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Full Text (PDF) Original Article Conditional Expression Demonstrates the Role of the Homeodomain Transcription Factor Pdx1 in Maintenance and Regeneration of ␤-Cells in the Adult Pancreas Andrew M. Holland,1 L. Jorge Go´n˜ ez,1 Gaetano Naselli,1 Raymond J. MacDonald,2 and Leonard C. Harrison1 The homeodomain transcription factor Pdx1 is essential increase its ␤-cell mass in response to physiological re- for pancreas development. To investigate the role of quirements, e.g., as in pregnancy (8) or in response to Pdx1 in the adult pancreas, we employed a mouse model injury (9). ␤-Cell neogenesis is presumed to depend on the in which transcription of Pdx1 could be reversibly re- existence of progenitor ␤-cells in the adult pancreas, pressed by administration of doxycycline. Repression of although the origin of progenitors from ductal (10–12), Pdx1 in adult mice impaired expression of insulin and islet (13,14), exocrine (15–19), or multilineage (20) cells glucagon, leading to diabetes within 14 days. Pdx1 remains contentious (21,22). In addition to its key role in repression was associated with increased cell prolifer- pancreas development, Pdx1 is implicated in ␤-cell neo- ation predominantly in the exocrine pancreas and up- genesis, being expressed in duct and duct-associated cells regulation of genes implicated in pancreas regenera- tion. Following withdrawal of doxycycline and derepres- in models of pancreas regeneration (20,23,24). It is un- sion of Pdx1, normoglycemia was restored within 28 days; clear, however, whether Pdx1 is required early for the during this period, Pdx1؉/Ins؉ and Pdx؉/Ins؊ cells were activation of a regenerative program or later for the observed in association with the duct epithelia. These differentiation of putative progenitor cells. Investigating findings confirm that Pdx1 is required for ␤-cell function the role of Pdx1 in the adult pancreas is precluded by the in the adult pancreas and indicate that in the absence of fact that pancreas development is arrested following spec- Pdx1 expression, a regenerative program is initiated with ification of the foregut endoderm in mice lacking Pdx1 the potential for Pdx1-dependent ␤-cell neogenesis. (1–3). To investigate the role of Pdx1 postdevelopment, we Diabetes 54:2586–2595, 2005 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 ␤-cell dx1 is a homeodomain transcription factor essen- function and regeneration in the adult pancreas. tial for pancreatic development (1–3). Early in mouse development (embryonic day 8.5), Pdx1 is RESEARCH DESIGN AND METHODS highly expressed in a region of the posterior P Generation of knock-in and transgenic mice. The basic elements of the foregut endoderm, from which the dorsal and ventral Pdx1 tet-off inducible gene repression system are shown schematically in Fig. pancreatic buds arise. Its expression then becomes pro- 1. Pdx1tTA knock-in and TgPdx1 transgenic mice were generated as previously gressively restricted to endocrine cells (4), and in the adult described (25). To obtain mice in which Pdx1 could be conditionally re- pressed, the Pdx1tTA/ϩ knock-in mice were crossed with TgPdx1 mice and pancreas Pdx1 regulates several genes including insulin, tTA/tTA Pdx1 glucose transporter-2, and glucokinase (5–7) integral to progeny intercrossed to derive Pdx1 ;Tg mice as well as genotype control mice (see RESULTS). ␤-cell function. The adult pancreas retains the capacity to 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 From the 1Autoimmunity and Transplantation Division, Walter and Eliza Hall Committee. Groups of mice were age and sex matched within each experi- 2 Institute of Medical Research, Parkville, Victoria, Australia; and the Depart- ment. Doxycycline was administered as a single intraperitoneal dose of 100 ment of Molecular Biology, University of Texas Southwestern Medical Center, mg/kg at time t ϭ 0 and maintained by addition to drinking water at 0.5 mg/ml. Dallas, Texas. Address correspondence and reprint requests to Leonard C. Harrison, BrdU (100 mg/kg) was administered intraperitoneally as a single dose 16 h Autoimmunity and Transplantation Division, Walter and Eliza Hall Institute of before the mice were killed. Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. E-mail: Glucose tolerance test. Mice were fasted overnight and challenged with 2 [email protected]. g/kg i.p. glucose. Glucose levels in retroorbital venous blood samples were Received for publication 3 March 2005 and accepted in revised form 3 June measured at 0, 15, 30, 60, 120, and 180 min postchallenge with an Accu-chek 2005. Advantage glucometer (Roche Diagnostics, Castle Hill, NSW, Australia). Ј Ј Ј DAPI, 4 ,6 -diamidine-2 -phenylindole dihydrochloride. Statistical analyses (Mann-Whitney tests) were performed using Graphpad © 2005 by the American Diabetes Association. Prism (GraphPad Software, San Diego, CA). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance Immunocytochemistry and immunofluorescence. Adult pancreata were with 18 U.S.C. Section 1734 solely to indicate this fact. fixed in Histochoice (Sigma, St. Louis, MO) or 4% paraformaldehyde for 3 h 2586 DIABETES, VOL. 54, SEPTEMBER 2005 A.M. HOLLAND AND ASSOCIATES 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. before processing, embedding in paraffin, and sectioning (5 ␮m). The follow- Analysis of the probe data were performed as follows. The perfect match ing primary antibodies were used: guinea pig anti-human insulin (Dako, probe intensities were background corrected and normalized across arrays Carpinteria, CA) at 1:1,000, rabbit anti-human amylase (Sigma) at 1:1,000, and then summarized for each probe set using robust multichip analysis rabbit anti-human glucagon (Dako) at 1:200, rabbit anti-human somatostatin (26,27). Once expression levels had been calculated, differentially expressed (Dako) at 1:100, rabbit anti-GLUT2 (a gift from Bernard Thorens, Universite´de genes were ranked and values expressed as fold change. Lausanne, Lausanne, Switzerland) at 1:100, and rabbit anti-human filtrin Quantitative real-time RT-PCR was performed on the Roche Lightcycler (Alpha Diagnostic, San Antonio, TX) at 1:20. Rabbit anti-Pdx1 (a gift from (Roche Diagnostics). For each gene to be assayed, a template was amplified Christopher Wright, Vanderbilt University Medical Center, Nashville, TN) was by PCR and subcloned into a pGEM-T vector (Promega, Madison, WI). The used at 1:500 and rabbit anti-Pdx1 antibody (purified IgG 1:25) was raised templates were used to generate standard curves for quantitative determina- against a glutathione S-transferase fusion protein containing amino acids tion of mRNA expression level in the individual RNAs described above. 14–284 of murine Pdx1. Fluorescein isothiocyanate–conjugated anti-guinea Expression levels were corrected relative to the expression of the of ␤-actin pig (ICN Biomedicals, Aurora, OH) or Alexa fluor 568–conjugated goat gene. Sequences of oligonucleotide primers used are available on request. anti-rabbit (Molecular Probes, Eugene, OR) immunoglobulins were used for Islet area determination. Islet area was determined for pancreata of mice immunofluorescence. Nuclei were counterstained with 4Ј,6Ј-diamidine-2Ј- that were either untreated or treated with doxycycline for 14 days (four mice phenylindole dihydrochloride (DAPI). BrdU-labeled cells were detected with per group). Three hematoxylin and eosin–stained sections from each pancreas a cell proliferation detection kit (Amersham, Buckinhamshire, U.K.) with were sampled, each section separated by ϳ250–300 ␮m. Nonoverlapping 40ϫ mouse monoclonal anti-BrdU primary and Alexa fluor 568–conjugated anti- magnification fields were captured with an Axiocam digital camera (Carl mouse secondary antibodies. Terminal deoxynucleotide transferase-mediated Zeiss), and the total tissue and islet area per field was determined using dUTP nick-end labeling was performed with an apoptag fluorescein apoptosis Axiovision 4.2 image analysis software (Carl Zeiss). All sections were scored detection kit (Serologicals, Norfolk, GA). Digital images were captured with blind with respect to treatment. Statistical significance was determined using an Axiocam camera from an Axioplan2 compound microscope (Carl Zeiss, a Mann-Whitney U test (GraphPad Software). Go¨ ttingen, Germany). Dual-color immunofluorescence images were compiled from two separate images of the same section using fluorochrome-specific filters. RESULTS BrdU-labeling index was determined
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