help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stoffers, D. A.
Right arrow Articles by Habener, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stoffers, D. A.
Right arrow Articles by Habener, J. F.
Endocrinology Vol. 140, No. 11 5374-5381
Copyright © 1999 by The Endocrine Society


ARTICLES

Developmental Expression of the Homeodomain Protein IDX-1 in Mice Transgenic for an IDX-1 Promoter/lacZ Transcriptional Reporter1

Doris A. Stoffers2, R. Scott Heller3, Christopher P. Miller4 and Joel F. Habener5

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of the homeodomain transcription factor IDX-1 (also known as IPF-1, STF-1, and PDX-1) is required for pancreas development, because disruption of the gene in mice and humans results in pancreatic agenesis. During embryonic development the idx-1 gene is first expressed in a localized region of foregut endoderm from which the duodenum and pancreas later develop. To more fully understand the role of IDX-1 in pancreas development, transgenic mice expressing the Escherichia coli lacZ gene under control of the 5'-proximal 4.6 kb of the idx-1 promoter were created as a reporter for the developmental expression of IDX-1. Here we show that the determinants for the developmental and tissue-specific expression of the endogenous idx-1 gene are faithfully reproduced by the 4.6-kb region of the idx-1 promoter. Expression of lacZ is detected in the development of the exocrine and endocrine pancreas in pancreatic ducts, common bile and cystic ducts, pyloric glands of the distal stomach, Brunner’s glands, the intestinal epithelium of the duodenum, and the spleen. The observed spatial and temporal pattern of lacZ expression directed by the IDX-1 promoter further supports an important role of IDX-1 in specifying the development of several endodermal structures within the midsegment of the body. An unexpected finding is that IDX-1 promoter-driven (transcriptional) lacZ activity does not always coincide with the localization of IDX-1 messenger RNA by in situ hybridization and IDX-1 protein by immunocytochemistry in adult rat duodenum, suggesting the existence of regulation of IDX-1 expression at the posttranscriptional level of expression of the idx-1 gene.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
AN UNDERSTANDING of the factors underlying the development of the endocrine and exocrine pancreas may provide insights into the pathogenesis of diabetes mellitus. The prevalence of diabetes is increasing worldwide in epidemic proportions (1). Diabetes is caused by an absolute (type 1) or a relative (type 2) deficiency of the pancreas to produce insulin in amounts sufficient to meet the body’s needs. A clearly established defect in the causation of both type 1 and type 2 diabetes is a reduction in the mass of ß-cells in the pancreas, which are required to produce insulin. Thus, studies that address the role of key factors, such as the transcription factor IDX-1/IPF-1/PDX-1 that is involved in pancreas development and the formation of ß-cells, may provide insights into the regulation of ß-cell mass. Here we present the results of studies that further elucidate the role of IDX-1 in mouse pancreas development.

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.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reporter DNA fusion gene construction and the creation of transgenic mice
The mouse idx-1 gene was cloned by screening a mouse genomic library (SV129, {lambda}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. 1Go). 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.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 1. Diagram of the recombinant DNA plasmid consisting of IDX-1 promoter and lacZ reporter transgene. NLS, Nuclear localization signal; SV40 poly-A, simian virus 40 polyadenylation signal. Cross-hatching (horizontal arrow) designates the coding sequence for ß-galactosidase.

 
X-Gal staining for expression of ß-galactosidase
Embryos and tissues were dissected at the designated ages, fixed for 45–60 min in 4% paraformaldehyde at 4 C, and cryoprotected in 30% sucrose in PBS overnight. After embedding in OCT medium (Miles, Elkhart, IL), cryosections were prepared. These sections were incubated with X-gal (Histomark kit, Kirkegaard & Perry Laboratories, Gaithersburg, MD) in a humidified chamber overnight at room temperature. Under these conditions, no endogenous background of ß-galactosidase activity was observed. Sections were postfixed in 4% paraformaldehyde for 10 min to promote adhesion to slides before mounting in Glycergel (DAKO Corp., Carpinteria, CA). Adjacent sections were stained with hematoxylin and eosin to aid in the identification of organ structures.

