further morphogenesis of the pancreas commences and before other pancreatic markers appear later in development (6, 7, 8, 9). Both exocrine acinar and endocrine islet cells arise from a common progenitor cell that appears in the pancreatic duct on about day 9 in the development of rat or mouse embryos (10). Notably, pancreatic pleuripotential cells persist into adult life as proliferation of endocrine cells in the ducts (neogenesis) is stimulated during regeneration in response to partial pancreatectomy (11), injury to the pancreas by ligation of the common pancreatic duct (12), cellophane wrapping of the pancreas (13), or administration of the ß-cell toxin streptozotocin during the immediate post-natal period (14). The ductal epithelium obtained from the pancreas of adult rats can be induced to form insulin/glucagon-positive cells by coculture with fetal pancreatic mesenchyme in vitro (10). These experimental models appear to recapitulate the ontological pathways of early pancreas development.
Several transcription factors are involved in the development of the pancreas and the commitment of pancreatic progenitor cells to become cells of either the exocrine or endocrine pancreas (15). These factors include the homeodomain and basic helix-loop-helix families of transcription factors. During early embryonic development, the homeodomain transcription factor IDX-1 (also known as IPF-1, STF-1, and PDX-1) is localized to a band of foregut endoderm from which the duodenum and pancreas develop (16, 17). IDX-1 is an essential transcription factor required for the development of the pancreas, because targeted disruption of the IDX-1 gene in mice results in the phenotype of pancreatic agenesis (18, 19). Furthermore, a human subject born with pancreatic agenesis is homozygous for an inactivating mutation of the human idx-1/ipf-1 gene (20). IDX-1 also is important in the regulation of transcription of the insulin gene via binding to the TA-rich A1 (P1) and A2/A3 (FLAT) elements of the rat insulin I promoter (16). Moreover, IDX-1 synergizes with the basic helix-loop-helix proteins, e12 and e47, to stimulate insulin gene transcription (21).
A further understanding of how IDX-1 regulates the expression of genes in the endocrine pancreas may contribute to an understanding of the complex pathways of pancreatic morphogenesis and the commitment of islet cell lineages. The regulation of pancreas development appears to be accomplished at several levels of idx-1 gene expression: transcription, translation, posttranslational modifications such as phosphorylation (22), and protein-protein interactions (21, 23).
At the transcriptional level, idx-1 gene expression in ß-cells is determined by enhancer sequences located within 6.5 kb of the rat idx-1 gene promoter (24) and 4.6 kb of the mouse ipf-1 promoter (25). To better understand the spatial and temporal patterns of expression of IDX-1 during development, we created transgenic mice expressing the Escherichia coli lacZ gene under the control of the 5'-proximal 4.6 kb of the IDX-1 promoter. The bacterial lacZ gene expresses the enzyme ß-galactosidase, which is not expressed in mammalian tissues, and thereby is a reliable reporter for the expression of transgenes in mice. Here we report that the determinants of both the developmental and tissue-type specificity of expression contained within this region of the promoter closely approximate developmental expression of the endogenous IDX-1 gene. Expression of the lacZ reporter driven by the IDX-1 promoter is observed in exocrine and endocrine pancreas, common and cystic bile ducts, and pancreatic ducts, as well as in the proximal duodenum, distal stomach, and spleen. Our findings support an important role for IDX-1 in determining the development not only of the pancreas but also of other endodermally derived organs located in the midthoracic segment of the body plan.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FIXII, Stratagene, La Jolla,
CA) with a 32P-labeled PCR-generated probe corresponding to
sequences near the 5'-end of the open reading frame in the rat IDX-1
complementary DNA. Three independent genomic clones of DNA were
isolated spanning 27 kb. Selected XbaI fragments were
subcloned and sequenced. To create the IDX-1 (-4.6 kb) promoter
fragment, an XbaI site was introduced into the DNA by
site-directed mutagenesis 19 bases upstream from the translation
initiation site, and the resulting XbaI/XbaI
4.6-kb fragment was subcloned into pBluescript
(Stratagene) containing the 3.4-kb E. coli lacZ
gene with an in-frame amino-terminal nuclear localization signal from
simian virus 40 large T antigen (26) and the 0.85-kb
XbaI/EcoRI simian virus 40 polyadenylation signal
(gift from G. Wong, Genetics Institute, Cambridge, MA;
Fig. 1
). The transgene fragment was
excised from the parental plasmid by digestion with NotI and
HindIII. The fragment was purified, injected into fertilized
FVB mouse oocytes, which were then transferred into pseudopregnant CD
females. Founder mice were identified by amplification (PCR) of DNA
prepared from tails.
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Day 9.5 embryos were fixed for 45–60 min in 4% paraformaldehyde at 4 C, rinsed three times in PBS, and directly incubated with X-gal (Histomark kit, Kirkegaard & Perry Laboratories) overnight at room temperature.
