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Functional Analysis of Pdx1 Overexpression in Nave Endoderm

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Functional Analysis of Pdx1 Overexpression in Nave Endoderm Functional Analysis of Pdx1 Overexpression in Naïve Endoderm Marco Gasparrini Faculty of Graduate Studies Division of Experimental Medicine McGill University Montréal, Québec, Canada August 2010 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Marco Gasparrini, 2010 ABSTRACT Pancreatic and duodenal homeobox 1 (Pdx1) was one of the first pancreas specific genes isolated. It is expressed in early pancreatic buds, throughout the duodenum and localized to insulin producing cells in the adult. Pdx1 plays a fundamental role in pancreas development as the loss-of-function of Pdx1 in mice and frogs result in absence of pancreatic tissue. In humans, Pdx1 homozygous mutations lead to pancreas agenesis, while heterozygous mutations result in type 2 diabetes. Our laboratory studies the role of Pdx1 in promoting ectopic pancreatic cell fates. Using Xenopus laevis as a model, we previously showed that the overexpression of a modified form of Pdx1, Pdx1-VP16, is sufficient to convert liver to pancreas. Whether Pdx1 is able to promote ectopic pancreas in naïve endoderm has yet to be determined. To achieve this, Pdx1 mRNA was overexpressed in the anterior endoderm. The overexpression resulted in ectopic tissue with reduced expression of exocrine and endocrine differentiation markers. In addition, stomach, duodenum and liver organogenesis was severely perturbed. To ascertain the identity of this ectopic tissue, microarray analysis was performed which confirmed the reduction in pancreatic endocrine and exocrine cells as well as the reduction in stomach, duodenum and hepatic tissue. Moreover, the genes highly upregulated suggest a pancreatic stellate cell phenotype. The information obtained from the gain-of-function analysis will help explain the role of this transcription factor in regulating the initial stages of pancreatic cell fate specification. 2 RÉSUMÉ Pancreatic and duodenal homeobox 1 (Pdx1) a été l’un des premiers gènes spécifiques du pancréas à être isolé. Pdx1 est exprimé tôt dans les bourgeons pancréatiques, à travers le duodénum et, à l’âge adulte, dans les cellules productrices d’insuline. Pdx1 joue un rôle fondamental quant au développement du pancréas car l’absence de ce gène chez les souris et les grenouilles empêche la formation et l’élaboration du tissue pancréatique. Chez les humains, les mutations homozygotes du gène Pdx1 engendrent l’agénésie du pancréas, tandis que les mutations hétérozygotes engendrent le diabète du type 2. Notre laboratoire étudie le rôle de Pdx1 et sa capacité à promouvoir le sort des cellules pancréatiques ectopiques. En utilisant Xenopus laevis comme modèle, nous avons précédemment démontré qu’une surexpression d’une forme modifié de Pdx1, Pdx1-VP16, est suffisante pour convertir le foie en pancréas. Il reste encore à savoir si Pdx1 est capable d’inciter la formation d’un pancréas ectopique parmi un endoderme naïf. Nous avons donc surexprimé l’ARNm Pdx1 parmi l’endoderme antérieur. La surexpression a conduit à du tissu ectopique avec une expression réduite des marqueurs de différenciations exocrines et endocrines. En outre, l’organogenèse de l'estomac, du duodénum et du foie a été gravement perturbée. Pour vérifier l’identité de ce tissue ectopique, une analyse du profil d’expression génétique par puce à ADN a été réalisée et a confirmée la réduction de cellules pancréatiques endocrines et exocrines, ainsi que la réduction des tissues de l'estomac, du duodénum et du foie. De plus, les gènes fortement induits suggèrent des cellules pancréatiques de phénotype stellaire. Les informations 3 obtenues par l'analyse de gain de fonction permettront d'expliquer le rôle de ce facteur de transcription dans la régulation de la phase initiale de la spécification du sort des cellules du pancréas. 4 TABLE OF CONTENTS ABSTRACT........................................................................................................... 2 RÉSUMÉ ............................................................................................................... 3 TABLE OF CONTENTS ..................................................................................... 5 LIST OF FIGURES .............................................................................................. 7 LIST OF TABLES ................................................................................................ 8 ACKNOWLEDGEMENTS ................................................................................. 9 Chapter 1: Introduction ..................................................................................... 10 1.1 Diabetes Mellitus....................................................................................... 