The homeodomain transcription factor Pdx1 is essential for pancreas development. To investigate the role of Pdx1 in the adult pancreas, we employed a mouse model in which transcription of Pdx1 could be reversibly repressed by administration of doxycycline. Repression of Pdx1 in adult mice impaired expression of insulin and glucagon, leading to diabetes within 14 days. Pdx1 repression was associated with increased cell proliferation predominantly in the exocrine pancreas and upregulation of genes implicated in pancreas regeneration. Following withdrawal of doxycycline and derepression of Pdx1, normoglycemia was restored within 28 days; during this period, Pdx1+/Ins+ and Pdx+/Ins cells were observed in association with the duct epithelia. These findings confirm that Pdx1 is required for β-cell function in the adult pancreas and indicate that in the absence of Pdx1 expression, a regenerative program is initiated with the potential for Pdx1-dependent β-cell neogenesis.

Pdx1 is a homeodomain transcription factor essential for pancreatic development (13). Early in mouse development (embryonic day 8.5), Pdx1 is highly expressed in a region of the posterior foregut endoderm, from which the dorsal and ventral pancreatic buds arise. Its expression then becomes progressively restricted to endocrine cells (4), and in the adult pancreas Pdx1 regulates several genes including insulin, glucose transporter-2, and glucokinase (57) integral to β-cell function. The adult pancreas retains the capacity to increase its β-cell mass in response to physiological requirements, e.g., as in pregnancy (8) or in response to injury (9). β-Cell neogenesis is presumed to depend on the existence of progenitor β-cells in the adult pancreas, although the origin of progenitors from ductal (1012), islet (13,14), exocrine (1519), or multilineage (20) cells remains contentious (21,22). In addition to its key role in pancreas development, Pdx1 is implicated in β-cell neogenesis, being expressed in duct and duct-associated cells in models of pancreas regeneration (20,23,24). It is unclear, however, whether Pdx1 is required early for the activation of a regenerative program or later for the differentiation of putative progenitor cells. Investigating the role of Pdx1 in the adult pancreas is precluded by the fact that pancreas development is arrested following specification of the foregut endoderm in mice lacking Pdx1 (13). To investigate the role of Pdx1 postdevelopment, we created a mouse model (25) in which the expression of Pdx1 can be reversibly repressed by administering the tetracycline analog, doxycycline. Here, we employ this model to investigate the requirement for Pdx1 in β-cell function and regeneration in the adult pancreas.

Generation of knock-in and transgenic mice.

The basic elements of the Pdx1 tet-off inducible gene repression system are shown schematically in Fig. 1. Pdx1tTA knock-in and TgPdx1 transgenic mice were generated as previously described (25). To obtain mice in which Pdx1 could be conditionally repressed, the Pdx1tTA/+ knock-in mice were crossed with TgPdx1 mice and progeny intercrossed to derive Pdx1tTA/tTA;TgPdx1 mice as well as genotype control mice (see results).

Animal care and treatment.

Animals were housed under standard 12-h light/dark conditions and fed and watered ad libitum. Experiments were approved by the Royal Melbourne Hospital Campus Animal Research Ethics Committee. Groups of mice were age and sex matched within each experiment. Doxycycline was administered as a single intraperitoneal dose of 100 mg/kg at time t = 0 and maintained by addition to drinking water at 0.5 mg/ml. BrdU (100 mg/kg) was administered intraperitoneally as a single dose 16 h before the mice were killed.

Glucose tolerance test.

Mice were fasted overnight and challenged with 2 g/kg i.p. glucose. Glucose levels in retroorbital venous blood samples were measured at 0, 15, 30, 60, 120, and 180 min postchallenge with an Accu-chek Advantage glucometer (Roche Diagnostics, Castle Hill, NSW, Australia). Statistical analyses (Mann-Whitney tests) were performed using Graphpad Prism (GraphPad Software, San Diego, CA).

Immunocytochemistry and immunofluorescence.

Adult pancreata were fixed in Histochoice (Sigma, St. Louis, MO) or 4% paraformaldehyde for 3 h before processing, embedding in paraffin, and sectioning (5 μm). The following primary antibodies were used: guinea pig anti-human insulin (Dako, Carpinteria, CA) at 1:1,000, rabbit anti-human amylase (Sigma) at 1:1,000, rabbit anti-human glucagon (Dako) at 1:200, rabbit anti-human somatostatin (Dako) at 1:100, rabbit anti-GLUT2 (a gift from Bernard Thorens, Université de Lausanne, Lausanne, Switzerland) at 1:100, and rabbit anti-human filtrin (Alpha Diagnostic, San Antonio, TX) at 1:20. Rabbit anti-Pdx1 (a gift from Christopher Wright, Vanderbilt University Medical Center, Nashville, TN) was used at 1:500 and rabbit anti-Pdx1 antibody (purified IgG 1:25) was raised against a glutathione S-transferase fusion protein containing amino acids 14–284 of murine Pdx1. Fluorescein isothiocyanate–conjugated anti-guinea pig (ICN Biomedicals, Aurora, OH) or Alexa fluor 568–conjugated goat anti-rabbit (Molecular Probes, Eugene, OR) immunoglobulins were used for immunofluorescence. Nuclei were counterstained with 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI). BrdU-labeled cells were detected with a cell proliferation detection kit (Amersham, Buckinhamshire, U.K.) with mouse monoclonal anti-BrdU primary and Alexa fluor 568–conjugated anti-mouse secondary antibodies. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling was performed with an apoptag fluorescein apoptosis detection kit (Serologicals, Norfolk, GA). Digital images were captured with an Axiocam camera from an Axioplan2 compound microscope (Carl Zeiss, Göttingen, Germany). Dual-color immunofluorescence images were compiled from two separate images of the same section using fluorochrome-specific filters.

BrdU-labeling index was determined by imaging 6–8 fields (200× magnification) of randomly selected pancreas sections from each of three untreated or 21-day doxycycline–treated, or six 14-day doxycycline–treated, mice per group. BrdU+ cells were counted with National Institutes of Health image software (National Institutes of Health, Bethesda, MD). Quantitative analysis of glucagon- and insulin-stained pancreata was performed with Axiovision 4.2 image analysis software (Carl Zeiss). Briefly, 4–8 nonoverlapping fields containing islets (coimmunostained with separate flourophores for insulin and glucagon and counterstained with DAPI) were imaged from pancreata of 2–3 animals for each treatment group. Insulin- and glucagon-stained areas were determined by performing identical semiautomated image analysis routines on each of the separate fluorescent image channels. Islet areas were determined by manually delineating the islets, and costained cells were counted manually on the merged immunofluorescent images.

