Tissue-Specific Transcriptional Activity of a Pancreatic Islet Cell-Specific Enhancer Sequence/Pax6-Binding Site Determined in Normal Adult Tissues in Vivo Using Transgenic Mice

Stephan Beimesche, Andrea Neubauer, Stephan Herzig, Rafal Grzeskowiak, Thomas Diedrich, Irmgard Cierny, Doris Scholz, Tahseen Alejel and Willhart Knepel

Department of Molecular Pharmacology University of Göttingen D-37075 Göttingen, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A pancreatic islet cell-specific enhancer sequence (PISCES) shared by the rat insulin-I, glucagon, and somatostatin genes binds the paired domain-containing transcription factor Pax6 and confers strong transcriptional activity in pancreatic islet cell lines. It was found recently that Pax6 plays a major role in islet development. In the present study, transgenic mice were used to investigate PISCES-mediated transcription in normal adult islets in vivo. In several independent mouse lines expressing a PISCES-luciferase reporter transgene, the PISCES motif directed gene expression in the adult eye, cerebellum, and discrete brain areas, consistent with the tissue distribution of Pax6. These tissues contain two Pax6 isoforms caused by alternative splicing, only one of which was found to bind the PISCES motif in electrophoretic mobility shift assays. No reporter gene expression was detected in adult pancreatic islets or in any other peripheral organ tested. RT-PCR analysis confirmed that Pax6 mRNA is present in adult islets. These results demonstrate that the PISCES motif is sufficient to direct highly tissue-specific gene expression in whole animals. The lack of PISCES-mediated transcription in adult islets indicates that the Pax6 protein(s) expressed in adult pancreatic islets function differently from the ones in the eye and cerebellum.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The peptide hormones insulin and glucagon are critical regulators of blood glucose concentration. Their inappropriate production and secretion causes diabetes mellitus. These hormones are synthetized within distinct cell types of the pancreatic islets (1). Gene inactivation by homologous recombination in mice has shown that various transcription factors are essential for endocrine cell differentiation in the developing pancreas, including Pdx1 (2), Isl1 (3), and Beta2/NeuroD (4). The lineage of the different endocrine cells may be defined by members of the Pax family of vertebrate genes, all of which contain a conserved sequence motif, the paired box, which encodes a DNA-binding domain (5, 6). Inactivation of Pax4 results in the absence of mature insulin- and somatostatin-producing cells (7), whereas Pax6 homozygous mutant mice lack glucagon-producing cells (8).

Some of these transcription factors are also expressed in mature endocrine cells and may, thus, contribute to maintain endocrine cells in a differentiated state. They may also directly regulate the expression of hormone genes in terminally differentiated islets. Pdx1 is produced in both ß- and {delta}-cells in the adult islet (9, 10, 11). It can bind to and transactivate the insulin gene promoter (12), particularly in combination with a heterodimer of the transcription factors E47 and Beta2, which binds to an adjacent site on the insulin promoter (13, 14). Isl1 is produced in all islet cells and can transactivate the glucagon promoter (15). Also, Pax genes are expressed in adult islets. Pax4 expression seems to be restricted largely to ß-cells, whereas Pax6 is expressed in mature {alpha}-, ß-, {gamma}-, and {delta}- endocrine cells (8). However, the role of all of these transcription factors in the regulation of hormone gene transcription in the differentiated endocrine pancreas is unclear, as evidence for activation of islet hormone gene transcription by these factors is based on results obtained from studies using islet tumor cell lines (12, 13, 14, 15). Although islet cell lines have retained many characteristics of normal islet cells and are extremely helpful for studies on islet cell biology, some features of islet cell lines are unique or more typical of immature islet cells (16, 17). Furthermore, genetic studies that could potentially address the mechanism of islet homone gene regulation in adult islets in vivo have not been informative, because null mutations in genes that encode transcription factors presumed to regulate islet hormone gene expression prevent normal pancreas development (2, 3, 4, 8).

