Essential Requirement for Pax6 in Control of Enteroendocrine Proglucagon Gene Transcription

Mary E. Hill, Sylvia L. Asa and Daniel J. Drucker

Department of Medicine (M.F.H., D.J.D.) The Banting and Best Diabetes Centre Toronto General Hospital Toronto, Ontario M5G 2C4, Canada
Department of Pathology (S.L.A.) The Mount Sinai Hospital University of Toronto Toronto, Ontario M5G 2C4, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The primary function of islet A cells is the synthesis and secretion of glucagon, an essential hormonal regulator of glucose homeostasis. The proglucagon gene is also expressed in enteroendocrine L cells of the intestinal epithelium, which produce glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2), regulators of insulin secretion and intestinal growth, respectively. We show here that Pax6, a critical determinant of islet cell development and proglucagon gene expression in islet A cells, is also essential for glucagon gene transcription in the small and large intestine. Pax6 is expressed in enteroendocrine cells, binds to the G1 and G3 elements in the proglucagon promoter, and activates proglucagon gene transcription. The dominant negative Pax6 allele, SEYNeu, represses proglucagon gene transcription in enteroendocrine cells. Mice homozygous for the SEYNeu mutation exhibit markedly reduced levels of proglucagon mRNA transcripts in the small and large intestine, and GLP-1 or GLP-2-immunopositive enteroendocrine cells were not detected in the intestinal mucosa. These findings implicate an essential role for Pax6 in the development and function of glucagon-producing cells in both pancreatic and intestinal endodermal lineages.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The islets of Langerhans contain distinct populations of specialized endocrine cells that synthesize and secrete insulin, glucagon, and somatostatin, hormones that regulate metabolic processes and, ultimately, glucose homeostasis. Characterization of the molecular factors important for islet development and islet hormone gene expression is of significant interest, in that inadequate development, dysfunction, or destruction of islet ß-cells produces diabetes mellitus in affected individuals. Isolation and analysis of insulin gene transcription factors have provided considerable information about the control of both insulin gene expression and islet cell development and differentiation, as many transcription factors play dual roles in regulating both insulin gene transcription and formation and organization of differentiated islet cell types in the developing pancreas (1, 2, 3, 4).

Given the central importance of the ß-cell and its product, insulin, for glucoregulation, the majority of studies of islet transcription factors have focused on the insulin gene. One of the first ß-cell transcription factors to be isolated, isl-1, does not appear to be essential for control of insulin gene transcription (5); however isl-1 does activate proglucagon gene transcription, and mice homozygous for a null isl-1 mutation exhibit defective islet development and A cell formation (6, 7). In contrast, the Pdx-1 transcription factor is required for insulin gene transcription in the adult ß-cell (4) and, remarkably, Pdx-1-/- mice fail to develop a pancreas (8). Furthermore, human subjects with heterozygous loss of function mutations in Pdx-1 develop maturity-onset diabetes and, similarly, heterozygous Pdx-1+/- mice develop glucose intolerance, providing important evidence for the central role of homeobox transcription factors for ß-cell function in vivo (4, 9).

Pdx-1 is a comparatively weak transactivator of insulin gene transcription and appears to function synergistically with BETA2/NeuroD, a helix-loop-helix protein that together with E47, binds to an adjacent site in the insulin gene promoter (3). Remarkably, BETA2/NeuroD is also essential for both insulin gene transcription and islet development. Mice with a null mutation in the BETA2/NeuroD gene exhibit severe abnormalities in islet cell formation and a marked reduction in the number of ß cells and rapidly develop diabetes and die in the neonatal period (10). Although isl-1, Pdx-1, and BETA2/NeuroD were originally isolated as insulin gene transcription factors, analysis of genetic mutations in transcription factors not previously implicated in the control of insulin gene expression has revealed an essential and unexpected role for specific genes in islet development.

Targeted inactivation of the gene encoding the Nkx2.2 homeodomain protein results in mice with arrested ß-cell development, leading to diabetes in the postnatal period (11). Mice with disruption of both Pax4 alleles fail to develop mature ß- and {delta}-cells and die within 72 h of birth due to growth retardation and dehydration. Intriguingly, pax4-/- mice have increased numbers of islet A cells, consistent with a possible block in islet cell differentiation (12). In contrast, islets from mice with a targeted inactivation in the Pax6 gene contain ß- and {delta}-cells but no A cells, suggesting that Pax6 is essential for the formation of the pancreatic A cell lineage (13). Furthermore, mice with the small eye SEYNeu Pax6 mutation also exhibit markedly reduced numbers of islet A cells and decreased levels of pancreatic glucagon (2). Taken together, these observations emphasize the essential importance of islet transcription factor genes for development and maturation of mature islet cells in vivo.