Differential gene expression and real-time PCR analyses.

Total RNA was isolated with RNAzol B (Tel-Test, Friendswood, TX) from the pancreata of age- and sex-matched Pdx1tTA/tTA;TgPdx1 mice either untreated (n = 3) or treated (n = 4) with doxycyline for 14 days. Equal amounts of RNA were pooled to create +dox and −dox groups for comparison on Affymetrix gene chip arrays (Affymetrix, Santa Clara, CA). A total of 30 μg total RNA was used to generate fragmented cRNA as per Affymetrix gene chip protocols. cRNA quality was assessed by hybridization of 5 μg fragmented cRNA to Affymetrix Test 3 arrays before hybridizing 20 μg fragmented cRNA to Affymetrix MGU74Av2 gene chip arrays. Chips were processed and scanned as per protocols in the Affymetrix gene chip expression analysis technical manual. Analysis of the probe data were performed as follows. The perfect match probe intensities were background corrected and normalized across arrays and then summarized for each probe set using robust multichip analysis (26,27). Once expression levels had been calculated, differentially expressed genes were ranked and values expressed as fold change.

Quantitative real-time RT-PCR was performed on the Roche Lightcycler (Roche Diagnostics). For each gene to be assayed, a template was amplified by PCR and subcloned into a pGEM-T vector (Promega, Madison, WI). The templates were used to generate standard curves for quantitative determination of mRNA expression level in the individual RNAs described above. Expression levels were corrected relative to the expression of the of β-actin gene. Sequences of oligonucleotide primers used are available on request.

Islet area determination.

Islet area was determined for pancreata of mice that were either untreated or treated with doxycycline for 14 days (four mice per group). Three hematoxylin and eosin–stained sections from each pancreas were sampled, each section separated by ∼250–300 μm. Nonoverlapping 40× magnification fields were captured with an Axiocam digital camera (Carl Zeiss), and the total tissue and islet area per field was determined using Axiovision 4.2 image analysis software (Carl Zeiss). All sections were scored blind with respect to treatment. Statistical significance was determined using a Mann-Whitney U test (GraphPad Software).

Conditional repression of Pdx1.

Two genetic modifications were required to achieve conditional repression of Pdx1 (25) (Fig. 1). First, the endogenous Pdx1 gene was inactivated by replacing its coding region with that of the tetracycline transactivator (tTA off) by homologous recombination. As a result, transcription of the tTA gene is directed by the endogenous Pdx1 transcriptional regulatory sequences. Mice with one inactivated Pdx1 allele develop normally (13) and can be maintained in the heterozygous state. Second, mice bearing a tTA-responsive Pdx1 gene were created by introducing a bicistronic transgene containing a Pdx1 mini-gene and an enhanced green fluorescence protein reporter gene under the control of a tTA-responsive promoter. When the two mouse lines were crossed, some of the progeny contained both the knocked-in tTA gene and the tTA-responsive transgene. Mice homozygous for the Pdx-tTA–off knock-in allele are dependent on tTA-mediated transcription of the transgene for expression of Pdx1. In this system, mice with the Pdx1tTA/tTA;TgPdx1 genotype develop normally because transgenic Pdx1 expression is driven, indirectly, by tTA expressed off the native Pdx1 promoter. Administration of doxycycline prevents the tTA from binding to the tTA-responsive promoter, thereby deactivating transcription of the transgene and depleting Pdx1 (Fig. 1). We previously reported (25) that Pdx1tTA/tTA;TgPdx1 mice survive to adulthood and maintain normal glucose homeostasis.

Doxycycline-mediated repression of Pdx1 progressively impairs β-cell function.

Adult mice were treated with doxycycline for 0, 7, 14, or 21 days, and β-cell function was assessed weekly by intraperitoneal glucose tolerance tests (Fig. 2A). Untreated Pdx1tTA/tTA;TgPdx1 mice had normal fasting blood glucose levels and responded normally to a glucose challenge, recovering to basal levels within 3 h. However, within 7 days of starting doxycycline, the early glucose response of Pdx1tTA/tTA;TgPdx1 mice was increased and their ability to recover after the glucose challenge was impaired, similar to that of the Pdx1tTA/+ mice. By 14 days of doxycycline treatment, Pdx1tTA/tTA;TgPdx1 mice had diabetes (Fig. 2A).

Although pancreas histology of Pdx1tTA/tTA;TgPdx1 mice treated with doxycycline for 14 days was grossly normal, the islets appeared to be smaller (Fig. 2B). Morphometric analysis confirmed a significant decrease (P < 0.05) in the islet area of treated animals (Fig. 2D). Transferase-mediated dUTP nick-end labeling staining was performed to determine whether impaired β-cell function in doxycycline-treated Pdx1tTA/tTA;TgPdx1 mice was associated with β-cell apoptosis. Transferase-mediated dUTP nick-end labeling–positive cells were detected at a similar low frequency in both control and doxycycline-treated mice, predominantly in the exocrine pancreas, labeled cells being rarely detected in the islets of either treated or untreated mice (data not shown). Staining for filtrin (28), a member of the nephrin family (29) whose expression is restricted to β-cells (28,30), revealed that β-cells, although lacking insulin (see below), were still present after 14 (Fig. 2C) and 28 days of doxycycline treatment. Quantitative RT-PCR demonstrated a decrease in Pdx1 and insulin transcripts and a corresponding increase in glucagon transcripts in pancreata of doxycycline-treated mice (Fig. 2E). In doxycycline-treated mice, histological analysis of the pancreas revealed total absence of Pdx1 (Fig. 3A, panel f) and a marked reduction of insulin, with only a few cells in each islet staining strongly (Fig. 3A, panel f). Glucagon-positive cells were prominent, as well as cells costaining for glucagon and insulin (Fig. 3A, panel h). Expression of the glucose transporter Glut2 was undetectable in the islets of Pdx1-repressed mice (Fig. 3A, panel i). Amylase was restricted to the exocrine pancreas, although its staining appeared to be reduced (Fig. 3A, panel j), consistent with our previous finding that the level of amylase mRNA is reduced in Pdx1-repressed mice (25). Untreated Pdx1tTA/tTA;TgPdx1 mice (Fig. 3A, panels a–e) and control Pdx1+/+;TgPdx1 mice (Fig. 3A, panels k–o) had normal expression of Pdx1, insulin, somatostatin, glucagon, Glut2, and amylase.