The origin of all islet endocrine cells from common progenitor cells (18) raises the possibility that fully differentiated islet cells may share transcriptional regulatory proteins directing insulin, glucagon, and somatostatin gene transcription. Indeed, a DNA sequence common to the insulin, glucagon, and somatostatin gene promoters, the pancreatic islet cell-specific enhancer sequence (PISCES), has been detected (19). Multiple copies of the PISCES motif are sufficient to selectively stimulate transcriptional activity of a luciferase reporter gene in various phenotypically distinct pancreatic islet cell lines without affecting expression in several nonislet cell lines (20). The PISCES motif shows high homology to the consensus recognition site of the Pax6 paired domain (19, 21), and Pax6 was found to bind and activate the PISCES motif (22). Because mutation studies have demonstrated that the PISCES element is important for the activity of each of the three hormone gene promoters (19, 20, 23), the PISCES element and its binding factor appear to be a key component of the transcription complexes coordinately directing insulin, glucagon, and somatostatin gene transcription in distinct islet cell types. However, these studies have again been performed using islet tumor cell lines. The transcriptional activity conferred by the PISCES element and its binding factor in mature endocrine cells of adult islets in vivo remained thus unclear. In the present study, PISCES-luciferase reporter transgenic mice were generated and used to study the transcriptional activity of the PISCES motif in normal mature tissues in vivo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PISCES Motif Directs Highly Tissue-Specific Expression in Mice
Four copies of a 16-bp oligonucleotide containing the PISCES motif as present in domain A of the glucagon G3 element were placed in front of the herpes simplex virus thymidine kinase promoter truncated at -81 and linked to the luciferase reporter gene (p4xG3AT81Luc). This construct has been shown to drive high level expression of the reporter gene after transfection into phenotypically distinct islet cell lines (19, 20, 23). Although homomultimeric repeats of transcriptional elements are artificial, this organization satisfies the requirement for multiple elements to generate a functional enhancer. This approach has been successful for characterizing the binding sites for single transcription factors by cell transfection and also in transgenic mice (24, 25, 26, 27, 28). For example, an oligomerized NFAT-binding motif that directs transcription of SV40 T-antigen in transgenic mice indicated cell populations exhibiting constitutive or inducible NFAT activity (25), and a minimal promoter containing three copies of a binding site for NF{kappa}B-related transcription factors indicated tisssue-specific and inducible transcriptional activities of distinct NF-{kappa}B/Rel proteins (26). To determine the tissue-specific transcriptional activity of the PISCES motif in mature tissues in vivo, transgenic mice were generated. The PISCES-luciferase reporter gene was microinjected as a linear fragment into pronuclei of one-cell embryos. Of six independently derived transgenic founder mice bearing from 2 to 15 4xG3AT81Luc transgenes, five expressed the luciferase reporter gene (Fig. 1Go). The absolute level of expression of the reporter gene was different in the independent transgenic lines; reporter enzyme activity was, respectively, 12,700, 466,100, 2,420,000, 14,500,000, and 21,200,000 light units/mg protein in the tissue with highest expression for each independent line (Fig. 1Go). However, all transgenic lines showed a similar pattern of reporter expression in adult animals (Figs. 1Go and 2Go). The PISCES motif directed gene expression in the eye and brain with highest levels in the cerebellum (Figs. 1Go and 2Go). Virtually no reporter enzyme activity was detected in pancreas and other peripheral organs including liver, spleen, stomach, intestine, kidney, lung, heart, and skeletal muscle (Figs. 1Go and 2Go). The level for pancreatic transgene expression in the five independent lines was, respectively, 0.0005, 0.0009, 0.003, 0.01, and 0.06% of the tissue with highest expression. Reporter expression was also not found in isolated pancreatic islets of Langerhans (not shown). These results show that a short oligonucleotide containing the PISCES motif is sufficient to direct tissue-specific gene expression in mature tissues in vivo; the PISCES motif does not direct pancreatic expression but directs expression in the eye and brain with highest levels in the cerebellum.