In contrast to the restricted expression of the insulin gene in the pancreatic ß-cell, the proglucagon gene is expressed in both pancreatic islets and enteroendocrine cells of the intestine. Although abnormalities in islet A cell development and proglucagon gene expression are evident in studies of mice with null mutations in islet transcription factors (2, 7, 13), considerably less information is available about the molecular determinants of enteroendocrine cell development and intestinal proglucagon gene expression in vivo. The finding that mice with targeted or naturally occurring Pax6 mutations develop striking abnormalities in the numbers of islet A cells and decreased pancreatic proglucagon gene expression prompted us to determine whether Pax6 might also be important for proglucagon gene expression in the intestine. We report here that Pax6 is an essential transcriptional regulator of proglucagon gene expression in enteroendocrine cells of the small and large bowel.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
As studies of Pax6 mutant and wild-type mice localized Pax6 expression to islet cells, including glucagon-producing A cells (2, 13, 14), we assessed whether Pax-6 is also expressed in proglucagon-producing intestinal enteroendocrine cells. Northern blot analysis using total and polyA+ RNA (Fig. 1Go) detected Pax6 mRNA transcripts in RNA from the intestinal GLUTag intestinal endocrine cell line and, with a longer exposure, InR1-G9 islet cells. After recent studies demonstrating that Pax6 activates islet proglucagon gene transcription through the proglucagon gene G3 enhancer element (2), we initially assessed the Pax6-dependent activation of the proglucagon gene promoter in BHK fibroblasts. A reporter plasmid that contains the G1-G3 proximal rat proglucagon promoter-enhancer elements in their natural genomic orientation (Fig. 2CGo), [-476]GLU-Luc, was significantly activated by cotransfection with wild-type Pax6, but not by the dominant negative Pax6 mutant allele, SEYNeu, in BHK fibroblasts (Fig. 2AGo). Remarkably, deletion of both the G3 and G2 enhancer sequences did not abrogate the Pax6 activation of proglucagon promoter activity, as both [–220]GLU-Luc (lacking G3) and [-168]GLU-Luc (lacking G3 and G2) were activated by Pax6 (Fig. 2AGo). Furthermore, deletion of the G3, G2, and G4 enhancer elements did not eliminate Pax6-dependent activation of proglucagon promoter activity, as a plasmid containing only G1 sequences, [-93]GLU-Luc, was still activated by Pax6 (Fig. 2AGo). These observations, taken together with the presence of a putative Pax6 binding site at approximately -90 to -60 of the proximal rat proglucagon gene promoter (15), suggested that the G1 element alone might support Pax6-dependent transcriptional activation. These results suggest that the G3 element may not be essential for pax6-dependent activation of the proglucagon promoter in fibroblasts.



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Figure 1. Northern Blot Analysis of Pax-6, Proglucagon, and 18s mRNA Transcripts in Total and polyA+ RNA from the Enteroendocrine GLUTag and Islet InR1-G9 Cell Lines

The times of exposure were 48 and 12 h for Pax-6 and glucagon blots, respectively. With a longer exposure (not shown), Pax-6 mRNA transcripts were detected in the InR1-G9 cell RNA. The relative migration positions of the 28s and 18s RNAs are shown.

 


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Figure 2. Relative Transcriptional Activity of Proglucagon Promoter Luciferase Plasmids in BHK Fibroblasts (A) InR1-G9 Islet Cells (B), and GLUTag Enteroendocrine Cells (C)

The structure of the proximal rat proglucagon promoter and the G1-G4 elements is shown in panel c. GLU-Luc plasmids contained 476, 220, 168, or 93 bp of rat proglucagon gene 5'-flanking sequences (15 ) fused to the luciferase coding region, or four copies of the G1 proglucagon gene enhancer region ligated upstream of a minimal proglucagon promoter [4G1]GLU-Luc. Relative luciferase activity (RLA) was normalized to the values obtained after transfection of the promoterless expression vector, SK-Luc, in the same experiments. Luciferase reporter plasmids were transfected alone with pBS (Bluescript, Stratagene, La Jolla, CA), with the promoterless expression vector pBAT14 alone, or with either the wild-type Pax-6 or the dominant negative Pax-6 SEYNeu cDNA. All transfections were done in quadruplicate, and the data shown represent the mean ± SEM from three different experiments. *, P <.05; **, P <.01; ***, P < 0.001.

 
As studies of islet A cells and pancreatic glucagon biosynthesis in Pax6 mutant mice infer a major role for Pax6 in the regulation of pancreatic proglucagon gene expression via the G3 element in islet A cells (2), we assessed the activity of proglucagon promoter-luciferase plasmids, with or without the G3 element, in InR1-G9 islet A cells. In keeping with the data obtained in BHK fibroblasts, Pax6 activated the proglucagon promoter in InR1-G9 cells, in a G3-independent manner (Fig. 2BGo). The greatest degree of Pax6-dependent proglucagon promoter activation was observed with [-168]GLU-Luc, a reporter that contains the G1 and G4, but not the G2 or G3 enhancer elements. Furthermore, Pax6 significantly increased the activity of [4G1]GLU-Luc in InR1-G9 cells, and the dominant negative SEYNeu cDNA markedly repressed the basal activity of [-93]GLU-Luc in islet cells (Fig. 2BGo). In contrast, the transcriptional activity of a minimal PRL promoter was not inhibited after cotransfection with the SEYNeu cDNA. These findings clearly demonstrate that Pax6 activates the proglucagon promoter, not only in transfected BHK cells but also in islet cells, in a G3-independent manner.