Quantitative image analysis of the pancreata of doxycycline-treated mice revealed that the decrease in insulin staining corresponded with an increase in glucagon staining (Fig. 3B) and with an increase in the number of islet cells coexpressing insulin and glucagon (Fig. 3C), confirming the quantitative RT-PCR results. These observations suggest that Pdx1 normally supresses expression of the glucagon gene in β-cells.

Doxycycline-induced diabetes is reversible.

A key feature of the tet-off system is the ability to reversibly control expression of the tTA-dependent transgene. Therefore, to demonstrate that derepression of the Pdx1 transgene would restore β-cell function, we withdrew doxycycline after 14 days administration to Pdx1tTA/tTA;TgPdx1 mice. Blood glucose levels of 8-h fasted mice were then measured every 7 days for 35 days. Blood glucose levels decreased significantly by 14 days and progressively until four of five mice were normoglycemic by 28 days, demonstrating that doxycycline-induced diabetes is reversible (Fig. 4A). Histological analysis revealed Pdx1 protein in some islet cells 7 days after doxycycline withdrawal, although insulin expression remained low (Fig. 4B). However, by 14 days, both Pdx1 and insulin were expressed homogeneously at pretreatment levels throughout the islets (Fig. 4B), and both the glucagon-positive islet area (Fig. 3B) and the number of insulin+/glucagon+ costained cells per islet (Figs. 3C and 4C) had also decreased.

Repression of Pdx1 initiates proliferation of pancreatic exocrine and duct-associated cells.

Foci of duct proliferation comprising small ductules embedded in connective tissue, similar to those observed in other models of pancreas regeneration (10,31), were present in mice treated with doxycycline for 14 days and became more prominent the longer the duration of doxycycline administration (Fig. 5A, upper panel). Staining for proliferating cell nuclear antigen confirmed the presence of proliferating cells within these ductal-like structures (Fig. 5A, lower panel). To quantitate cellular proliferation, Pdx1tTA/tTA;TgPdx1 mice either untreated or treated with doxycycline for 14 or 21 days were pulsed with BrdU overnight before they were killed. Immunofluorescence analysis revealed increased nuclear labeling in the pancreata of Pdx1-repressed mice at both 14 (P < 0.0005) and 21 (P < 0.0001) days (Fig. 5B). BrdU-positive cells were more frequent within exocrine tissue, although they were also observed proximate to ducts, within foci of duct proliferation and in islets (Fig. 5B).

Derepression of Pdx1 leads to the appearance of duct-associated insulin+ cells.

Following withdrawal of doxycycline, insulin+ cells were primarily detected in islets or smaller islet-like cell clusters (Fig. 5C). Pdx1 was observed at high levels in islets and islet-like cell clusters in insulin+ cells (Fig. 5C). In addition, insulin+ cells expressing Pdx1 were observed embedded in or closely associated with ducts (Fig. 5C). Pdx1+/insulin cells were also observed associated with ducts (Fig. 5C, panel b), but the Pdx1 staining in these cells was generally of lower intensity than in the insulin+ cells.

Repression of Pdx1 upregulates genes implicated in pancreas regeneration.

To further characterize the molecular events associated with Pdx1 repression and potentially identify novel Pdx1-regulated genes, pancreatic RNA was isolated from Pdx1tTA/tTA;TgPdx1 mice that had been treated with doxycycline for 14 days and from untreated mice, and gene expression profiles were compared on Affymetrix MGU74Av2 gene chip arrays. To minimize variation, the gene chip analyses were performed on pooled RNA samples from treated and untreated mice. The experiment was replicated with independent cohorts of mice, and the results obtained were essentially identical. Pdx1 repression was associated with upregulation of 97 transcripts and downregulation of 29 transcripts by at least 1.8-fold (Table 1). Gene ontology associations revealed that >50% of the upregulated genes were involved in metabolism or cell growth and/or maintenance (Fig. 6A). The results were validated by real-time RT-PCR of several genes in individual mouse RNA samples that had been pooled for the gene chip experiments (Figs. 2E and 6B). Despite variation between individual samples, expression levels determined by real-time RT-PCR correlated well with the fold change determined from the gene chip analysis of pooled RNA. Of the downregulated genes, the most notable were the insulin genes ins1 and ins2, validating the results of the gene chip strategy. Upregulated genes notably included several previously identified by differential gene expression screens in models of pancreas regeneration (32,33), in particular the regenerating islet–derived (Reg) family (Reg2, Reg3α, Reg3β, and Reg3γ) that plays a role in the maintenance of progenitor cell populations and endocrine differentiation via the notch signal transduction pathway and transducer of ErbB2.1 (Tob1) involved in the regulation of cell proliferation and transformation. Novel transcripts were also identified in the screen. One of these, 1810015C04Rik (Entrez Gene ID 66270) was confirmed by RT-PCR to be upregulated in all of the Pdx1-repressed mice (Fig. 6B) and found to encode a highly conserved protein of unknown function. It has been previously identified in a number of tissues, including embryonic pancreas (Unigene Mm.25311), suggesting a potential role for this gene in early pancreas development. RT-PCR analysis revealed expression of 1810015C04Rik in two pancreatic β-cell lines, Min1046 and βTC-3, as well as in kidney and liver but not in the α-cell line, αTC-1 (Fig. 6C).

Using genetically modified mice in which Pdx1 transcription can be reversibly repressed, we confirm the critical role of Pdx1 in maintaining β-cell function. Furthermore, we provide evidence that Pdx1 normally suppresses glucagon expression in β-cells and that repression of Pdx1 leads to enhanced proliferation of presumptive β-cell progenitors.