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 {alpha}-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).


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Six independent lines of mice expressing the IDX-1-(-4.6 kb) lacZ transgene were examined for tissue-specific lacZ expression (Table 1Go). 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.


View this table:
[in this window]
[in a new window]
 
Table 1. Distribution of lacZ expression in six independent lines of mice

 
lacZ expression in midgestation embryos
On e12.5, lacZ was expressed in the majority of cells of the pancreatic epithelium (Fig. 2Go, B and D). A cluster of epithelial cells in the distal stomach (Fig. 3Go, A–D) also expressed the transgene as did epithelial cells lining the common bile duct and cystic duct (Figs. 2FGo and 3HGo). 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. 3Go, F–K).



View larger version (94K):
[in this window]
[in a new window]
 
Figure 2. Developmental expression of IDX(-4.6)lacZ on e12.5. Expression is targeted to the majority of cells in pancreatic epithelium (P) at this stage. A–D, Epithelial cells lining the distal stomach (S), common bile duct (cbd), and cystic duct (cd) express the transgene (E and F). A, C, and E, Hematoxylin and eosin stain; B, D, and F, ß-galactosidase staining of adjacent sections. For reference, liver (L) and heart (H) are marked.

 


View larger version (116K):
[in this window]
[in a new window]
 
Figure 3. Anatomical distribution of the expression of IDX(-4.6)lacZ in 8-µm serial sections through an e12.5 embryo. Every 10th section was stained with X-gal to detect ß-galactosidase activity. Staining was observed in the distal stomach (A–D), pancreas (D–I), common bile duct (H), and duodenum (F–K). S, Stomach; DP, dorsal pancreas; VP, ventral pancreas; pd, pancreatic duct; cbd, common bile duct; D, duodenum. Approximate locations of sagittal sections examined are shown by the vertical lines through the embryo diagram (L).

 
Coexpression of IDX-1(-4.6) lacZ with markers of the endocrine pancreas in late gestation and at 1 month of age
The idx-1(-4.6 kb) lacZ gene is clearly expressed in both dorsal and ventral pancreata on e9.5 before the rotation and fusion of the two pancreatic buds (Fig. 4AGo). On e12.5, all of the pancreatic epithelial cells expressed the lacZ gene (red), including nests of insulin-expressing ß-cells (yellow green) (Fig. 4BGo). 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.



View larger version (79K):
[in this window]
[in a new window]
 
Figure 4. Expression of IDX(-4.6)lacZ in the pancreas during late gestation and in adulthood. A, Whole mount lacZ expression in a transgenic e9.5 embryo; B, dual fluorescence of lacZ (Cy3, red) and insulin (Cy2, green) immunoreactivity in the same frozen sections of e12.5 pancreas. Colocalization of IDX-1 and insulin is shown as yellow (C) dual fluorescence immunostaining of postnatal day 1 mouse pancreas as in B. D, X-Gal staining of exocrine pancreas (E) and an islet (I) of a 1-month-old IDX(-4.6)lacZ mouse (magnification, x200). E, IDX-1 immunocytochemical staining of adult pancreas (rat), exocrine pancreas (E) and an islet (I) are indicated (magnification, x400).

 
By postnatal day one, expression of the lacZ gene was restricted predominantly to the insulin-producing cells, with occasional expression in nuclei of the exocrine pancreas (Fig. 4CGo). 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. 4DGo). By immunostaining, IDX-1 protein was restricted to islets of the adult rat pancreas (Fig. 4EGo).

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. 5Go). 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. 5CGo). 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.



View larger version (129K):
[in this window]
[in a new window]
 
Figure 5. Expression of IDX(-4.6)lacZ in the pancreatic duct system of the adult pancreas. A and B, Transgene expression in the common pancreatic duct (CPD). C and D, Expression in the main ducts (M. D.). E and F, lacZ expression in a subset of the smaller ducts (SD). Occasional cells exhibiting intense levels of transgene expression are observed in the main ducts (arrows in C).