To determine whether Pax6 also regulates proglucagon gene transcription in intestinal endocrine cells, we carried out transfection experiments using the GLUTag enteroendocrine cell line. Pax6 significantly activated the transcriptional activity of proglucagon promoter plasmids in GLUTag cells (Fig. 2CGo), demonstrating that Pax6 is transcriptionally active in intestinal enteroendocrine cells, and not only in islet cell types. Furthermore, the Pax6-dependent activation of -[220]GLU-Luc (Fig. 2CGo) demonstrates that the G3 element is not absolutely required for Pax6 activation in enteroendocrine cells. Moreover, transfection of the dominant negative SEYNeu allele significantly inhibited the transcriptional activity of both -[93]GLU-Luc and [4G1]GLU-Luc, consistent with a role for the G1 element in Pax6-dependent transcriptional activity in GLUTag enteroendocrine cells.

The finding that Pax6 activated proglucagon gene transcription via both the G3 (2) and G1 elements prompted us to assess whether Pax6 was capable of binding both these promoter elements in electrophoretic mobility shift assay (EMSA) studies in vitro. After transfection of Pax6 into BHK cells, a unique DNA-protein complex was detected using the G1 element probe, which was not detected in wild-type BHK cells or cells transfected with the expression vector alone (Fig. 3AGo, complex G1B). Furthermore, an identically migrating prominent G1B complex was observed in GLUTag nuclear extracts, and a similar, although less abundant complex, was detected using InR1-G9 extract (not shown). Using a G3 element probe containing a previously identified Pax6 binding site (2), a major DNA-protein complex (G3C) was detected in nuclear extracts prepared from Pax6-transfected BHK cells. A similar G3C complex was detected in GLUTag nuclear extracts (Fig. 3AGo).



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Figure 3. EMSA Experiments with the Proglucagon Gene G1 and G3 Elements

A, Nuclear extracts from wild-type BHK fibroblasts (with or without transfected Pax-6) and GLUTag enteroendocrine cells were incubated with either G1 or G3 probes and analyzed as described in Materials and Methods. The slightly different migration positions of the G3C complexes are attributable to specific electrophoresis conditions, as evidenced by comparable migration abnormalities of the free probe in the various lanes. Specific complexes are designated by arrows. FP, Free probe. B, Competition for G1 and G3 complex formation in GLUTag cells using increasing (10- to 1000-fold) molar amounts of excess unlabeled homologous competitor oligonucleotide. C, EMSA experiments with Pax-6 or cdx-2/3 antisera in GLUTag cell extracts. G1 or G3 probes were incubated with no extract (lanes 1 and 5) nuclear extract only (-) or extract plus either Pax-6 (lanes 3 and 7) or cdx-2/3 (lanes 4 and 8) antisera. D and E, EMSA experiments using GLUTag extracts and either the G3 (panel D) or G1 (panel E) elements as probes, and the G3, rat proglucagon gene promoter Gluc sequence (-110/-95), G1, insulin (Ins), or somatostatin (SMS) PISCES elements as competitor oligonucleotide sequences. FP, Free probe.

 
All 3 GLUTag complexes, G1A–C, were diminished after competition for G1 binding with increasing amounts of unlabeled G1 competitor oligonucleotides (Fig. 3BGo), but no competition was observed with heterologous nonspecific competitor DNA (not shown). Similarly, the three major complexes observed using GLUTag extracts and the G3 probe, G3A-C, were markedly reduced after competition with increasing amounts of unlabeled G3 sequences (Fig. 3BGo). Taken together, these experiments strongly suggest that Pax6 forms a complex with both the G1 and G3 elements in GLUTag enteroendocrine cells.

To provide additional evidence for the presence of Pax6 in GLUTag DNA-protein complexes detected with either the G1 or G3 probes, EMSA experiments were carried out in the presence or absence of Pax6 antisera (14). The G1B complex was markedly diminished after preincubation of GLUTag extracts with Pax6 antisera (Fig. 3CGo), strongly suggesting that endogenous Pax6 binds to the proglucagon promoter via the G1 element in enteroendocrine cells. Similarly, the GLUTag G3C complex was almost completely eliminated after preincubation with Pax6 antisera (Fig. 3CGo). In contrast, neither the GLUTag G1B nor G3C complexes were diminished after preincubation with antisera against cdx-2/3 or after incubation with nonimmune sera (not shown).

The finding that Pax6 binds to both the G3 and G1 elements suggests that Pax6 recognizes DNA sequences common to both G3 and G1. Furthermore, a core DNA sequence designated the PISCES (pancreatic islet cell-specific enhancer sequence) element has been identified in the 5'-flanking region of the insulin, somatostatin, and glucagon genes (16, 17). To ascertain whether DNA sequences corresponding to putative PISCES elements could compete for the binding to the G3 and G1 elements, a series of EMSA competition experiments were performed (Fig. 3Go, D and E). An oligonucleotide from the rat glucagon gene (-110/-95) 5'-flanking region just 5'- to the boundary of G1 did not compete for complex formation with either the G3 or G1 probes. In contrast, the G1 element modestly diminished the formation of the G3C complex, whereas the insulin gene PISCES element almost completely prevented the formation of the G3C complex (Fig. 3DGo). Similarly, both the insulin gene PISCES element and the G3 sequence competed for the formation of the G1B complex, whereas the somatostatin element was a comparably ineffective competitor for Pax6 binding to G3 (Fig. 3DGo) or G1 (not shown). Taken together, these findings illustrate that the core element common to the G3, G1, and insulin gene PISCES element is capable of competing for Pax6 binding in GLUTag extracts in vitro.