Mutations of Pdx1 leading to impaired β-cell function underlie maturity-onset diabetes of the young type 4 in humans, and several studies (57,34,35) have demonstrated a dose-dependent requirement for Pdx1 in the maintenance of normal glucose homeostasis in mice. These studies initially examined pancreatic function in adult mice with one Pdx1 allele inactivated (Pdx1+/−) and more recently used transgenic approaches to conditionally inactivate (5) or inducibly repress Pdx1 either with antisense Pdx1 (6) or Pdx1-specific ribozyme (7) strategies. However, these models are limited by their dependence on expression of molecules under the control of the insulin promoter, which is downregulated following repression of Pdx1, resulting in relatively mild impairment of glucose tolerance (6,7) or the late onset of diabetes in the case of RIP-Cre-Pdx1 mice (5). In our model, the coding sequence for the tet-off regulatory protein (tTA) is incorporated into the Pdx1 locus so that normal temporal and spatial regulation of the gene is maintained, thereby overcoming a problem inherent in many transgenic systems that utilize partial regulatory sequences out of their genomic context. The genetically modified mice are morphologically and functionally normal, yet doxycycline treatment of adult mice repressed Pdx1 expression and induced marked hyperglycemia associated with reduced expression of insulin and the glucose transporter, Glut2. These effects were reversible upon withdrawal of doxycycline. This is therefore a truly inducible gene knock-out model in which the role of the gene can be studied at any time during development or in mature animals. β-Cells were still present in Pdx1-repressed mice, and some contained residual insulin, which is not surprising as Pdx1 is only one of a number of transcription factors involved in the regulation of insulin transcription (36). Indeed, mice heterozygous for Pdx1 have normal pancreatic insulin content, although their ability to secrete insulin and clear glucose is impaired (5,37). We confirmed that the expression of several genes involved in the maintenance of glucose homeostasis, including insulin (38), Glut2 (39), and glucokinase (40), as is regulated by Pdx1 in the inducible antisense Pdx1 repression model of Lottmann et al. (6). Similarly, we were unable to detect Glut2 in the islets of Pdx1-repressed mice. It is likely that the loss of Glut2 leads to impaired glucose sensing and contributes to the diabetic phenotype of the Pdx1-repressed mice. In addition, we observed a reduction in islet area in Pdx1-repressed mice, indicating loss of the ability to maintain β-cell mass in the absence of Pdx1. It has been recently reported that haploinsufficiency of Pdx1 may be responsible for increased apoptosis of islet cells in Pdx1+/− mice (41). We did not find evidence for increased β-cell apoptosis in Pdx1-repressed mice. Nevertheless, as apoptosis is a rapid and transient event, it is possible that a population of apoptosis-sensitive β-cells may have been ablated before the 7-day time point at which our first observations were made.

In addition to the progressive loss of insulin expression in β-cells during Pdx1 repression, we observed a concomitant increase in glucagon expression and in the number of cells expressing both insulin and glucagon in islets. These observations are consistent with studies demonstrating that glucagon expression is repressed by Pdx1 in β-cell lines (42,43) and that the α-cell mass is increased in β-cell–specific Pdx1 knock-down mouse models (5,7). Similarly, ectopic expression of Pdx1 in α-cell lines has been shown to repress glucagon gene expression (44), although this probably requires cell line–specific cofactors (45).

We performed microarray analysis to characterize the response to Pdx1 repression. A number of upregulated genes were involved in metabolism, consistent with studies (37,46) in which defects in mitochondrial metabolism have been linked to diminished expression of Pdx1. Several of the most differentially upregulated genes (Reg2, Reg3α, Reg3β, and Reg3γ) were members of the regenerating islet-derived gene family originally identified in models of β-cell regeneration (4749). In addition, several other upregulated genes, including Numb and Tob, are likely to be involved in islet cell development. Numb is a modulator of the notch signaling pathway involved in the maintenance of embryonic neural progenitor cells (32). It is also expressed in early pancreatic progenitors and mature β-cells and has been implicated in the differentiation of β-cells from duct cells (33). The epidermal growth factor receptor ErbB2 is expressed in the developing pancreas and is upregulated in a model of duct-cell proliferation and islet neogenesis (50). Tob is an ErbB2 interacting protein implicated in the regulation of cell proliferation and differentiation (51). We also identified a novel highly conserved gene, 1810015C04Rik, which is expressed in the developing pancreas and mature β- but not α-cells. Further investigation is required to determine the role of 1810015C04Rik and other novel genes identified in this screen in β-cell viability and regeneration. In addition, it will also be important to determine whether these changes in gene expression are due to Pdx1 repression per se or are secondary to impaired β-cell function and hyperglycemia. Nevertheless, the initiation of a regenerative program by Pdx1 repression is a novel observation and a basis for further understanding β-cell neogenesis in the adult pancreas.

The adult pancreas has the capacity to increase its β-cell mass during times of increased physiological need or in response to injury. However, the nature of putative β-cell progenitors is a subject of ongoing debate. Numerous studies (911) support the existence of duct-associated progenitors in models of pancreatic injury, whereas a more recent lineage-tracing study (13) indicates that β-cell progenitors derive from β-cells themselves in islets. These findings are not mutually exclusive and may reflect different means to an end, depending on circumstances. Our model of reversible Pdx1 repression reveals a potential duct-associated progenitor cell population. First, during Pdx1 repression, we observed focal regions of duct proliferation, recognized as a hallmark of pancreas regeneration (10,31). Second, we observed an increase in cell replication, proportional to the duration of Pdx1 repression, primarily in the exocrine pancreas but also associated with duct-like structures as well as in islets. Third, we found that genes known to be associated with β-cell regeneration were upregulated during Pdx1 repression. Finally, after reversal of Pdx1 repression we observed cells that were Pdx1+/insulin+ or Pdx1+(low)/Ins closely associated with duct epithelium.

In rodent models of pancreas injury (20,23,24), Pdx1 expression has been associated with β-cell neogenesis. The proliferation we observed in response to repression of Pdx1 could be secondary to hyperglycemia rather than the loss of Pdx1 itself, as observed in other models of induced hyperglycemia (31,52). However, our findings show that proliferation of progenitors occurs in the absence of Pdx1 and that Pdx1 is required for their differentiation potentially into β-cells. Gu et al. (53) found that Pdx1 was rarely expressed in the ductal cells of the adult pancreas and were likely to be present only in differentiating cells in the process of delaminating from the ducts. In a separate study (R.J.M., unpublished data), it was shown that Pdx1 is not required for the continued proliferation of duct cells in the developing pancreas but is absolutely required for the differentiation of duct-like cells of the embryonic epithelium into endocrine and exocrine lineages. As suggested by Waguri et al. (20), β-cells could arise from both intraislet and ductal progenitors, depending on the circumstance. During periods of acute hyperglycemia, it may be sufficient for β-cells to enter the cell cycle and thereby increase the β-cell mass. However, following pancreatic injury or chronic hyperglycemia, a more primitive regenerative program may recruit duct-associated β-cell progenitors. Possibly consistent with evidence from lineage tracing (13), we also observed occasional islet cells labeled with BrdU in Pdx1-repressed mice but were unable to determine to what extent these cells contribute to β-cell regeneration. It is worth noting that in Pdx1-repressed mice, filtrin staining revealed the presence of β-cells, but the islet area was decreased, suggesting that Pdx1 may be required for the maintenance of intraislet progenitors. An important caveat, however, is that we have not been able to discriminate between restoration of β-cell function in pre-existing β-cells and β-cell neogenesis.