 
Localization of the expression to Brunner’s glands and pyloric glands of the proximal duodenum and distal stomach
Later in the development of the rat embryo, the IDX-1 promoter-driven expression of lacZ in the gut became restricted to Brunner’s glands of the proximal duodenum and pyloric glands of the distal stomach (Fig. 6Go, A–H) and coincided with the expression of IDX-1 mRNA as detected by in situ histohybridization (Fig. 6EGo). In the more distal duodenum, lacZ expression was limited to cells within the submucosa (Fig. 6IGo). 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. 6JGo), but parallels the distribution of IDX-1 mRNA observed by in situ hybridization (30).



View larger version (89K):
[in this window]
[in a new window]
 
Figure 6. Extrapancreatic expression of IDX-1 and lacZ transgene in adult mice. Extrapancreatic expression of IDX-1 in the foregut. A and B, Transgene expression in the pyloric glands of the distal stomach; phase contrast (A) and light micrograph (B) of X-gal-stained tissue are shown. C–F, Expression of IDX-1(-4.5)/lacZ and IDX-1 mRNA in Brunner’s glands of the proximal duodenum. Phase contrast (C) and light micrograph (D) of X-gal-stained tissue are shown. E, In situ hybridization with an IDX-1 antisense riboprobe. F, An adjacent control section hybridized with a somatostatin antisense riboprobe. G–J, Expression of IDX-1 in the duodenum. G, In situ hybridization with an IDX-1 antisense riboprobe. H, Adjacent control section hybridized with a somatostatin antisense riboprobe; I, X-gal staining in distal duodenum. J, IDX-1 immunostaining in adult rat duodenum. K and L, Expression of IDX-1 in the spleen. K, IDX-1(-4.6)lacZ transgene. L, IDX-1 immunostaining in a subpopulation of extrafollicular cells in the rat spleen.

 
Expression of IDX-1 in the spleen
In one line of mice, expression of IDX-1(-4.6)lacZ was observed in the spleen (Fig. 6KGo). Immunostaining of IDX-1 was seen in the nuclei of a subpopulation of extrafollicular cells in the spleen (Fig. 6LGo). 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
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
These studies further show that the 4.5-kb 5'-promoter sequence of the idx-1 gene is sufficient to faithfully recapitulate the developmental expression of the IDX-1 gene in mice. In an earlier study the lacZ gene was driven by the endogenous IDX-1 promoter (19). On e12.5, lacZ, driven by the IDX-1 promoter, is expressed throughout the pancreatic epithelium. As development of the pancreas proceeds, expression of the lacZ reporter gene in the endocrine pancreas becomes progressively more spatially restricted to insulin-positive ß-cells within the islets. We also observed a low level of lacZ expression in epithelial cells within the ducts of the exocrine pancreas in adult animals. This finding is consistent with the idea that IDX-1 is expressed in progenitor cells located within ducts that give rise to both exocrine and endocrine cell types (17). Previously reported observations consistent with this idea are the findings of increased levels of IDX-1 in pancreatic ducts during regeneration of the pancreas after partial pancreatectomy (28). We observed strong expression of lacZ in epithelial cells of the distal stomach and proximal duodenum with gradually decreasing expression along the distal duodenum as well as in the cuboidal epithelium of the common bile duct and cystic duct, which are lined by GLUT2-positive cells (32). Recent data suggest that IDX-1 may regulate GLUT2 expression (33).

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.


    Acknowledgments
 
We thank S. Bonner-Weir for advice regarding ductal expression of the reporter transgene, H. Hermann and V. Stanojevic for excellent experimental assistance, and T. Budde for preparation of the manuscript.