The results of the RNA, transfection, and EMSA experiments demonstrated that Pax6 is expressed in intestinal GLUTag cells and activates enteroendocrine proglucagon gene transcription via binding to and activation of the G3 and G1 enhancer elements. To explore the relevance of Pax6 for intestinal proglucagon gene expression in vivo, we analyzed enteroendocrine cell populations in mice with a dominant negative Pax6 SEYNeu mutation. These mice have previously been shown to exhibit disordered islet formation, markedly reduced numbers of islet A cells, and decreased levels of pancreatic glucagon (2). As SEYNeu homozygous mice die shortly after birth, live SEYNeu -/- mice and both +/- and +/+ littermates were obtained on neonatal day 1, generally within 1 h after birth, euthanized, and genotyped (Fig. 4AGo) using PCR and restriction enzyme analysis as described previously (18). We first analyzed proglucagon mRNA transcripts in the small and large bowel of wild-type and mutant mice by RT-PCR. Although proglucagon mRNA transcripts were comparable in RNA from +/+ and +/- mice, the levels were markedly reduced in both small and large bowel RNA from SEYNeu -/- mice (Fig. 4BGo).



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Figure 4. Glucagon Gene Expression in SEYNeu Mice

A, PCR analysis of genotyping at the Pax-6 locus in +/+ wild-type, +/- heterozygous, and -/- SEYNeu mice. The SEYNeu allele contains a unique HindII site that generates 140- and 80-bp fragments after enzymatic digestion. B, RT-PCR analysis of intestinal gene expression in +/+ and SEYNeu mice. Intestinal RNA from neonatal day 1 age- and sex-matched +/+, +/-, and -/- SEYNeu littermates. RNA from small and large bowel was reverse transcribed and analyzed by RT-PCR using primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proglucagon, over a range of cycle numbers from 19–25, as described in Materials and Methods.

 
We next analyzed the distribution and morphology of endocrine cells in the islets and gut of mice with the homozygousSEYNeu mutation. Consistent with previously reported findings (2), islets from SEYNeu mice were small and poorly developed with abnormal distribution of hormone-containing cells (Fig. 5Go). Cells containing glucagon, although reduced in number, were readily identified (Fig. 5Go) but did not show the usual pattern of distribution at the periphery of islets as seen in control wild-type animals. Insulin immunoreactivity was reduced and was found most prominently in single cells and small clusters adjacent to ducts rather than in well formed islets (Fig. 5Go). Somatostatin-containing cells were reduced in number in SEYNeu mice compared with controls. Staining for pancreatic polypeptide was not markedly different in SEYNeu and control mice. Cells containing peptide YY (PYY) were numerous in the pancreases of SEYNeu mice but were not distributed in normal islets as they were in control animals (Fig. 5Go).



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Figure 5. Pancreatic Islet Morphology in +/+ Control and SEYNeu Mice

Glucagon immunoreactivity (G) is present in the islets of neonatal day 1 SEYNeu mice, but the distribution of the immunoreactive cells is distorted compared with the normal peripheral location of glucagon in control (+/+) animals. Insulin positivity (I) is reduced in SEYNeu mice, and it is localized predominantly in scattered individual cells, whereas insulin-containing cells are found in well formed islets in +/+ controls (d). Somatostatin-containing (S) cells are fewer in the pancreas of SEYNeu mice than in controls. Staining for pancreatic polypeptide (PP) is not different in SEYNeu and control mice. Cells containing PYY are numerous in both SEYNeu and control animals, but the distribution within well formed islets is not seen in SEYNeu mice.

 
The intestinal morphology of the small and large intestine in neonatal day 1 SEYNeu mice appeared normal, as assessed by conventional light microscopy. However, immunolocalization studies of intestinal endocrine cells showed total absence of glucagon-like peptide 1 (GLP-1) and GLP-2 immunoreactivity in both the small and large bowel (Fig. 6Go) whereas enteroendocrine cells containing these peptides were readily identified in control +/+ mice (Fig. 6Go). In contrast to the absence of intestinal endocrine cells expressing GLP-1 or GLP-2, endocrine cells containing immunopositivity for PYY, cholecystokinin, serotonin, and secretin, were found in the usual distribution in the small and large bowel of SEYNeu mice (Fig. 7Go). As GLP-2 stimulates growth of the mucosal epithelium in mice (19), we analyzed intestinal morphology of the small bowel in -/- mice. No significant differences in small bowel crypt plus villus height were observed after assessment of multiple histological sections of small bowel from +/+ andSEYNeu mice (data not shown).



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Figure 6. Absence of GLP-1 and GLP-2 Immunoreactivity in the Gut of SEYNeu Mice

GLP-1 immunostaining yields entirely negative results in the bowel of a neonatal day 1 SEYNeu mouse, whereas a comparable area in the gut of a +/+ control mouse has numerous positive cells. GLP-2 immunostaining is also completely negative in the bowel of a SEYNeu mouse but several GLP-2-immunoreactive cells are present in the bowel of a +/+ control mouse. No GLP-1- or GLP-2-immunopositive cells were seen in several dozen histological sections from the small and large bowel of three different SEYNe mice.