Combining the conditional Pdx1 expression model with lineage tracing or cell-specific ablation models could further elucidate the origins of β-cell progenitors and contribute to the goal of renewing β-cells for the treatment of type 1 diabetes.

FIG. 1.

The inducible Pdx1 knock-out mouse model. Schematic view of the Pdx1tTA knock-in and TgPdx1 transgene constructs. The tetracycline transactivator gene (tTA off) was knocked-in to the Pdx1 locus such that the tTA gene is expressed under the control of the endogenous Pdx1 promoter (Pdx1tTA mice). Mice carrying a transgene encoding the Pdx1 gene (TgPdx1) and a reporter gene (enhanced green fluorescence protein [EGFP] under the control of a tTA responsive promoter (tetO) were bred with Pdx1tTA mice. In transgenic mice homozygous for the tTA off gene (Pdx1tTA/tTA;TgPdx1), Pdx1 is transcribed from the transgene. After administration of doxycycline, tTA binding to the tetO is blocked, abolishing expression of Pdx1.

FIG. 1.

The inducible Pdx1 knock-out mouse model. Schematic view of the Pdx1tTA knock-in and TgPdx1 transgene constructs. The tetracycline transactivator gene (tTA off) was knocked-in to the Pdx1 locus such that the tTA gene is expressed under the control of the endogenous Pdx1 promoter (Pdx1tTA mice). Mice carrying a transgene encoding the Pdx1 gene (TgPdx1) and a reporter gene (enhanced green fluorescence protein [EGFP] under the control of a tTA responsive promoter (tetO) were bred with Pdx1tTA mice. In transgenic mice homozygous for the tTA off gene (Pdx1tTA/tTA;TgPdx1), Pdx1 is transcribed from the transgene. After administration of doxycycline, tTA binding to the tetO is blocked, abolishing expression of Pdx1.

FIG. 2.

Doxycycline-mediated repression of Pdx1 progressively impairs β-cell function. A: Pdx1tTA/tTA;TgPdx1 mice were treated with doxycycline for 0 (○), 7 (•), 14 (□), and 21 (▪) days. β-Cell function was assessed by intraperitoneal glucose tolerance test and measurement of blood glucose (means ± SE). The responses of Pdx1tTA/+ (▵) and Pdx1+/+;TgPdx1 (▴) mice without doxycycline treatment are shown for reference. Absolute values of blood glucose for 14- and 21-day treatment groups at the 15-, 30-, and 60-min time points may be higher, as *3/3, †4/6, and ‡1/6 animals had levels above the upper limit of the glucometer (i.e., >33.3 mmol/l). B: Hematoxylin and eosin–stained sections of pancreata from Pdx1tTA/tTA;TgPdx1 mice untreated or treated with doxycyline for 14 days (+dox 14d). Bar = 200 μm. C: Immunofluorescence analysis of filtrin expression in pancreas sections from Pdx1tTA/tTA;TgPdx1 mice either untreated or treated with doxycycline for 14 days. Bar = 40 μm. D: Morphometric analysis of islet area in untreated and 14-day doxycycline-treated Pdx1tTA/tTA;TgPdx1 mice (*P < 0.05). E: Real-time RT-PCR analysis of Pdx1, insulin (Ins1/Ins2), and glucagon RNA in pancreata from PdxtTA/tTA;TgPdx1 mice either untreated (n = 3; A–C) or treated (n = 4; D–G) with doxycycline for 14 days. Expression is shown relative to the highest level in the cohort. The PCR for insulin used oligonucleotide primers designed to highly conserved sequences in the Ins1 and Ins2 genes.

FIG. 2.

Doxycycline-mediated repression of Pdx1 progressively impairs β-cell function. A: Pdx1tTA/tTA;TgPdx1 mice were treated with doxycycline for 0 (○), 7 (•), 14 (□), and 21 (▪) days. β-Cell function was assessed by intraperitoneal glucose tolerance test and measurement of blood glucose (means ± SE). The responses of Pdx1tTA/+ (▵) and Pdx1+/+;TgPdx1 (▴) mice without doxycycline treatment are shown for reference. Absolute values of blood glucose for 14- and 21-day treatment groups at the 15-, 30-, and 60-min time points may be higher, as *3/3, †4/6, and ‡1/6 animals had levels above the upper limit of the glucometer (i.e., >33.3 mmol/l). B: Hematoxylin and eosin–stained sections of pancreata from Pdx1tTA/tTA;TgPdx1 mice untreated or treated with doxycyline for 14 days (+dox 14d). Bar = 200 μm. C: Immunofluorescence analysis of filtrin expression in pancreas sections from Pdx1tTA/tTA;TgPdx1 mice either untreated or treated with doxycycline for 14 days. Bar = 40 μm. D: Morphometric analysis of islet area in untreated and 14-day doxycycline-treated Pdx1tTA/tTA;TgPdx1 mice (*P < 0.05). E: Real-time RT-PCR analysis of Pdx1, insulin (Ins1/Ins2), and glucagon RNA in pancreata from PdxtTA/tTA;TgPdx1 mice either untreated (n = 3; A–C) or treated (n = 4; D–G) with doxycycline for 14 days. Expression is shown relative to the highest level in the cohort. The PCR for insulin used oligonucleotide primers designed to highly conserved sequences in the Ins1 and Ins2 genes.

FIG. 3.

Doxycycline-mediated repression of Pdx1 impairs β-cell gene expression. A: Immunofluorescence analysis of insulin (Ins), Pdx1, somatostatin (Som), glucagon (Glu), Glut-2, and amylase (Amy) in pancreas sections from Pdx1tTA/tTA;TgPdx1 mice either untreated (−dox, panels a–e) or treated (+dox 14d, panels f–j) with doxycycline for 14 days and from control Pdx1+/+;TgPdx1 mice treated (+dox 14d, panels k–o) with doxycycline for 14 days. Arrows in panel h indicate colocalization of insulin and glucagon. Dashed lines in panel i demarcate islet. Bar = 50 μm. B: Morphometric analysis of islet area (means ± SE) stained with insulin (□) or glucagon (▪) in Pdx1tTA/tTA;TgPdx1 mice. Mice were untreated, treated with doxycycline for 14 days (+dox), or treated with doxycycline for 14 days before withdrawing doxycycline for 7 or 14 days (−dox). Data are shown as percentage of islet area stained for each hormone. C: Morphometric analysis of the number of insulin+/glucagon+-costained cells per islet area in Pdx1tTA/tTA;TgPdx1 mice treated as in B.