Immunocytochemistry
Cryosections of the pancreas and intestinal tissues were
postfixed with 4% paraformaldehyde in PBS for 10 min at room
temperature and stained with anti-IDX-1 at a 1:750 dilution, with
antiinsulin at a 1:100 dilution (guinea pig; ICN Biomedicals, Inc., Costa Mesa, CA), or with anti-ß-galactosidase at a 1:100
dilution (mouse; a gift from Dr. G. W. Aponte, University of
California-Berkeley). Primary antisera were visualized with
biotinylated secondary antibodies (Vector Laboratories, Inc., Burlingame, CA) and avidin-biotin (Vectastain
ABC Kit, Vector Laboratories, Inc.), followed by
development with 
-ethyl carbazole. Alternatively, dual fluorescence
immunostaining was performed.
After several rinses in PBS, tissue sections were permeablized with cold methanol for 10 min at -20 C. Nonspecific binding was blocked with normal donkey serum (3%) for 30 min. Rabbit IDX-1 antiserum (1:1000 dilution) and guinea pig insulin antiserum (1:2000 dilution) were added to the sections and incubated overnight at 4 C. Primary antibodies were washed off in PBS, and slides were blocked with normal donkey serum for 10 min at room temperature. Donkey antirabbit indocarbocyanine (Cy3) and goat antiguinea pig (Cy2) conjugated (dilution, 1:1500) were added for 30 min. Slides were rinsed with PBS and mounted in fluorescence mounting medium (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Images were obtained using a Carl Zeiss epifluorescence microscope (Carl Zeiss, New York, NY) equipped with an Optronics TEC-470 CCD camera (Optronics Engineering, Goleta, CA) interfaced with a Power Macintosh 7100 installed with IP Lab Spectrum analysis software (Signal Analytics, Vienna, VA).
In situ hybridization
Rat duodenum was excised and fixed overnight in 4%
paraformaldehyde in PBS. Tissue samples were dehydrated and embedded in
paraffin. Sections were cut, and in situ hybridizations were
performed with 35S-labeled complementary DNA or
complementary RNA probes for rat somatostatin (control) or IDX-1
messenger RNA (mRNA) (27).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Strong expression of lacZ was observed in the pancreatic
islets in all six lines of transgenic mice. Expression of
lacZ was also observed in the exocrine pancreas and
pancreatic ducts. Three lines (BG4, BG13, and BG15) also showed
expression in the duodenum and distal stomach. One line (BG4) expressed
the lacZ transgene in the spleen.
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, B
and D). A cluster of epithelial cells in the distal stomach (Fig. 3, A–D) also expressed the transgene as
did epithelial cells lining the common bile duct and cystic duct
(Figs. 2F and 3H). Examination of lacZ expression in
serial sections of mouse embryos demonstrated a gradient of
transgene expression in the structures derived from the foregut. The
strongest expression of lacZ was observed proximally in the
foregut and gradually tapered to lower levels of expression in the
distal duodenum (Fig. 3, F–K).
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). On e12.5, all
of the pancreatic epithelial cells expressed the lacZ gene
(red), including nests of insulin-expressing ß-cells
(yellow green) (Fig. 4B). Immunostaining of adjacent
sections of e12.5 pancreas with antisera to ß-galactosidase and IDX-1
showed that all of the pancreatic epithelial cells were positive for
both antisera (data not shown). Thus, idx-1 gene expression
(lacZ expression) and IDX-1 protein colocalize to the
epithelial cells of the mouse pancreas on e12.5.
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). In 1-month-old mice, significant
expression of ß-galactosidase was observed within the nuclei of
exocrine cells, but only within a relatively restricted subset of
acinar lobules of the pancreas (Fig. 4D). By immunostaining, IDX-1
protein was restricted to islets of the adult rat pancreas (Fig. 4E).
Expression of IDX-1(-4.6) lacZ in the mature pancreatic ductal
system
It is believed that the islet precursor cells in the pancreatic
ducts of the adult pancreas are marked by IDX-1 expression (28);
therefore, lacZ expression was evaluated throughout the
pancreatic ductal network of adult IDX-1(-4.6 kb) lacZ
mice. Staining was observed at low levels (compared with that in
islets) in all classes of pancreatic ducts, including the common
pancreatic duct, main ducts, large intralobular ducts, and
small intralobular ducts (Fig. 5).
Notably, occasional cells expressing high levels of IDX-1(-4.6 kb)
lacZ reporter activity, comparable to levels observed within
the islets, were observed in the larger pancreatic ducts (Fig. 5C).
These cells are reminiscent of the occasional islet hormone-positive
cells previously noted in the ductal epithelium and are widely believed
to represent the early stages of islet neogenesis (29). All larger
ducts examined had some expression of the IDX-1(-4.6 kb)
lacZ reporter. In contrast, only a small percentage of ducts
within the extensive network of smaller ducts were stained.
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, A–H) and coincided with the expression of IDX-1 mRNA as detected by
in situ histohybridization (Fig. 6E). In the more distal
duodenum, lacZ expression was limited to cells within the
submucosa (Fig. 6I). This finding of lacZ expression in the
distal duodenum contrasts significantly with the expression of IDX-1
protein throughout the mucosal epithelial cells of the duodenum (Fig. 6J), but parallels the distribution of IDX-1 mRNA observed by in
situ hybridization (30).