 


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Figure 7. Localization of Enteroendocrine Cells in the Gut of SEYNeu Mice

In the bowel of neonatal day 1 SEYNeu mice, enteroendocrine cells are detected that exhibit immunoreactivity for PYY, cholecystokinin (CCK), serotonin (SER), and secretin (S) in a distribution that is unchanged from control +/+ mice (not shown).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Enteroendocrine cells of the small and large bowel exhibit a regional distribution along the small and large bowel that is unique to specific hormonal subtypes (20). As enteroendocrine cells and pancreatic islets are both believed to arise from endodermal origin, it is not surprising that several endocrine genes, including secretin, gastrin, and PYY, are transiently expressed in the fetal endocrine pancreas, after which expression in adult islets is extinguished but is maintained in gastrointestinal endocrine cells throughout adult life (21, 22). Although enteroendocrine cells appear to be derived from a common pluripotential crypt precursor capable of giving rise to multiple differentiated intestinal cell lineages (23), little is known about the molecular determinants of intestinal endocrine cell formation. Furthermore, the hierarchy and complexity of transcription factors that regulate endocrine cell lineage in the gut remain poorly understood.

Analysis of mice with a targeted disruption of the helix-loop-helix transcription factor BETA2 has provided important insights into the genetic control of endocrine cell lineages in the gut (10). The secretin gene, a transcriptional target for BETA2, is transiently expressed in fetal islet ß-cells, after which expression in the adult is restricted to enteroendocrine cells (24). BETA2-/- mice exhibit striking abnormalities in islet morphogenesis with a severe reduction in mature islet ß-cells, and a complete absence of secretin- and cholecystokinin-producing intestinal enteroendocrine cells; in contrast, the numbers of enteroendocrine cells expressing the proglucagon and somatostatin genes were normal despite the lack of BETA2 expression (10). The findings reported here extend our understanding of enteroendocrine cell biology by demonstrating that Pax6 is essential for expression of the proglucagon gene in the intestinal epithelium. Whether the absence of Pax6 primarily perturbs endocrine cell formation or, specifically, hormone gene transcription is difficult to determine, as cell lineage markers for the proglucagon enteroendocrine cell population have not been identified. Nevertheless, proglucagon mRNA transcripts were not completely absent from the intestine of -/- mice, suggesting that SEYNeu mice contain at least a few putative enteroendocrine cells that support proglucagon gene transcription in vivo.

The finding that Pax6 plays an essential role in expression of both pancreatic and intestinal proglucagon gene expression further supports the existence of shared mechanisms for regulating hormone gene expression in both islet and intestinal endocrine cells. DNA-binding protein(s) from islet cell lines were originally identified that bound to highly similar promoter sequences, designated the PISCES motif, present in the 5'-flanking regions of the insulin, somatostatin, and proglucagon gene promoters (Refs. 16, 25 and Fig. 8Go). Multiple DNA-protein complexes have been detected using the glucagon gene G3 element as a probe in EMSA experiments, as shown here and in previous studies (16). Furthermore, the somatostatin and insulin PISCES elements compete for binding with multiple DNA complexes formed by G3 (16). The demonstration that Pax6 binds to the PISCES element in the insulin, proglucagon, and somatostatin promoters, taken together with the competition for DNA-protein complex formation on G3 using PISCES elements from islet hormone promoters, is consistent with the hypothesis that these elements recognize one or more highly similar, if not identical, transcription factor complexes (16). The major reduction in islet hormone gene expression in Pax6 SEYNeu mice strongly implicates Pax6 as a PISCES-binding protein that is functionally essential for transcriptional regulation in the endocrine pancreas (2). Our results extend the functional concept of the PISCES motif to include regulation, via Pax6, of both islet and enteroendocrine gene expression.



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Figure 8. GLP-1-Immunopositive Cells in the Islets and Intestine of a Newborn Wild-Type (+/+) and SEYNeu (-/-) Mouse

The organization and number of GLP-1-immunopositive islet cells is abnormal, and GLP-1- or GLP-2-immunopositive intestinal endocrine cells are not detected in the SEYNeu mouse.

 
Pax-6 immunoreactive cells have been detected in the mouse fore/midgut endoderm as early as embryonic day 9 (E9.0) (2), and by day E9.5, a few glucagon-positive cells are detected at the time of the appearance of the pancreatic bud that also appear to coexpress Pax-6. At E15.5, numerous cells in the endocrine pancreas express Pax-6 and either glucagon, insulin, somatostatin, or pancreatic polypeptide. The ontogeny of Pax6 expression in the developing gut has not been reported. The murine intestinal epithelium undergoes a complex pattern of cytodifferentiation beginning at approximately E15, and distinct enteroendocrine cell populations are detectable in the small intestine by E17 (26). Similarly, endocrine cells are first detected in the mouse colon by E15.5, with the earliest cell type detected expressing PYY, followed by the appearance of additional hormonal phenotypes between E17.5 and E18.5 (27). Glucagon-immunopositive cells are infrequent before E19 in the developing small bowel, but detectable by E16.5 in the murine colon (27). Whether intestinal Pax6 expression is exclusively localized to the glucagon-immunopositive enteroendocrine cell lineage in the developing and adult gut remains to be determined.