10 1.1.1 Global Epidemic .................................................................................. 10 1.1.2 Three Types of Diabetes Mellitus........................................................ 10 1.1.3 Current Treatment Therapies ............................................................... 12 1.2 Xenopus to Study Pancreas Organogenesis ............................................ 14 1.3 Xenopus Pancreas Morphogenesis........................................................... 15 1.4 The Mature Vertebrate Pancreas............................................................ 16 1.5 Pancreatic and Duodenal Homeobox 1 ................................................... 18 1.5.1 Pdx1 Temporal and Spatial Expression ............................................... 18 1.5.2 Pdx1 Loss-of-functional Analysis........................................................ 19 1.5.3 Clinical Significance............................................................................ 21 1.5.4 Pdx1 Gain-of-function Analysis .......................................................... 22 1.5.5 Pdx1 Direct Gene Targets.................................................................... 25 1.6 The Pancreatic Stellate Cell ..................................................................... 25 1.6.1 PaSC Properties ................................................................................... 25 1.6.2 Activated PaSC .................................................................................... 26 1.6.3 Embryonic Origin of PaSC .................................................................. 27 1.7 Hypothesis and Objectives ....................................................................... 28 Chapter 2: Materials and Methods ................................................................... 29 In Vitro Fertilization and Microinjection ..................................................... 29 Probe Synthesis and Whole-mount In situ Hybridization .......................... 30 Pax4 Cloning with RACE............................................................................... 34 Cloning Pdx1-GR Fusion Constructs ............................................................ 35 RNA Extraction, Purification and Microarray Analysis ............................ 36 Microarray Validation by RT-PCR .............................................................. 37 Chapter 3: Results............................................................................................... 40 5 Early Pdx1 Overexpression Caused Gastrulation Defects .......................... 40 Established Temporal Regulation of Pdx1 Overexpression........................ 41 Pdx1 Overexpression Resulted in Ectopic Tissue Formation ..................... 42 Ectopic Tissue Displayed Reduced Exocrine Differentiation ..................... 44 Ectopic Tissue Displayed Reduced Endocrine Differentiation................... 45 Ectopic Tissue Disrupted Foregut Organ Development.............................. 48 Identifying Ectopic Tissue Formation by Microarray ................................ 51 Reduction in Pancreas, Liver and Intestinal Markers Confirmed by Microarray....................................................................................................... 53 Upregulated Genes were Common between Treatment Groups................ 59 Validation of Microarray by RT-PCR and In Situ Hybridization............. 59 Functional Categories of Differentially Expressed Genes........................... 63 Microarray Suggests Stellate Cell Formation .............................................. 67 Chapter 4: Discussion......................................................................................... 70 Early Pdx1 Overexpression Caused Gastrulation Defects .......................... 70 Reduced Foregut Differentiation Markers................................................... 71 Microarray Analysis on Ectopic Tissue Formation..................................... 74 Conclusions and Future Direction................................................................. 82 BIBLIOGRAPHY ............................................................................................... 84 6 LIST OF FIGURES Figure 1: Early Pdx1 overexpression resulted in gastrulation defects ….……….41 Figure 2: Pdx1-GR overexpression caused ectopic tissue formation ……………43 Figure 3: Ectopic tissue displayed reduced exocrine markers …………………..45 Figure 4: Ectopic tissue displayed reduced endocrine differentiation …………..46 Figure 5: Reduction in initial endocrine cell differentiation …………………….48 Figure 6: Ectopic tissue perturbed stomach, duodenum and liver development ..49 Figure 7: Transient activation did not result in differentiation of ectopic tissue ..51 Figure 8: Experimental design of microarray analysis ……………………...…..52 Figure
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