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). Immunostaining of IDX-1 was seen in
the nuclei of a subpopulation of extrafollicular cells in the spleen
(Fig. 6L). The identity of these cells and their role are unknown, but
their presence may reflect the close relationship between the pancreas
and the spleen during development (31). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Earlier studies of the expression of IDX-1 demonstrated a colocalization of IDX-1 immunoreactivity with amylase during embryonic development, but an apparent absence of IDX-1 in the exocrine tissue of adult rats (17). In the present study, we found expression of the IDX-1 promoter-driven lacZ gene in some, but not all, lobules of exocrine tissue during adult life. The level of lacZ expression varied from one lobule to the next and tended to be similar within a given lobule. Two possible explanations for these observations arise. 1) The sensitivity of X-gal staining enabled detection of a previously unappreciated low level of IDX-1 expression in exocrine tissue. We have also observed low levels of IDX-1 expression in nuclei of pancreatic exocrine cells in normal rats and mice using a sensitive immunofluorescence technique (data not shown). 2) An exocrine-specific silencer element of the idx-1 gene may exist outside the boundaries of the 4.6-kb IDX-1 promoter fragment employed in these studies to drive expression of the lacZ gene.
In the adult mouse, extrapancreatic expression of lacZ occurred in pyloric glands of the distal stomach and Brunner’s glands of the proximal duodenum. The role of IDX-1 in the function of these glandular organs is unknown; however, the complete absence of Brunner’s glands was noted in mice homozygous for the targeted disruption of the idx-1 gene, suggesting a requirement for IDX-1 in the early development of pyloric and Brunner’s glands (19). Furthermore, the abnormalities in the duodenal epithelium of these mice (19) and the expression of lacZ in the spleen in one of our transgenic mouse lines underscore the important role of IDX-1 in the patterning of this foregut segment of the body plan. The timing of expression of the IDX-1/lacZ transgene in the spleen of one line of mice was unexpected.
Although an insertional effect could account for the observation of splenic lacZ expression, it is important to note that the spleen arises from the ventral mesenchyme just posterior to the endoderm of the foregut that gives rise to the ventral pancreas. Further, mice made transgenic to overexpress the growth factor sonic hedgehog under the control of the IDX-1 promoter have splenic agenesis (34). This is an important finding, because the suppression of sonic hedgehog expression in the dorsal mesenchyme at the location of the gut tube in the embryonic development of the dorsal pancreas is required for further development of the pancreas. In addition, the misexpression of transforming growth factor-ß directed by the IDX-1 promoter results in a dysmorphogenesis of development in which the spleen is fused to the pancreas (Miller, C. P., unpublished observations). Finally, a recent report describes the appearance of islet hormone-expressing cells in the spleen of mice with a homozygous targeted disruption of exocrine-specific transcription factor p48 (35). Taken together, these observations indicate that the expression of IDX-1 has some as yet unexplained role in the development of the spleen as well as in the development of the pancreas and Brunner’s glands of the proximal duodenum and pyloric glands in the distal stomach.
In the distal duodenum, the expression of lacZ as well as that of mRNA encoding IDX-1 detected by in situ hybridization are restricted to the submucosa, whereas IDX-1 immunoreactivity is localized exclusively to epithelial cells of the gut mucosa. These findings imply the existence of a cell-specific difference between the expression of IDX-1 mRNA and protein. Translation may be linked to the stability of the IDX-1 mRNA, and the stability of the IDX-1 protein may be such that it survives the lifetime of the duodenal epithelium. A discordance among IDX-1 promoter activity, mRNA levels, and protein has also been observed in islets in which the promoter activity of IDX-1 is strong, IDX-1 mRNA is undetectable in islets of adult rodents and is difficult to detect in total pancreatic RNA by Northern blot analysis (30), whereas the IDX-1 protein measured by immunoreactivity in islets is abundant (30, 36). In contrast, mRNA is highly abundant in ducts where promoter activity and immunoreactivity are only detected at low levels in scattered cells within the duct. The discordant levels suggest complexity at the level of the posttranscriptional control of the expression of IDX-1.
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Acknowledgments |
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Footnotes |
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2 Recipient of a USPHS Mentored Clinical Scientist Development Award.
Present address: Division of Endocrinology, Diabetes and Metabolism,
University of Pennsylvania School of Medicine, Clinical Research
Building 626, 415 Curie Boulevard, Philadelphia, Pennsylvania
19104.
3 Present address: Department of Developmental Biology, Hagedorn
Research Institute, Niels Steenensvej 6, DK 2820 Gentofte, Denmark.
4 Present address: Genetics Institute, 87 Cambridge Park
Drive, Cambridge, Massachusetts 02140.
5 Investigator with the Howard Hughes Medical Institute.
Received July 23, 1999.
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