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Figure 1. Reporter Transgene Expression in Adult Tissues

Luciferase reporter enzyme activity was measured in the indicated tissues from five independent transgenic lines carrying the 4xG3AT81Luc transgene. Values are light units x 10-6 per mg protein. Mesenc., Mesencephalon; Telenc., telencephalon; Bulbus olf., bulbus olfactorius; Medulla obl., medulla oblongata; Dienc., diencephalon; Pituitary gl., pituitary gland.

 


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Figure 2. Relative Reporter Transgene Expression in Adult Tissues

Luciferase reporter enzyme activity was measured in the indicated tissues from five independent transgenic lines carrying the 4xG3AT81Luc transgene. For each independent transgenic line, luciferase activity measured in the tissue with highest activity is set as 100 and luciferase activity measured in the other tissues is expressed relative to that. Mesenc., Mesencephalon; Telenc., telencephalon; Bulbus olf., bulbus olfactorius; Medulla obl., medulla oblongata; Dienc., diencephalon; gl., gland.

 
To verify that this tissue specificity is conferred by the PISCES motif, we analyzed the expression of a transgene containing a mutated G3A homomultimer (4xG3AmutT81Luc). This mutation functionally inactivates the PISCES motif as shown in protein-DNA binding and cell transfection experiments (19, 20, 23). In four independent transgenic lines bearing from 1 to 10 transgenes, reporter enzyme expression was very low. Figure 3Go shows the expression of the mutated transgene in several tissues of two independent transgenic lines. The expression of the mutated transgene was low, and its pattern of expression different from that of the wild-type transgene (Fig. 3Go, compare with Fig. 1Go). These results support the conclusion that the PISCES motif directs expression in vivo in the mature eye and brain.



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Figure 3. Expression of a Mutated Transgene in Adult Tissues

Luciferase reporter enzyme activity was measured in the indicated tissues from two independent transgenic lines carrying the 4xG3AmutT81Luc transgene. Values are light units x 10-6 per mg protein. The corresponding figures for pancreatic expression of the mutated transgene are 0.000003 and 0.00001, respectively. Mesenc., Mesencephalon; Telenc., telencephalon; Bulbus olf., bulbus olfactorius; Medulla obl., medulla oblongata; Dienc., diencephalon; Pituitary gl., pituitary gland.

 
The expression of the PISCES-luciferase transgene in the adult brain was further analyzed in tissue sections of the brain of the second highest expressing line at the macroscopic level using a Molecular Light Imager (EG&G Berthold, Bad Wildbad, Germany). The sagittal sections shown in Fig. 4Go confirm that the PISCES motif directs expression to highly restricted areas in the brain. No reporter expression was found in the cerebral cortex, hippocampus, or caudate putamen (Fig. 4Go). The strongest luminescence signals were detected over the cerebellum (Fig. 4Go, top panel), the optic chiasma, and the superior colliculus (Fig. 4Go, bottom panel). In additional sections, some signals were also found in the olfactory bulb, septal area, zona incerta, and amygdala (not shown).



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Figure 4. Imaging of Transgene Expression in Sagittal Sections of the Brain of a 4xG3AT81Luc Transgenic Mouse

Luciferase luminescence signals are superimposed in pseudocolor onto the brightfield image. Red, High intensity; blue, low intensity. The strongest luminescence signals are detected over the cerebellum (top panel) and, in a section closer to the midline, over the superior colliculus (S.C.) and the optic chiasma/optic tract (O.C.) (lower panel).

 
The expression of the PISCES-luciferase transgene was studied also in the mouse embryo, using the line with the highest transgene expression in the adult brain. On day 13.5 of embryonic development, strong transgene expression was found in the brain but not in the embryonic pancreas. Reporter enzyme activity in extracts of the head was 1,474,165 ± 352,555 light units/mg protein (n = 6), whereas no activity was detected in extracts from the embryonic pancreas. Likewise, strong transgene expression in the head, but not the pancreas, was detected on days 15.5, 16.5, 17.5, and 18.5 postcoitus (p.c.) (not shown).