As the levels of proglucagon mRNA transcripts were markedly reduced in SEYNeu mice, these mutant mice provide an opportunity to assess the importance of the proglucagon-derived peptides for fetal growth and development. Although intestinal-derived GLP-2 has been recently identified as a potent intestinal growth factor in adult mice (19), the role, if any, of GLP-2 (or GLP-1) in the development of the fetal gut is not known. The observation that the small and large bowel appear histologically normal in SEYNeu mice despite the marked reduction in intestinal proglucagon gene expression (and hence GLP-2 biosynthesis) suggests that normal levels of GLP-2 are not essential for growth and differentiation of the intestinal mucosal epithelium during fetal development in vivo. In summary, the results presented here demonstrate that a single transcription factor, Pax6, is essential for both islet cell formation and islet and enteroendocrine gene transcription, via interaction with a DNA element common to the promoters of genes expressed in the endocrine cells of the pancreas and gut.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The plasmids pBat14, pBat14.mPax6, and pBat14.mPax6 SEYNeu were kindly provided by M. S. German (University of California, San Francisco, CA). These plasmids are eukarykotic expression vectors under the control of the cytomegalovirus (CMV) promoter (2). The 5'-deleted rat proglucagon gene promoter sequences, subcloned in the promoterless plasmid SK-Luc immediately adjacent to the coding sequence of the firefly luciferase reporter gene, have been previously described (28, 29). Synthetic oligonucleotides corresponding to specific rat proglucagon gene G1 sequences (15) were annealed, separated by agarose gel electrophoresis, gel purified, and ligated into the [-82]GLU-Luc plasmid (28). This new plasmid was designated [4G1]GLU-Luc and contained four copies of the G1 sequence in a 5'-3' orientation adjacent to the -82 to +58 fragment of the proximal rat proglucagon promoter. The plasmid [-36]PRL-Luc was a kind gift from H. P. Elsholtz (University of Toronto, Toronto, Ontario, Canada). The CMV promoter plasmid, CMV-Luc, and the promoterless plasmid, SK-Luc, were used as positive and negative controls, respectively, in each transfection experiment.

Cell Culture and Transfections
All cell culture reagents used were obtained from Life Technologies/Gibco BRL (Toronto, Ontario, Canada). Cell lines were maintained in DMEM (4.5 g glucose/liter). Baby hamster kidney (BHK) fibroblasts and InR1-G9 islet cells (30) were grown in DMEM supplemented with 5% calf serum; mouse enteroendocrine GLUTag cells (31) were grown in DMEM supplemented with 10% FCS. BHK cells and InR1-G9 cells were transfected by the calcium phosphate precipitation method with 5 µg reporter plasmid DNA + 5 µg expression plasmid DNA per 60-mm dish. GLUTag cells were electroporated with 10 µg reporter plasmid + 10 µg expression plasmid. All cells were harvested 16–20 h after transfection for analysis of luciferase activity as described previously (28, 32), and values were normalized relative to the background luciferase activity obtained after transfection of SK-Luc in the same experiment. Statistical analysis was performed using Student’s t test.

RNA Analysis
RNA was prepared from cells and tissue by the acid ethanol precipitation method previously described (33). Poly A+ RNA was isolated using a Qiagen Poly A Kit (Mississauga, Ontario, Canada). Electrophoresis of RNA, transfer to nylon membrane, hybridization, and washing were carried out as previously described (34). For RT-PCR, first-strand cDNA synthesis was generated from total RNA using the SuperScript Preamplification System from Life Technologies. Target cDNA was then amplified using specific oligonucleotide primer pairs by the PCR method. Primers for mouse proglucagon were 5'-TGAAGACCATTTACTTTGTGGCT-3' and 5'-CTGGTGGCAAGATTGTCCAGAAT-3'; primers for glyceraldehyde-3-phosphate dehydrogenase were 5'TCCACCACCCTGTTGCTGTAG-3' and 5'-GACCACAGTCCATGACATCACT-3'. Reactions were denatured at 94 C for 1 min and then annealed at 66 C for 45 sec, followed by extension at 72 C for 1 min for 19–25 cycles.

Mice and Genotyping
Small eye SEYNeu +/- mice were kindly provided by Brigid Hogan (Vanderbilt University, Nashville, TN) and maintained in the animal facility of the Toronto Hospital. SEYNeu +/- were mated to generate homozygous, heterozygous, and wild-type offspring, which were distinguished by PCR and restriction enzyme analysis of the amplification product as previously described (18).

EMSAs
Nuclear proteins from GLUTag, InR1-G9, and BHK cells were prepared as previously described (28, 32). Synthetic oligonucleotides corresponding to specific proglucagon gene G1 and G3 sequences (15) were annealed, labeled with [32P]ATP using Klenow enzyme, and purified in a G50 spin column. EMSAs were performed as described previously (28, 32). For supershift experiments, nuclear extracts were preincubated with 1:10 diluted anti-Pax6 antiserum (a generous gift from Dr. S. Saule, Lille, France) or nonimmune antisera at 4 C for 30 min before addition of 32P-labeled DNA probe and subsequent incubation at 30 C for 30 min. All reaction mixes were loaded onto a 5% nondenaturing polyacrylamide gel, and after electrophoresis, the gel was exposed to x-ray film for 24–48 h. Oligonucleotide competitor sequences from the rat insulin and glucagon gene PISCES elements were synthesized as per Fig. 4AGo in Ref. 16 .