    Footnotes
 
1 This work was supported in part by USPHS Grant DK-30457 (to J.F.H.). Back

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. Back

3 Present address: Department of Developmental Biology, Hagedorn Research Institute, Niels Steenensvej 6, DK 2820 Gentofte, Denmark. Back

4 Present address: Genetics Institute, 87 Cambridge Park Drive, Cambridge, Massachusetts 02140. Back

5 Investigator with the Howard Hughes Medical Institute. Back

Received July 23, 1999.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. O’Rahilly S 1997 Non-insulin dependent diabetes mellitus: the gathering storm. Br Med J 314:955–959[Free Full Text]
  2. Pictet R, Rutter WJ 1972 Development of the embryonic endocrine pancreas. In: Steiner DF, Freinkel N (eds) Handbook of Physiology. American Physiology Society, Washington DC, vol 1:25–66
  3. Slack JMW 1995 Developmental biology of the pancreas. Development 121:1569–1580[Abstract]
  4. Gittes GK, Galante PE, Hanahan D, Rutter WJ, Debas HT 1996 Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors. Development 122:439–447[Abstract]
  5. Golosow N, Grobstein C 1962 Epitheliomesenchymal interactions in pancreatic morphogenesis. Dev Biol 4:242–255[CrossRef][Medline]
  6. Alpert S, Hanahan D, Teitelman G 1988 Hybrid insulin genes reveal a developmental lineage for pancreatic endocrine cells and imply a relationship with neurons. Cell 53:295–308[CrossRef][Medline]
  7. Herrera P-L, Huarte J, Sanvito F, Meda P, Orci P, Vasalli JD 1991 Embryogenesis of the murine endocrine pancreas: early expression of pancreatic polypeptide gene. Development 113:1257–1265[Abstract]
  8. Gittes GK, Rutter WJ 1992 Onset of cell-specific expression in the developing mouse pancreas. Proc Natl Acad Sci USA 89:1128–1132[Abstract/Free Full Text]
  9. Teitelman G, Alpert S, Polak JM, Mertinez A, Hanahan D 1993 Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon, and the neuronal proteins tyrosine hydroxylase and neuropeptide Y but not pancreatic polypeptide. Development 118:1031–1039[Abstract]
  10. Dudek RW, Lawrence IE, Hill RS, Johnson RC 1991 Induction of islet cytodifferentiation by fetal mesenchyme in adult pancreatic ductal epithelium. Diabetes 40:1014–1048
  11. Bonner-Weir S 1994 Regulation of pancreatic ß-cell mass in vivo. Recent Prog Horm Res 49:91–104
  12. Wang RN, Kloppel G, Bouwens L 1995 Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 38:1405–1411[CrossRef][Medline]
  13. Rosenberg L, Vinik AI 1993 In vitro stimulation of hamster pancreatic duct growth by an extract derived from the "wrapped" pancreas. Pancreas 8:255–260[Medline]
  14. Bonner-Weir S, Trent DF, Honey RN, Weir GC 1981 Responses of neonatal rat islets to streptozotocin: limited B-cell regeneration and hyperglycemia. Diabetes 30:64–69[Abstract]
  15. Stoffers DA, Thomas MK, Habener JF 1997 Homeodomain protein IDX-1: a master regulator of pancreas development and insulin gene expression. Trends Endocrinol Metab 8:145–151
  16. Ohlsson H, Karlsson K, Edlund T 1993 IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J 12:4251–4259[Medline]
  17. Guz Y, Montminy MR, Stein R, Leonard J, Gamer LW, Wright CVE, Teitelman G 1995 Expression of murine STF-1, A putative insulin gene transcription factor, in ß cell of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny. Development 121:11–18[Abstract]
  18. Jonsson J, Carlsson L, Edlund T, Edlund H 1994 Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371:606–609[CrossRef][Medline]
  19. Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BLM, Wright CVE 1996 PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122:983–995[Abstract]