FIG. 3.

Doxycycline-mediated repression of Pdx1 impairs β-cell gene expression. A: Immunofluorescence analysis of insulin (Ins), Pdx1, somatostatin (Som), glucagon (Glu), Glut-2, and amylase (Amy) in pancreas sections from Pdx1tTA/tTA;TgPdx1 mice either untreated (−dox, panels a–e) or treated (+dox 14d, panels f–j) with doxycycline for 14 days and from control Pdx1+/+;TgPdx1 mice treated (+dox 14d, panels k–o) with doxycycline for 14 days. Arrows in panel h indicate colocalization of insulin and glucagon. Dashed lines in panel i demarcate islet. Bar = 50 μm. B: Morphometric analysis of islet area (means ± SE) stained with insulin (□) or glucagon (▪) in Pdx1tTA/tTA;TgPdx1 mice. Mice were untreated, treated with doxycycline for 14 days (+dox), or treated with doxycycline for 14 days before withdrawing doxycycline for 7 or 14 days (−dox). Data are shown as percentage of islet area stained for each hormone. C: Morphometric analysis of the number of insulin+/glucagon+-costained cells per islet area in Pdx1tTA/tTA;TgPdx1 mice treated as in B.

FIG. 4.

Doxycycline-induced diabetes is reversible. A: Time course study of 8-h fasting blood glucose levels (means ± SE) in Pdx1tTA/tTA;TgPdx1 mice (n = 5) recovering from doxycycline-induced diabetes (▪) compared with untreated Pdx1tTA/tTA;TgPdx1 controls (□). Diabetes was induced by doxycycline administration for 14 days. Four of five mice returned to pretreatment blood glucose levels by 28 days postwithdrawal of doxycycline. *Note that 2/5 mice had blood glucose levels above the upper limit of the glucometer (i.e., >33.3mmol/l), and these values were taken as 33.4 mmol/l for the purpose of illustrating the data. B: Restoration of Pdx1 expression in derepressed Pdx1tTA/tTA;TgPdx1 mice. Immunofluorescence analysis reveals expression of insulin (green) and Pdx1 (red) in islets 7 or 14 days following cessation of 14-day treatment with doxycycline. Nuclei are counterstained with DAPI (blue). C: Normal expression of glucagon is restored following derepression of Pdx1. Immunofluorescence analysis reveals expression of insulin (green) and glucagon (red) in islets 7 or 14 days following cessation of 14-day treatment with doxycycline. Insets show insulin+/glucagon+ cells in recovering pancreata. Abnormal glucagon expression in an islet from a mouse treated with doxycycline for 28 days is shown for comparison. Bar = 20 μm.

FIG. 4.

Doxycycline-induced diabetes is reversible. A: Time course study of 8-h fasting blood glucose levels (means ± SE) in Pdx1tTA/tTA;TgPdx1 mice (n = 5) recovering from doxycycline-induced diabetes (▪) compared with untreated Pdx1tTA/tTA;TgPdx1 controls (□). Diabetes was induced by doxycycline administration for 14 days. Four of five mice returned to pretreatment blood glucose levels by 28 days postwithdrawal of doxycycline. *Note that 2/5 mice had blood glucose levels above the upper limit of the glucometer (i.e., >33.3mmol/l), and these values were taken as 33.4 mmol/l for the purpose of illustrating the data. B: Restoration of Pdx1 expression in derepressed Pdx1tTA/tTA;TgPdx1 mice. Immunofluorescence analysis reveals expression of insulin (green) and Pdx1 (red) in islets 7 or 14 days following cessation of 14-day treatment with doxycycline. Nuclei are counterstained with DAPI (blue). C: Normal expression of glucagon is restored following derepression of Pdx1. Immunofluorescence analysis reveals expression of insulin (green) and glucagon (red) in islets 7 or 14 days following cessation of 14-day treatment with doxycycline. Insets show insulin+/glucagon+ cells in recovering pancreata. Abnormal glucagon expression in an islet from a mouse treated with doxycycline for 28 days is shown for comparison. Bar = 20 μm.

FIG. 5.

Repression of Pdx1 initiates proliferation of pancreatic exocrine and duct-associated cells. A: Hematoxylin and eosin–stained section (H&E) and proliferating cell nuclear antigen (red)- and insulin (green)-costained sections of ductal foci in pancreas from Pdx1tTA/tTA;TgPdx1 mice treated with doxycyline for 28 days. Note proliferation of cells (red staining) in central focal region (dashed outline). Nuclei counterstained with DAPI. Bar = 50 μm. B: Immunofluorescence staining of BrdU-positive cells in representative pancreas sections from an untreated Pdx1tTA/tTA;TgPdx mouse (−dox) or following treatment with doxycycline for 14 (+dox 14d) or 21 days (+dox 21d). A focus of ductal proliferation is shown in the 14-day treatment group, and a single BrdU-labeled cell can be seen in the islet (circled). Quantitative analysis of BrdU-labeling index is shown as number of labeled cells/field. Bar = 50 μm. C: Insulin and Pdx1 expression in mice treated with doxycycline for 14 days before cessation of treatment and after 14 days recovery. Note duct-associated insulin+ cells and smaller islet-like cell clusters (panel a) and Pdx1+/Ins+(*) and Pdx1+/Ins (arrow) cells associated with ducts (panel b). Bars = 50 μm (a) and 25 μm (b).

FIG. 5.

Repression of Pdx1 initiates proliferation of pancreatic exocrine and duct-associated cells. A: Hematoxylin and eosin–stained section (H&E) and proliferating cell nuclear antigen (red)- and insulin (green)-costained sections of ductal foci in pancreas from Pdx1tTA/tTA;TgPdx1 mice treated with doxycyline for 28 days. Note proliferation of cells (red staining) in central focal region (dashed outline). Nuclei counterstained with DAPI. Bar = 50 μm. B: Immunofluorescence staining of BrdU-positive cells in representative pancreas sections from an untreated Pdx1tTA/tTA;TgPdx mouse (−dox) or following treatment with doxycycline for 14 (+dox 14d) or 21 days (+dox 21d). A focus of ductal proliferation is shown in the 14-day treatment group, and a single BrdU-labeled cell can be seen in the islet (circled). Quantitative analysis of BrdU-labeling index is shown as number of labeled cells/field. Bar = 50 μm. C: Insulin and Pdx1 expression in mice treated with doxycycline for 14 days before cessation of treatment and after 14 days recovery. Note duct-associated insulin+ cells and smaller islet-like cell clusters (panel a) and Pdx1+/Ins+(*) and Pdx1+/Ins (arrow) cells associated with ducts (panel b). Bars = 50 μm (a) and 25 μm (b).

FIG. 6.

Repression of Pdx1 upregulates genes implicated in pancreas regeneration. A: Gene ontology associations of genes upregulated >1.8-fold. B: Real-time RT-PCR analysis of selected genes identified by microarray. Expression levels of Reg3α, Reg3β, Tob1, and 1810015C04Rik were determined in the pancreata of PdxtTA/tTA;TgPdx1 mice either untreated (n = 3; A–C) or treated (n = 4; D–G) with doxycycline for 14 days. Expression is shown relative to the highest level in the cohort. C: RT-PCR of 1810015C04Rik in epithelial tissues and pancreas cell lines. Note that expression was not detected in α-TC1 cells. M, DRIgest III molecular weight marker; Sm. intest, small intestine.

FIG. 6.

Repression of Pdx1 upregulates genes implicated in pancreas regeneration. A: Gene ontology associations of genes upregulated >1.8-fold. B: Real-time RT-PCR analysis of selected genes identified by microarray. Expression levels of Reg3α, Reg3β, Tob1, and 1810015C04Rik were determined in the pancreata of PdxtTA/tTA;TgPdx1 mice either untreated (n = 3; A–C) or treated (n = 4; D–G) with doxycycline for 14 days. Expression is shown relative to the highest level in the cohort. C: RT-PCR of 1810015C04Rik in epithelial tissues and pancreas cell lines. Note that expression was not detected in α-TC1 cells. M, DRIgest III molecular weight marker; Sm. intest, small intestine.

TABLE 1

Differential gene expression in the pancreas of 14-day doxycycline–treated mice

Upregulated
Downregulated
Affymetrix probe set IDGene nameFold changeAffymetrix probe set IDGene nameFold changeAffymetrix probe set IDGene nameFold change
96009_s_at Pap (Reg3b) 6.15 96680_at Dnajb9 2.53 101331_f_at Igk-V8 −5.07 
92712_at 1810009J06Rik 6.08 97890_at Sgk 2.51 102823_at IgHg −4.98 
96064_at Reg3g 5.98 160366_at BC031181 2.51 97658_f_at Ins1 −4.06 
99532_at Tob1 5.69 160230_at Cad 2.49 93927_f_at Igh-4 −3.96 
103954_at Reg3a 5.46 100574_f_at Gpi1 2.48 97008_f_at — −3.87 
160124_r_at Atp6v1c1 4.66 94478_at Rab5a 2.47 101870_at Igh-4 −3.86 
160676_at — 4.08 95518_at 1810015C04Rik 2.47 102824_g_at IgHg −3.83 
97205_at Eif3s1 3.99 94007_at Tex189 2.46 97574_f_at Igh-VJ558 −3.81 
103667_at — 3.91 96594_at Hspa4 2.45 100150_f_at Ins2 −3.35 
99500_at Slc12a2 3.73 100718_at Ptma 2.44 100376_f_at Igh-VJ558 −3.10 
161890_f_at Pap (Reg3b) 3.66 93254_at Mapk1 2.44 102157_f_at Igk-V8 −3.01 
99191_at Cri1 3.51 100151_at Tde1 2.43 97564_f_at — −3.01 
96083_s_at Hnrpdl 3.45 93488_at Srr 2.42 97659_r_at Ins1 −2.97 
160417_at Kif5b 3.45 101973_at Cited2 2.41 100721_f_at LOC56304 −2.85 
103891_i_at Ell2 3.43 101441_i_at Itpr5 2.40 102156_f_at Igk-V21 −2.77 
95657_f_at Atp6v1f 3.43 103695_f_at C330007P06Rik 2.39 97197_r_at — −2.63 
94489_at Ptp4a1 3.37 96959_at Ube2n 2.37 97563_f_at — −2.62 
94343_at Dnajc3 3.29 94319_at Rab18 2.37 100362_f_at MGC60843 −2.61 
103534_at Hbb-b2 3.24 160119_at Mglap 2.35 96973_f_at — −2.60 
95786_at Reg2 3.17 104343_f_at pla2g12a 2.35 100377_f_at Igh-VJ558 −2.59 
101955_at Hspa5 3.15 103873_i_at 2310015N07Rik 2.32 100299_f_at Igk-V28 −2.49 
100323_at Amd2 3.12 96949_at Ythdf1 2.32 97576_f_at Igh-4 −2.47 
93081_at Rbbp7 3.10 95749_at Armet 2.31 98063_at Glycam1 −2.33 
101002_at Oazin 3.05 95064_at Acc2 2.30 93086_at Igk-V8 −2.31 
161912_r_at Numb 2.98 95516_at Rab9 2.29 162169_r_at Hist1h2bc −2.29 
96633_s_at Morf4l2 2.93 96007_at Ssr3 2.29 92866_at H2-Aa −2.18 
94267_i_at Ubl5 2.86 104119_at 2610024E20Rik 2.28 94725_f_at — −2.10 
93773_f_at Zfp265 2.84 93082_at Za20d3 2.27 160478_r_at Rpl32 −2.03 
160395_at D11Ertd603e 2.83 96281_at Atp6v1g1 2.27 161143_r_at Dnpep −1.89 
99575_at Mgst3 2.82 103370_at Lin7c 2.24    
94469_at Mat2b 2.82 160240_at 1110003E01Rik 2.22    
97207_f_at Lypla1 2.81 102002_at Ubqln2 2.20    
96187_at Pkp4 2.81 160499_at Tra1 2.19    
161314_r_at 1810009A16Rik 2.80 94077_f_at Rpn2 2.19    
103429_i_at AL024210 2.76 93337_at Vps4b 2.18    
93208_at Trygn16 2.74 101461_f_at Pja1 2.17    
161214_r_at BC037034 2.73 104070_at Pcaf 2.15    
160111_at Eif1ay 2.73 94017_s_at Sfrs2 2.15    
160257_at Fkbp1a 2.72 94433_at Slc38a2 2.14    
93310_at Psmc6 2.69 102647_g_at — 2.14    
160082_s_at Arf4 2.65 160360_at Sep15-pending 2.14    
98625_s_at Adh5 2.64 97839_at Snx6 2.14    
101475_at Bmi1 2.62 99579_at Atp1b3 2.08    
104249_g_at Ssr3 2.61 100123_f_at Itgb1 2.04    
104241_at Mef2d 2.60 100144_at Ncl 2.03    
160543_at Snx3 2.60 161864_f_at Ptdss1 2.02    
93512_f_at Adk 2.59 103925_at Mllt3 1.97    
104419_at Fndc3 2.58 93547_at Cbfb 1.77    
95613_at 2010200I23Rik 2.54       
Upregulated
Downregulated
Affymetrix probe set IDGene nameFold changeAffymetrix probe set IDGene nameFold changeAffymetrix probe set IDGene nameFold change
96009_s_at Pap (Reg3b) 6.15 96680_at Dnajb9 2.53 101331_f_at Igk-V8 −5.07 
92712_at 1810009J06Rik 6.08 97890_at Sgk 2.51 102823_at IgHg −4.98 
96064_at Reg3g 5.98 160366_at BC031181 2.51 97658_f_at Ins1 −4.06 
99532_at Tob1 5.69 160230_at Cad 2.49 93927_f_at Igh-4 −3.96 
103954_at Reg3a 5.46 100574_f_at Gpi1 2.48 97008_f_at — −3.87 
160124_r_at Atp6v1c1 4.66 94478_at Rab5a 2.47 101870_at Igh-4 −3.86 
160676_at — 4.08 95518_at 1810015C04Rik 2.47 102824_g_at IgHg −3.83 
97205_at Eif3s1 3.99 94007_at Tex189 2.46 97574_f_at Igh-VJ558 −3.81 
103667_at — 3.91 96594_at Hspa4 2.45 100150_f_at Ins2 −3.35 
99500_at Slc12a2 3.73 100718_at Ptma 2.44 100376_f_at Igh-VJ558 −3.10 
161890_f_at Pap (Reg3b) 3.66 93254_at Mapk1 2.44 102157_f_at Igk-V8 −3.01 
99191_at Cri1 3.51 100151_at Tde1 2.43 97564_f_at — −3.01 
96083_s_at Hnrpdl 3.45 93488_at Srr 2.42 97659_r_at Ins1 −2.97 
160417_at Kif5b 3.45 101973_at Cited2 2.41 100721_f_at LOC56304 −2.85 
103891_i_at Ell2 3.43 101441_i_at Itpr5 2.40 102156_f_at Igk-V21 −2.77 
95657_f_at Atp6v1f 3.43 103695_f_at C330007P06Rik 2.39 97197_r_at — −2.63 
94489_at Ptp4a1 3.37 96959_at Ube2n 2.37 97563_f_at — −2.62 
94343_at Dnajc3 3.29 94319_at Rab18 2.37 100362_f_at MGC60843 −2.61 
103534_at Hbb-b2 3.24 160119_at Mglap 2.35 96973_f_at — −2.60 
95786_at Reg2 3.17 104343_f_at pla2g12a 2.35 100377_f_at Igh-VJ558 −2.59 
101955_at Hspa5 3.15 103873_i_at 2310015N07Rik 2.32 100299_f_at Igk-V28 −2.49 
100323_at Amd2 3.12 96949_at Ythdf1 2.32 97576_f_at Igh-4 −2.47 
93081_at Rbbp7 3.10 95749_at Armet 2.31 98063_at Glycam1 −2.33 
101002_at Oazin 3.05 95064_at Acc2 2.30 93086_at Igk-V8 −2.31 
161912_r_at Numb 2.98 95516_at Rab9 2.29 162169_r_at Hist1h2bc −2.29 
96633_s_at Morf4l2 2.93 96007_at Ssr3 2.29 92866_at H2-Aa −2.18 
94267_i_at Ubl5 2.86 104119_at 2610024E20Rik 2.28 94725_f_at — −2.10 
93773_f_at Zfp265 2.84 93082_at Za20d3 2.27 160478_r_at Rpl32 −2.03 
160395_at D11Ertd603e 2.83 96281_at Atp6v1g1 2.27 161143_r_at Dnpep −1.89 
99575_at Mgst3 2.82 103370_at Lin7c 2.24    
94469_at Mat2b 2.82 160240_at 1110003E01Rik 2.22    
97207_f_at Lypla1 2.81 102002_at Ubqln2 2.20    
96187_at Pkp4 2.81 160499_at Tra1 2.19    
161314_r_at 1810009A16Rik 2.80 94077_f_at Rpn2 2.19    
103429_i_at AL024210 2.76 93337_at Vps4b 2.18    
93208_at Trygn16 2.74 101461_f_at Pja1 2.17    
161214_r_at BC037034 2.73 104070_at Pcaf 2.15    
160111_at Eif1ay 2.73 94017_s_at Sfrs2 2.15    
160257_at Fkbp1a 2.72 94433_at Slc38a2 2.14    
93310_at Psmc6 2.69 102647_g_at — 2.14    
160082_s_at Arf4 2.65 160360_at Sep15-pending 2.14    
98625_s_at Adh5 2.64 97839_at Snx6 2.14    
101475_at Bmi1 2.62 99579_at Atp1b3 2.08    
104249_g_at Ssr3 2.61 100123_f_at Itgb1 2.04    
104241_at Mef2d 2.60 100144_at Ncl 2.03    
160543_at Snx3 2.60 161864_f_at Ptdss1 2.02    
93512_f_at Adk 2.59 103925_at Mllt3 1.97    
104419_at Fndc3 2.58 93547_at Cbfb 1.77    
95613_at 2010200I23Rik 2.54       

Genes discussed in text are bold.

This work was supported by a Juvenile Diabetes Research Foundation (JDRF) Postdoctoral Fellowship (to A.M.H.) and by a partnership Program Grant from the JDRF and the National Health and Medical Research Foundation of Australia (to L.C.H.). R.J.M. was supported by National Institutes of Health Grant DK55266.

The authors thank Ken Simpson for technical assistance with microarray analysis and Fang-Xu Jiang for helpful discussions.

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