Histology and Immunocytochemistry
Tissues were processed for immunohistochemistry and/or intestinal morphometry as previously described (35, 36). Formalin-fixed, paraffin-embedded tissue was sectioned at 4 µm for imunohistochemistry using the streptavidin-biotin-peroxidase complex technique. Primary antisera and antibodies were directed against the following antigens and were used at the specified dilutions: insulin (monoclonal antibody from BioGenex Laboratories, Inc., San Ramon, CA) 1:40 for 30 min; glucagon (polyclonal antiserum from Immunon, Pittsburg, PA) 1:200 for 30 min; GLP-1 (polyclonal antiserum prepared by D. J. Drucker) 1:1500 for 30 min; GLP-2 (polyclonal antiserum prepared by D. J. Drucker) 1:2500 for 30 min; PYY (polyclonal antiserum from Peninsula Laboratories Inc., Belmont CA) 1:2000 for 30 min; somatostatin (polyclonal antiserum from DAKO Corp., Carpinteria, CA) prediluted preparation further diluted 1:40 overnight; pancreatic polypeptide (polyclonal antiserum from DAKO Corp.) 1:6000 for 30 min; cholecystokinin (polyclonal antiserum from Serotec, Oxford, U.K.) 1:1000 for 30 min; serotonin (monoclonal antibody from DAKO Corp.) 1:50 for 60 min; secretin (polyclonal antiserum from BioGenex Laboratories, Inc., San Ramon, CA) prediluted preparation further diluted 1:5 overnight; gastrin (polyclonal antiserum from Immunostain, Los Angeles, CA) prediluted preparation further diluted 1:20 overnight; vasoactive intestinal peptide (polyclonal antiserum from Zymed Laboratories, Inc., San Francisco, CA) prediluted preparation further diluted 1:10 overnight. The reactions were visualized using 3,3'-diaminobenzidine and hydrogen peroxide. Appropriate positive and negative controls were performed in each case. The gastrointestinal tract from newborn +/+, +/- and SEYNe -/- mice was removed and fixed in 10% buffered formalin. Intestinal micrometry was performed with the use of a Leitz (Westar, Germany) microscope with a video camera connected to a computer monitor. The microscope was calibrated at x10 magnification, and crypt plus villus height in the small bowel was measured by examining at least 10 longitudinally oriented villi from each animal and is expressed in micrometers (mean ± SEM).


    FOOTNOTES
 
Address requests for reprints to: Dr. D. Drucker, The Toronto Hospital, 200 Elizabeth Street CCRW3–838, Toronto, Ontario M5G 2C4, Canada.

This work was supported by an operating grant to D.J.D. from the Medical Research Council (MRC) of Canada. D.J.D. is a Senior Scientist of the MRC.