  20. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF 1997 Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 coding region. Nat Genet 15:106–110[CrossRef][Medline]
  21. Peers B, Leonard J, Sharma S, Teitelman G, Montminy MR 1994 Insulin expression in pancreatic islet cells relies on cooperative interactions between the helix loop helix factor E47 and the homeobox factor STF-1. Mol Endocrinol 8:1798–1806[Abstract/Free Full Text]
  22. MacFarlane W, Read ML, Gilligan M, Bujalska I, Docherty K 1994 Glucose modulates the binding activity of the ß cell transcription factor IUF-1 in a phosphorylation-dependent manner. Biochem J 303:625–631
  23. Peers B, Sharma S, Johnson T, Kamps M, Montminy M 1995 The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain. Mol Cell Biol 15:7091–7097[Abstract]
  24. Sharma S, Leonard J, Lee S, Chapman HD, Leiter EH, Montminy MR 1996 Pancreatic islet expression of the homeobox factor STF-1 relies on an E-box motif that binds USF. J Biol Chem 271:2294–2299[Abstract/Free Full Text]
  25. Wu KL, Gannon M, Peshavaria M, Offield MF, Henderson E, Ray M, Marks A, Gamer LW, Wright CV, Stein R 1997 Hepatocyte nuclear factor 3ß is involved in pancreatic ß-cell-specific transcription of the pdx-1 gene. Mol Cell Biol 17:6002–6013[Abstract]
  26. Yoneda Y, Semba T, Kaneda Y, Noble RL, Matsuoka Y, Kurihara T, Okada Y, Imamoto N 1992 A long synthetic peptide containing a nuclear localization signal and its flanking sequences of SV40 T-antigen directs the transport of IgM into the nucleus efficiently. Exp Cell Res 201:313–320[CrossRef][Medline]
  27. Simmons DM, Arriza JL, Swanson LW 1989. J Histotechnol 12:169–181
  28. Sharma A, Zangen DH, Reitz P, Taneja M, Lissauer ME, Miller CP, Weir GC, Habener JF, Bonner-Weir S 1999 The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreas regeneration. Diabetes 48:507–513[Abstract]
  29. Madden ME, Sarras MPJ 1989 The pancreatic ductal system of the rat: cell diversity, ultrastructure, and innervation. Pancreas 4:472–485[Medline]
  30. Miller CP, McGehee R, Habener JF 1994 IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene. EMBO J 13:1145–1156[Medline]
  31. Skandalakis LJ, Gray SW, Ricketts R, Skandalakis JE The spleen. In: Skandalakis JE, Gray SW (eds) Embryology for Surgeons: The Embryological Basis for the Treatment of Congenital Anomalies. Williams & Wilkins, Baltimore, pp 334–365
  32. Pang K, Mukonoweshuro C, Wong GG 1994 Beta cells arise from glucose transporter type 2 (Glut2)-expressing epithelial cells of the developing rat pancreas. Proc Natl Acad Sci USA 91:9559–9563[Abstract/Free Full Text]
  33. Waeber G, Thompson N, Nicod P, Bonny C 1996 Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol Endocrinol 10:1327–1334[Abstract/Free Full Text]
  34. Apelqvist A, Ahlgren U, Edlund H 1997 Sonic hedgehog directs specialised mesoderm differentiation in the intestine and pancreas. Curr Biol 7:801–804[CrossRef][Medline]
  35. Krapp A, Knofler M, Ledermann B, Burki K, Berney C, Zoerkler N, Hagen-buchle O, Wellauer PK 1998 The bHLH protein PTF1–p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas. Genes Dev 12:3752–3763[Abstract/Free Full Text]
  36. Stoffers DA, Heller RS, Miller CP, Habener JF Expression of homeodomain factor IDX-1 in pancreas and duodenum: discordance among expression of an IDX-1 ß-galactosidase transcriptional reporter, IDX-1 mRNA and protein. 10th International Congress of Endocrinology, 1996, San Francisco, CA, p P2–35 (Abstract)