PISCES-Binding Proteins (PISCES-BPs) in Cerebellum and Eye
A PISCES-BP has been characterized in nuclear extracts from islet cell lines (19, 20, 23). This PISCES-BP has recently been identified as the transcription factor Pax6 (22). To characterize PISCES-BP(s) in the cerebellum and eye of adult mice, the electrophoretic mobility shift assay was used. Labeled G3A was incubated with whole-cell extracts from the cerebellum or eye. A protein complex that bound specifically to labeled G3A and that was competed for by G3A, but not by G3Amut, was detected in both extracts (Fig. 5Go, compare lane 7 with 8, and lane 11 with 12, respectively). This protein complex in cerebellum and eye extracts comigrated with PISCES-BP from an islet cell line (Fig. 5Go, compare lanes 12 and 8 with lane 4) and was abolished by the addition of a specific anti-Pax6 antiserum (not shown). Pax6 is known to be expressed in the cerebellum and eye of adult mice (29). These results provide no evidence that the cerebellum and eye express specific PISCES-BPs other than Pax6. One additional, prominent complex of slower mobility that forms on labeled G3A was detected (Fig. 5Go, lanes 5 and 9); however, because this complex also binds labeled G3Amut (Fig. 5Go, lanes 6 and 10), which is not active in cell transfection and transgenic mice tests, it is not relevant to the function of the PISCES motif. We were unable to detect PISCES-BPs in extracts from pancreas or isolated pancreatic islets (not shown).



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Figure 5. PISCES-BPs in the Eye and Cerebellum as Revealed by Electrophoretic Mobility Shift Assay

Nuclear extracts (lane 1) or whole-cell extracts (lanes 2–12) from the eye, cerebellum (Cerebel.) or HIT islet cell line were incubated with the probes indicated. The competitors were added at a 500-fold molar excess. The oligonucleotide G3A contains the PISCES motif, whereas the PISCES motif is mutated in G3Amut. F, Free probe.

 
Differential PISCES Binding by Pax6 Splice Variants
The eye and brain of adult mice are known to express the Pax6 gene as at least two isoforms (6, 30, 31). An alternatively spliced exon (exon 5a) in vertebrate Pax6 genes is included variably in the mature mRNA transcript, resulting in the insertion of a 14-amino acid peptide in the paired domain (6). This protein exhibits unique DNA-binding properties (21). To investigate the ability of these splice variants to bind the PISCES motif, the two Pax6 paired domains (denoted Pax6 and Pax6–5a) were expressed in Escherichia coli as glutathione S-transferase fusion proteins and used in an electrophoretic mobility shift assay. As shown in Fig. 6Go, Pax6 bound very well to labeled oligonucleotides containing the glucagon G3 enhancer-like element (G3) or domain A of the G3 element (G3A) with the PISCES motif, whereas the extended paired domain (Pax6–5a) did not. These results show that of the two alternative Pax6 proteins, only the one without the 14-amino acid insertion in the paired domain can bind the PISCES motif.



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Figure 6. Pax6, but Not the Splice Variant Pax6–5a Paired Domain, Binds the PISCES Motif as Revealed by Electrophoretic Mobility Shift Assay

The bacterially expressed Pax6 or Pax6–5a paired domains were incubated with the probes indicated. The oligonucleotides G3 and G3A contain the PISCES motif. The oligonucleotides P6Con and 5aCon contain high-affinity consensus binding sites for the Pax6 and Pax6–5a paired domains, respectively (21 ), and served as positive controls. An oligonucleotide (SCE) containing the cAMP response element of the rat somatostatin gene was used as negative control. F, Free probe. Specific complexes formed by Pax6 and Pax6–5a are indicated by arrows on the left and right, respectively. Pax6–5a has been shown to form several complexes when incubated with labeled 5aCon (21 ).

 
Pax6 Gene Expression in Pancreatic Islets as Revealed by RT-PCR
Pax6 gene expression was studied in pancreatic islets by RT-PCR. Two primer pairs were used, one of which flanks the alternatively spliced exon 5a (Fig. 7Go). With poly (A)+ RNA extracted from the adult pancreas (Fig. 7Go) or from isolated pancreatic islets (not shown), the primer pair that targets exon 2 and exon 6 detected pax6 transcripts with and without exon 5a (499- and 457-bp fragment, respectively, Fig. 7Go). The primer pair that targets exon 6 and exon 13 (Fig. 7Go) generated a single product of the expected size (933-bp fragment, Fig. 7Go). The RT-PCR products were verified by subcloning and sequencing. These results confirm (6, 8, 22, 32) that the Pax6 gene is expressed in adult pancreatic islets. They indicate that the transcripts in the islets are fully consistent with the known Pax6 cDNA, with and without the alternatively spliced exon 5a. Similar results were obtained with poly (A)+ RNA extracted from the embryonic pancreas on days 11.5/12.0 p.c. (Fig. 7Go). With poly (A)+ RNA extracted from the eye and cerebellum, both primer pairs generated products of the expected size for both splice variants (not shown), consistent with published reports of the expression of both splice variants in the eye and brain (21, 30, 31, 33).



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Figure 7. Pax6 Gene Expression in Mouse Pancreas as Revealed by RT-PCR

The upper part of the figure shows the relative positions of the two primer pairs used. The Pax6 cDNA is shown with the exons of the human gene. UTR, Untranslated region. The sizes of the expected fragments are 499 and 457 bp with the primer pair that targets exon 2 and exon 6 (with and without exon 5a) and 933 bp with the primer pair that targets exon 6 and exon 13. Poly (A)+ RNA from adult or embryonic (day 11.5/12.0 p.c.) pancreas was used. The RT-PCR products were verified by subcloning and sequencing.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study transgenic mice were used to investigate the transcriptional activity of the PISCES motif, which binds the transcription factor Pax6, in normal mature tissues in vivo. Although the Pax6 gene is expressed in the adult eye, brain, and pancreatic islets, this study shows that the PISCES motif directs expression in the eye and brain but not in pancreatic islets, suggesting that the Pax6 protein(s) in normal pancreatic islets function differently.

This study shows that four copies of a short 16-bp oligonucleotide containing the PISCES motif are sufficient to direct highly cell-specific expression in transgenic mice. Reporter gene expression was selectively activated by the PISCES motif in the eye and discrete areas within the central nervous system of the adult mouse, with highest levels in the cerebellum and colliculi superiores and low activity in the olfactory bulb, septal area, zona incerta, and amygdala. No reporter expression was found in the cerebral cortex, hippocampus, caudate putamen, or any peripheral organ tested. The PISCES-binding transcription factor Pax6 is known to be expressed in mature eye tissues including the retina, lens, corneal epithelia, and iris (34). In the cerebellar cortex of the adult brain, the granular cell layer expresses high levels of the Pax6 gene (29). In general, the reporter gene expression pattern observed in the present study agrees with the known tissue distribution of Pax6 (29), suggesting that transcriptional activity of the PISCES motif is conferred by the transcription factor Pax6. A possible exception could be the superior colliculus, where Pax7 rather than Pax6 is expressed (29). However, in this region most of the fibers of the optic nerve terminate (35), and thus the luminescence signal detected over the superior colliculus and optic chiasma/optic tract could be generated by reporter enzyme localized within projections of Pax6-expressing retinal ganglion cells (30, 34, 36). Pax6 is expressed early in development, and as is indicated by severe malformations of spontaneous mutants in Drosophila (eyeless), mouse (Small eye), and man (aniridia), Pax6 is essential for the formation of the eye, nose, and central nervous system (5, 6, 29, 30, 37). Pax6 is down-regulated in most cell types as they become postmitotic and differentiate. Expression is maintained, however, within several cell types of the mature eye and in discrete subregions of the adult brain (see above). The PISCES-mediated transcriptional activity observed in the present study in the adult mouse suggests that Pax6 protein(s) expressed in the adult eye and brain possess independent transcriptional activity, supporting a role for Pax6 in the differentiation and maintenance of mature eye tissues and specific brain cell subtypes (29, 34, 38).

The gross distribution of glucagon (39, 40, 41) and somatostatin gene products (42, 43) in the adult eye and brain partially overlap with the expression pattern of Pax6, raising the possibility that Pax6 could contribute to their regulation. Other genes with identified Pax6-binding sites are the mouse {alpha}A-crystallin gene (44) and the gene encoding the neural cell adhesion molecule (45). Even so, the Pax6 target genes in the mature eye and brain remain to be defined. It is noteworthy that the homomultimeric PISCES minienhancer could serve to direct the expression of proteins under study to defined regions of the eye and brain.

The PISCES is a sequence motif of the rat glucagon, insulin-I, and somatostatin genes (19). The finding of the present study that a regulatory sequence of islet hormone genes is able to direct expression in the brain exemplifies that the endocrine cells of the pancreas are related to neurons. Although of endodermal origin, the pancreatic islets have several characteristics in common with the brain (1, 18, 39), which may be explained at the molecular level by the fact that pancreatic islets share several transcription factors with the brain including Beta2/NeuroD (4), brain4 (46), as well as Pax6. Pax6 is expressed early during pancreatic development (8, 22), and several lines of evidence suggest that the Pax6 gene is expressed also in mature, normal pancreatic islets. Using RT-PCR, Turque et al. (32) detected exon 7-containing pax6 transcripts in islets of juvenile 4- to 5-week-old mice, and in the present study exon 2 to 13-containing pax6 transcripts were found in adult mouse islets. The mRNAs of two splice variants, with and without exon 5a, were detected, although only the paired domain without exon 5a, when expressed in bacteria, was found to bind the PISCES motif. Sander et al. (22) showed immunofluorescence staining of adult mouse pancreatic islets with an antiserum directed against the paired domain of quail Pax6. Finally, a mutated Pax6 gene of a disrupted allele lacking the region from exon 4 to 6 was found to be transcriptionally active in the islets of Langerhans in newborn mice (8). Although these data indicate that the Pax6 gene is expressed in normal pancreatic islets, they define neither the protein(s) formed nor their functional activity. Transgenesis provides the most comprehensive and rigorous test of cell-specific transcriptional activity for DNA control elements. The present study shows that the PISCES motif by itself does not possess transcriptional activity in normal pancreatic islets of the adult or embryonic mouse, suggesting that the Pax6 protein(s) in normal islets function differently from the ones in the eye, the brain, and pancreatic islet cell lines in which the PISCES motif confers strong transcriptional activity (Refs. 19, 20, 23 and the present study).

The lack of PISCES-mediated transcription in normal pancreatic islets could be explained by unique posttranslational modifications of Pax6, the absence in islets of essential coactivators, or the presence of modulatory proteins, as exemplified by the homeodomain protein Engrailed-1 in quail neuroretina, which inhibits the DNA binding of Pax6 through direct protein-protein interaction (47). It is noteworthy that engrailed may also be expressed in islets (48). The lack of activity of Pax6 at the isolated PISCES motif in normal pancreatic islets may not be unique, since Pax6 has been shown to repress ß-crystallin gene transcription through a Pax6-binding site of the ßB1-crystallin promoter in chicken primary lens epithelial cells (49). Based on the expression of the Pax6 gene in normal islets and based on the activation of G3-mediated transcription by overexpression of a Pax6 isoform in a non-glucagon-expressing cell line, it has been suggested that Pax6, acting through the PISCES motif, may be required for pancreatic islet hormone gene transcription in vivo (22). The lack of PISCES-mediated transcriptional activity in normal islets in vivo, as determined in the present study using transgenic mice, could indicate that islet hormone gene transcription is maintained in normal islets independently of Pax6. Alternatively, Pax6 may be essential for islet hormone gene expression in pancreatic islets and act through the PISCES motif, if a specific chromatin positioning or sequences in addition to the PISCES element are necessary for activation of transcription by Pax6 in pancreatic islets. Thus, Pax6 protein(s) may act through the PISCES motif in the context of the islet hormone gene promoters. The regulation of the POMC gene gives an example of a case in which transgenes containing regulatory sequences were expressed in one of the expected tissues but not in others, until additional DNA sequences were included (50). The identification of the sequences necessary and the proteins that bind to them will be required before conclusions can be drawn about the role of Pax6 in hormone gene transcription in normal pancreatic islets.

The results obtained in Pax6-/- mice clearly establish a role of Pax6 for endocrine cell differentiation in the developing pancreas (8). With the exception of islet hormone genes, Pax6 target genes through which Pax6 directs pancreatic islet formation have not yet been identified. In those genes, Pax6 or Pax6–5a could act through PISCES-related or other DNA sites as well as through protein-protein interaction. Developmental regulation of the splicing of 5'-exons of the Pax6 gene has been shown in Drosophila, where the form of the eyeless transcript expressed in the adult stage is distinct from the embryonic form and also from the larval form (51). An alternative exon {alpha} replacing the exons 0–3 (corresponding to the mouse exons 1–4) has been found in the quail (52). However, no evidence for development-specific formation of pax6 transcripts in mouse islets was obtained by RT-PCR in the present study. The nature and relative roles of the Pax6 protein(s) derived from the Pax6 and Pax6–5a mRNAs during pancreatic islet development remain to be defined.

In summary, the present study shows that the PISCES motif is sufficient to direct expression in the eye and brain, but is not sufficient to direct expression in pancreatic islets of transgenic mice, suggesting that the Pax6 protein(s) in normal islets function differently from the ones in the eye, the brain, and islet cell lines. Designed to fulfill islet-specific functions, the Pax6 protein(s) in normal islets could thus expand the functional diversity of the Pax6 gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation and Analysis of Transgenic Mice
The construction of the PISCES-luciferase reporter gene plasmids (p4xG3AT81Luc, p4xG3AmutT81Luc) has been described previously (20). Transgenic mice were generated according to standard procedures (53). Reporter fusion genes free of vector sequences were obtained by digestion with BamHI and ApaI, purified, and microinjected into the male pronucleus of fertilized eggs (FVB/N). Microinjected eggs were transferred to the oviducts of foster mothers (CBA/CaJ). Genomic (tail) DNA from the founder mice and offspring was digested with BglII, electrophoresed, and subjected to Southern blot analysis using a 1.6-kb XbaI luciferase fragment as probe [Megaprime DNA labeling system, (Amersham, Arlington Heights, IL) using [{alpha}-32P]dCTP]. Reporter gene expression was determined by measuring reporter enzyme activity in tissue extracts or on cryostat sections. The tissues were homogenized (200 mg wet weight per ml ice-cold 100 mM K phosphate buffer, pH 7.8, containing 1 mM dithiothreitol, 4 mM EGTA, 4 mM EDTA, 0.7 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml pepstatin, and 5 µg/ml aprotinin), subjected to three cycles of freeze-thawing, and centrifuged. Luciferase activity was measured in the supernatants as described (54, 55). Protein was measured using a commercial kit (Bio-Rad Laboratories, Inc., Munich, Germany). Sections (40 µm) were cut with a cryostat at -26 C and transferred onto slides (Super Frost*/Plus microscope slides, Menzel-Gläser, Braunschweig, Germany). Sections were covered with luciferin substrate solution (Luciferase Assay Kit, Berthold Detection Systems, Pforzheim, Germany), and the luminescence signal was measured with a Molecular Light Imager (EG&G Berthold). All animal studies were conducted according to the "Guidelines for Care and Use of Experimental Animals" and approved by the Committee on Animal Care und Use of the local institution and state.

Electrophoretic Mobility Shift Assays
Oligonucleotides with 5'-GATC overhangs were labeled by a fill-in reaction using [{alpha}-32P]dCTP and Klenow enzyme (56). The oligonucleotides used as probes or competitors have been described previously: G3, G3A, and G3Amut (23); SCE (57); and P6Con and 5aCon (21). Nuclear extracts from the insulin-producing pancreatic islet cell line HIT were prepared as described (57). Whole-cell extracts from the eye and cerebellum of transgenic mice (~150 and 260 mg wet weight per 200 µl extraction buffer) as well as from HIT cells were prepared following the protocol of Kornhauser et al. (58). Using 15 µg of nuclear protein or 10 µl of whole-cell extracts in 25 µl (total volume) reaction buffer containing 18 mM HEPES, pH 7.9, 50 mM NaCl, 90 mM KCl, 2 mM MgCl2, 0.08 mM EDTA, 0.4 mM EGTA, 5 mM dithiothreitol, 0.4 mM NaF, 2 mM spermidin, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 µg/ml leupeptin, 0.28 µg/ml pepstatin, 16 µg/ml bestatin, 0.4 µg/ml aprotinin, 25 µg of BSA, 2 µg of poly(dI-dC), 12% glycerol, and 0.1% NP-40, the assay was performed as described (23). The Pax6 and Pax6–5a paired domains were expressed in E. coli as glutathione-S-transferase fusion proteins, purified, and used in an electrophoretic mobility shift assay as described (21, 57).

RT-PCR
Pancreatic islets were isolated as described (59). Poly (A)+ RNA was extracted from the adult eye, cerebellum, pancreas, or isolated pancreatic islets using commercial kits (Micro Fast Track or Fast Track 2.0, Invitrogen, San Diego, CA). Poly (A)+ RNA from embryonic mouse pancreas (day 11.5/12.0 p.c.) was a generous gift from Dr. Luc St. Onge (DeveloGen, Göttingen, Germany). RT-PCR was performed using a commercial kit (Gene Amp Thermostable rTth Reverse Transcriptase RNA PCR Kit, Roche Molecular Systems, Branchburg, NJ) with primers and PCR reaction conditions as follows. Primer pair 2/6: upstream primer 5'-ACGAAAGAGAGGATGCCTC-3' (exon 2), downstream primer 5'-CCCAAGCAAAGATGGAAG-3' (exon 6); 30 sec at 95 C, 1 min at 58 C, 3 min at 72 C, for 40 cycles; the expected products are 457 and 499 bp long (without and with exon 5a) (30). Primer pair 6/13: upstream primer 5'-CATCTTTGCTTGGGAAATC-3' (exon 6), downstream primer 5'-AACTTGGACGGGAACTGAC-3' (exon 13); 30 sec at 95 C, 1 min at 59 C, 2 min at 72 C, for 40 cycles; the expected product is 933 bp long (30). After agarose gel electrophoresis, the products obtained were verified by extraction, subcloning (pCR 2.1, Invitrogen), and cycle sequencing (Thermo Sequenase fluorescent-labeled primer cycle sequencing kit, Amersham, Braunschweig, Germany; M13 fluorescent primer).


    ACKNOWLEDGMENTS
 
We thank Richard L. Maas (Harvard Medical School, Boston, MA) for pGEX-Pax6 and pGEX-Pax6–5a, Simon Saule (Institut Pasteur, Lille, France) for anti-Pax6 antiserum, Klaus Becker, Ksenia Sovic, and Arnold Hasselblatt (University of Göttingen, Göttingen, Germany) for islet preparation, and Rainer Kembügler (EG&G Berthold, Bad Wildbad, Germany) for his help with the Molecular Light Imager. We thank Luc St. Onge (DeveloGen, Göttingen, Germany) for the generous gift of poly (A)+ RNA from embryonic pancreas and for instructing us how to dissect pancreatic tissue from mouse embryos.


    FOOTNOTES
 
Address requests for reprints to: Willhart Knepel, Department of Molecular Pharmacology, University of Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. E-mail: wknepel{at}med.uni-goettingen.de

This work was supported by the Deutsche Forschungsgemeinschaft, SFB402/A3.

Received for publication May 8, 1998. Revision received February 1, 1999. Accepted for publication February 3, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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