Received for publication February 3, 1999. Revision received May 5, 1999. Accepted for publication May 25, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Sander M, German MS 1997 The ß cell transcription factors and development of the pancreas. J Mol Med 75:327–340[CrossRef][Medline]
  2. Sander M, Neubuser A, Kalamaras J, Ee HC, Martin GR, German MS 1997 Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development. Genes Dev 11:1662–1673[Abstract]
  3. Naya FJ, Stellrecht CMM, Tsai M-J 1995 Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes Dev 9:1009–1019[Abstract]
  4. Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H 1998 ß-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the ß-cell phenotype and maturity onset diabetes. Genes Dev 12:1763–1768[Abstract/Free Full Text]
  5. German MS, Wang J, Chadwick RB, Rutter WJ 1992 Synergistic activation of the insulin gene by a LIM-homeo domain protein and a basic helix-loop-helix protein: building a functional insulin minienhancer complex. Genes Dev 6:2165–2176[Abstract]
  6. Wang M, Drucker DJ 1995 The LIM domain homeobox gene isl-1 is a positive regulator of islet cell-specific proglucagon gene transcription. J Biol Chem 270:12646–12652[Abstract/Free Full Text]
  7. Ahlgren U, Pfaff SL, Jessell TM, Edlund T, Edlund H 1997 Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells. Nature 385:257–260[CrossRef][Medline]
  8. 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]
  9. Stoffers DA, Ferrer J, Clarke WL, Habener JF 1997 Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet 17:138–139[Medline]
  10. Naya FJ, Huang H, Qiu Y, Mutoh H, DeMayo F, Leiter AB, Tsai M-J 1997 Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice. Genes Dev 11:2323–2334[Abstract/Free Full Text]
  11. Sussel L, Kalamaras J, Hartigan-O’Connor DJ, Meneses JJ, Pedersen RA, Rubenstaein JL, German MS 1998 Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic ß cells. Development 125:2213–2221[Abstract/Free Full Text]
  12. Sosa-Pineda B, Chowdhury K, Torres M, Oliver G, Gruss P 1997 The Pax4 gene is essential for differentiation of insulin-producing ß cells in the mammalian pancreas. Nature 386:399–402[CrossRef][Medline]
  13. St-Onge L, Sosa-Pineda B, Chowdhury K, Mansouri A, Gruss P 1997 Pax6 is required for differentiation of glucagon-producing {alpha}-cells in mouse pancreas. Nature 387:406–409[CrossRef][Medline]
  14. Turque N, Plaza S, Radvanyi F, Carriere C, Saule S 1994 Pax-QNR/Pax-6, a paired box and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells. Mol Endocrinol 8:929–938[Abstract]
  15. Philippe J, Drucker DJ, Knepel W, Jepeal L, Misulovin Z, Habener JF 1988 Alpha-cell-specific expression of the glucagon gene is conferred to the glucagon promoter element by the interactions of DNA-binding proteins. Mol Cell Biol 8:4877–4888[Medline]
  16. Knepel W, Vallejo M, Chafitz JA, Habener JF 1991 The pancreatic islet-specific glucagon G3 transcription factors recognize control elements in the rat somatostatin and insulin-I genes. Mol Endocrinol 5:1457–1466[Abstract]
  17. Diedrich T, Furstenau U, Knepel W 1997 Glucagon gene G3 enhancer: evidence that activity depends on combination of an islet-specific factor and a winged helix protein. Biol Chem 378:89–98[Medline]
  18. Quinn JC, West JD, Hill RE 1996 Multiple functions for Pax6 in mouse eye and nasal development. Genes Dev 10:435–446[Abstract]
  19. Drucker DJ, Ehrlich P, Asa SL, Brubaker PL 1996 Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Natl Acad Sci USA 93:7911–7916[Abstract/Free Full Text]
  20. Roth KA, Hertz JM, Gordon JI 1990 Mapping enteroendocrine cell populations in transgenic mice reveals an unexpected degree of complexity in cellular differentiation within the gastrointestinal tract. J Cell Biol 110:1791–1801[Abstract]
  21. Brand SJ, Fuller PJ 1988 Differential gastrin gene expression in rat gastrointestinal tract and pancreas during neonatal development. J Biol Chem 263:5341–5347[Abstract/Free Full Text]
  22. Upchurch BH, Apone GW, Leiter AB 1994 Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor. Development 120:245–252[Abstract/Free Full Text]
  23. Cheng H, Leblond CP 1974 Origin, differentiation, and renewal of the four main epithelial cell types in the mouse small intestine. Am J Anat 141:537–562[Medline]
  24. Wheeler MB, Nishitani J, Buchan AMJ, Kopin AS, Chey WY, Chang T-M, Leiter AB 1992 Identification of a transcriptional enhancer important for enteroendocrine and pancreatic islet cell-specific expression of the secretin gene. Mol Cell Biol 12:3531–3539[Abstract]
  25. Wrege A, Diedrich T, Hochhuth C, Knepel W 1995 Transcriptional activity of domain A of the rat glucagon G3 element conferred by an islet-specific nuclear protein that also binds to similar pancreatic islet cell-specific enhancer sequences (PISCES). Gene Expression 4:205–216[Medline]
  26. Roth KA, Rubin DC, Birkenmeier EH, Gordon JI 1991 Expression of liver fatty acid-binding protein/human growth hormone fusion genes within the enterocytes and enteroendocrine cell populations of fetal transgenic mice. J Biol Chem 266:5949–5954[Abstract/Free Full Text]
  27. Upchurch BH, Fung BP, Rindi G, Ronco G, Leiter AB 1996 Peptide YY expression is an early event in colonic endocrine cell differentiation: evidence from normal and transgenic mice. Development 122:1157–1163[Abstract/Free Full Text]
  28. Jin T, Drucker DJ 1996 Activation of proglucagon gene transcription through a novel promoter element by the caudal-related homeodomain protein cdx-2/3. Mol Cell Biol 16:19–28[Abstract]
  29. Jin T, Trinh DKY, Wang F, Drucker DJ 1997 The caudal homeobox protein cdx-2/3 activates endogenous proglucagon gene expression in InR1–G9 islet cells. Mol Endocrinol 11:203–209[Abstract/Free Full Text]
  30. Drucker DJ, Philippe J, Mojsov S 1988 Proglucagon gene expression and posttranslational processing in a hamster islet cell line. Endocrinology 123:1861–1867[Abstract]
  31. Drucker DJ, Jin T, Asa SL, Young TA, Brubaker PL 1994 Activation of proglucagon gene transcription by protein kinase A in a novel mouse enteroendocrine cell line. Mol Endocrinol 8:1646–1655[Abstract]
  32. Jin T, Drucker DJ 1995 The proglucagon gene upstream enhancer contains positive and negative domains important for tissue-specific proglucagon gene transcription. Mol Endocrinol 9:1306–1320[Abstract]
  33. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Chem 162:156–159
  34. Drucker DJ, Brubaker PL 1989 Proglucagon gene expression is regulated by a cyclic AMP-dependent pathway in rat intestine. Proc Natl Acad Sci USA 86:3953–3957[Abstract]
  35. Tsai C-H, Hill M, Asa SL, Brubaker PL, Drucker DJ 1997 Intestinal growth-promoting properties of glucagon-like peptide 2 in mice. Am J Physiol 273:E77–E84
  36. Asa SL, Lee YC, Drucker DJ 1996 Development of colonic and pancreatic endocrine tumors in mice expressing a glucagon-SV40 T antigen transgene. Virchows Arch [A] 427:595–605