This article has been cited by other articles:


Home page
Mol. Cell. Biol.Home page
T. Kitamura, Y. I. Kitamura, M. Kobayashi, O. Kikuchi, T. Sasaki, R. A. DePinho, and D. Accili
Regulation of Pancreatic Juxtaductal Endocrine Cell Formation by FoxO1
Mol. Cell. Biol., August 15, 2009; 29(16): 4417 - 4430.
[Abstract] [Full Text] [PDF]


Home page
Endocr Relat CancerHome page
V. Fendrich, R. Ramerth, J. Waldmann, K. Maschuw, P. Langer, D. K Bartsch, E. P Slater, A. Ramaswamy, and M. Rothmund
Sonic hedgehog and pancreatic-duodenal homeobox 1 expression distinguish between duodenal and pancreatic gastrinomas
Endocr. Relat. Cancer, June 1, 2009; 16(2): 613 - 622.
[Abstract] [Full Text] [PDF]


Home page
DevelopmentHome page
A. G. Cole, F. Rizzo, P. Martinez, M. Fernandez-Serra, and M. I. Arnone
Two ParaHox genes, SpLox and SpCdx, interact to partition the posterior endoderm in the formation of a functional gut
Development, February 15, 2009; 136(4): 541 - 549.
[Abstract] [Full Text] [PDF]


Home page
DiabetesHome page
A. Ackermann Misfeldt, R. H. Costa, and M. Gannon
{beta}-Cell Proliferation, but Not Neogenesis, Following 60% Partial Pancreatectomy Is Impaired in the Absence of FoxM1
Diabetes, November 1, 2008; 57(11): 3069 - 3077.
[Abstract] [Full Text] [PDF]


Home page
Genes Dev.Home page
J. M. Oliver-Krasinski and D. A. Stoffers
On the origin of the {beta} cell
Genes & Dev., August 1, 2008; 22(15): 1998 - 2021.
[Abstract] [Full Text] [PDF]


Home page
CarcinogenesisHome page
J. Ma, M. Chen, J. Wang, H. H.X. Xia, S. Zhu, Y. Liang, Q. Gu, L. Qiao, Y. Dai, B. Zou, et al.
Pancreatic duodenal homeobox-1 (PDX1) functions as a tumor suppressor in gastric cancer
Carcinogenesis, July 1, 2008; 29(7): 1327 - 1333.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
P. O. Wiebe, J. D. Kormish, V. T. Roper, Y. Fujitani, N. I. Alston, K. S. Zaret, C. V. E. Wright, R. W. Stein, and M. Gannon
Ptf1a Binds to and Activates Area III, a Highly Conserved Region of the Pdx1 Promoter That Mediates Early Pancreas-Wide Pdx1 Expression
Mol. Cell. Biol., June 1, 2007; 27(11): 4093 - 4104.
[Abstract] [Full Text] [PDF]


Home page
DevelopmentHome page
M. J. Doyle, Z. L. Loomis, and L. Sussel
Nkx2.2-repressor activity is sufficient to specify {alpha}-cells and a small number of {beta}-cells in the pancreatic islet
Development, February 1, 2007; 134(3): 515 - 523.
[Abstract] [Full Text] [PDF]


Home page
Mol. Endocrinol.Home page
H. Zhang, A. M. Ackermann, G. A. Gusarova, D. Lowe, X. Feng, U. G. Kopsombut, R. H. Costa, and M. Gannon
The FoxM1 Transcription Factor Is Required to Maintain Pancreatic {beta}-Cell Mass
Mol. Endocrinol., August 1, 2006; 20(8): 1853 - 1866.
[Abstract] [Full Text] [PDF]


Home page
Genes Dev.Home page
T. Miyatsuka, H. Kaneto, T. Shiraiwa, T.-a. Matsuoka, K. Yamamoto, K. Kato, Y. Nakamura, S. Akira, K. Takeda, Y. Kajimoto, et al.
Persistent expression of PDX-1 in the pancreas causes acinar-to-ductal metaplasia through Stat3 activation
Genes & Dev., June 1, 2006; 20(11): 1435 - 1440.
[Abstract] [Full Text] [PDF]


Home page
Mol. Endocrinol.Home page
J. L. Volinic, J. H. Lee, K. Eto, V. Kaur, and M. K. Thomas
Overexpression of the Coactivator Bridge-1 Results in Insulin Deficiency and Diabetes
Mol. Endocrinol., January 1, 2006; 20(1): 167 - 182.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
J. C. Van Velkinburgh, S. E. Samaras, K. Gerrish, I. Artner, and R. Stein
Interactions between Areas I and II Direct pdx-1 Expression Specifically to Islet Cell Types of the Mature and Developing Pancreas
J. Biol. Chem., November 18, 2005; 280(46): 38438 - 38444.
[Abstract] [Full Text] [PDF]


Home page
DiabetesHome page
H. Kaneto, Y. Nakatani, T. Miyatsuka, T.-a. Matsuoka, M. Matsuhisa, M. Hori, and Y. Yamasaki
PDX-1/VP16 Fusion Protein, Together With NeuroD or Ngn3, Markedly Induces Insulin Gene Transcription and Ameliorates Glucose Tolerance
Diabetes, April 1, 2005; 54(4): 1009 - 1022.
[Abstract] [Full Text] [PDF]


Home page
J EndocrinolHome page
S. S. Maria-Engler, M. L. C Correa-Giannella, L. Labriola, K. Krogh, C. Colin, F. H. Lojudice, C. A. M. Aita, E. M. C. de Oliveira, T. C S. Correa, I. C. da Silva, et al.
Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures
J. Endocrinol., December 1, 2004; 183(3): 455 - 467.
[Abstract] [Full Text] [PDF]


Home page
GutHome page
H Sakai, Y Eishi, X-L Li, Y Akiyama, S Miyake, T Takizawa, N Konishi, M Tatematsu, M Koike, and Y Yuasa
PDX1 homeobox protein expression in pseudopyloric glands and gastric carcinomas
Gut, March 1, 2004; 53(3): 323 - 330.
[Abstract] [Full Text] [PDF]


Home page
DiabetesHome page
D. Melloul, S. Marshak, and E. Cerasi
Regulation of pdx-1 Gene Expression
Diabetes, December 1, 2002; 51(90003): S320 - 325.
[Abstract] [Full Text] [PDF]


Home page
J. Clin. Endocrinol. Metab.Home page
H. Stingl, W. J. Schnedl, M. Krssak, E. Bernroider, M. G. Bischof, T. Lahousen, G. Pacini, and M. Roden
Reduction of Hepatic Glycogen Synthesis and Breakdown in Patients with Agenesis of the Dorsal Pancreas
J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4678 - 4685.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
S. E. Samaras, M. A. Cissell, K. Gerrish, C. V. E. Wright, M. Gannon, and R. Stein
Conserved Sequences in a Tissue-Specific Regulatory Region of the pdx-1 Gene Mediate Transcription in Pancreatic {beta} Cells: Role for Hepatocyte Nuclear Factor 3{beta} and Pax6
Mol. Cell. Biol., July 1, 2002; 22(13): 4702 - 4713.
[Abstract] [Full Text] [PDF]


Home page
DiabetesHome page
L. Rachdi, L. El Ghazi, F. Bernex, J.-J. Panthier, P. Czernichow, and R. Scharfmann
Expression of the Receptor Tyrosine Kinase KIT in Mature {beta}-Cells and in the Pancreas in Development
Diabetes, September 1, 2001; 50(9): 2021 - 2028.
[Abstract] [Full Text] [PDF]


Home page
EndocrinologyHome page
M. K. Thomas, J. H. Lee, N. Rastalsky, and J. F. Habener
Hedgehog Signaling Regulation of Homeodomain Protein Islet Duodenum Homeobox-1 Expression in Pancreatic {{beta}}-Cells
Endocrinology, March 1, 2001; 142(3): 1033 - 1040.
[Abstract] [Full Text] [PDF]


Home page
ScienceHome page
N. Lumelsky, O. Blondel, P. Laeng, I. Velasco, R. Ravin, and R. McKay
Differentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similar to Pancreatic Islets
Science, May 18, 2001; 292(5520): 1389 - 1394.
[Abstract] [Full Text]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stoffers, D. A.
Right arrow Articles by Habener, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stoffers, D. A.
Right arrow Articles by Habener, J. F.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals