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

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 9: Venn diagram displaying downregulated microarray genes ………….53

Figure 10: Venn diagram displaying upregulated microarray genes ……………59

Figure 11: Validation of microarray results by RT-PCR ………………………..60

Figure 12: Validation of microarray results by in situ hybridization ……………63

Figure 13: Ectopic tissue expressed stellate cell markers ……………...………..68

Figure 14: Ectopic tissue formation marked for stellate cells …………………...69

7 LIST OF TABLES

Table 1: Generated antisense RNA probes for in situ hybridization ……..……..31

Table 2: Designed PCR primers for cloning genes for in situ hybridization ……32

Table 3: Designed primers for RT-PCR ………………………………………...38

Table 4: Downregulated microarray genes present in pancreas ………..……….55

Table 5: Downregulated microarray genes present in liver ……………..………56

Table 6: Downregulated microarray genes present in stomach/duodenum .…….57

Table 7: Functional categories of upregulated microarray genes ……………….65

8 ACKNOWLEDGEMENTS

I would like to first and foremost thank my supervisor Dr. Marko E. Horb.

He provided me with an incredible opportunity to perform research in an excellent laboratory at a renowned institution. Dr. Horb’s dedication to the field inspires all who meet him to strive and become excellent researchers. His suggestions, support, financial contributions and guidance have helped me conduct great research and achieve my goals throughout the completion of my degree.

I would also like to acknowledge all the members of the Horb laboratory.

Lori Horb and Zeina Jarikji are great laboratory technicians and researchers.

Thank you for helping me conduct my experiments and for squeezing the frogs.

Dr. Esther Pearl, Cassandra Bilogan, Akash Srivastava and Daniel Oropeza have all supported me throughout my degree through endless scientific discussions, by giving me advice and for having a memorable time while doing it.

Finally I would love to thank my family. Without their love and support I wouldn’t have been able to do this. To my beautiful girlfriend Lisa, thank you for being patient these past few years and for believing in me more then myself.

9 Chapter 1: Introduction

1.1 Diabetes Mellitus

1.1.1 Global Epidemic

Diabetes mellitus is a serious disease whereby the body is unable to correctly regulate blood glucose levels. Inappropriate control of blood sugar can lead to long term complications including cardiovascular disease, kidney disease, retinopathy, neuropathy, erectile dysfunction, amputation and depression (Nathan,

1993). According to the World Health Organization (WHO), there is currently

200 million people world wide suffering from diabetes. This number is projected to rise to 366 million by 2030 (WHO, 2010). Based on 2005 data, over 2 million people were diagnosed with diabetes in Canada. Moreover, in 2005 alone, diabetes treatment has cost Canada’s public healthcare system 5.6 billion dollars

(C.D.A., 2008). Such costs in Canada and around the world will only continue to rise unless more research is devoted to understanding the causes of the disease and in the development of effective therapeutics.

1.1.2 Three Types of Diabetes Mellitus

Diabetes mellitus is manifested in three distinct forms: type 1, type 2 and neonatal diabetes. Type 1 diabetes, also known as insulin dependent diabetes or juvenile diabetes, accounts for 5% to 10% of total cases of diabetes worldwide

(ADA, 2009). It develops due to the immune system’s inability to recognize self

10 from non-self. Type 1 diabetes is characterized by the T-cell mediated autoimmune destruction of pancreatic insulin producing cells. The loss of β-cells causes a reduction in endogenous insulin production resulting in an increase in blood glucose levels. Therefore an absolute requirement for insulin is needed. The precise etiology of the disease is not known but genetic and environmental factors are involved (Bach and Chatenoud, 2001; Haller et al., 2005). Recent genetic etiology studies in humans have established a polygenic model for the pathogenesis of type 1 diabetes. Genes encoding MHC HLA class II antigen presenting molecules have been shown to be responsible for the improper display of β-cell antigens to T-cells (Todd, 2010).

Neonatal diabetes mellitus is often mistaken for the more common type 1 diabetes. This rare condition occurs in one to 400 000 live births and hyperglycemia is present within the first 6 months of life (Shield, 2000). This differs from type 1 diabetes where the incidence of hyperglycemia occurs later in childhood/ adulthood. In 50% of those diagnosed with neonatal diabetes, their condition is permanent. In the remaining cases, diabetes is transient; disappearing within three months but reappearing in adolescence and early adulthood (Mackay and Temple, 2010). Neonatal diabetes is a genetic rather than an autoimmune disorder. The causes of this diabetes are monogenic in origin affecting β-cell function and thus insulin secretion (Greeley et al., 2010).

Type 2 diabetes, also known as non-insulin dependent diabetes or adult onset diabetes, is often associated with obese and sedentary individuals. It is not an autoimmune disorder; instead it a metabolic condition resulting from defects in insulin action and β-cell dysfunction (Reaven et al., 1984). The dysfunction of β-

11 cells is characterized by a decrease in insulin production and β-cell loss (Silva et al., 2000; Butler et al., 2003). Apoptosis, defects in β-cell replication and the dedifferentiation of β-cells into a progenitor state have all been linked to the causes of β-cell loss (Jonas et al., 1999; Butler et al., 2003; Guillausseau et al.,

2008). Similar to type 1 diabetes, the precise etiology of type 2 diabetes is not known. However, certain lifestyle factors are believed to play an important role in the development of type 2 diabetes. In a recent study, individuals who maintained a high level of physical activity, a healthy diet rich in fiber, never smoked, consumed alcohol in moderation and had a body mass index of less than 25, had an 89% lower incidence of diabetes (Mozaffarian et al., 2009).

1.1.3 Current Treatment Therapies

The current treatment strategy for diabetes involves insulin injections or the administration of oral hypoglycemic agents. More often then not, it is the combination of oral and injection therapies that provide the greatest means to control glycemic levels (Nelson et al., 2006). However, despite the amelioration of some symptoms, patients continue to suffer from long term complications

(Nathan, 1993).

Whole pancreatic organ and islet cell transplantation has provided a means to restore homeostatic insulin secretion (Kelly at al., 1967; Scharp and Lacy,

1990). In turn, patients were capable of regulating blood glucose levels independent of exogenous insulin injections (Shapiro et al., 2000). However, a 5 year post-islet transplantation study revealed that only 10% of patients maintained insulin independence (Ryan et al., 2005). In addition, the availability of donors,

12 surgical complications and long term effects of immunosuppressive regimens suggest that this therapeutic approach is not available or suitable for all patients

(Vardanyan et al., 2010).

Current research is directed towards generating replacement β-cells from different cell types that can secrete insulin and mimic the glucose-responsiveness of normal pancreatic β-cells (Scharfmann, 2003). One strategy involves differentiating human embryonic stem cells (hESC) into functional β-cells. The complex procedure involves converting hESC into definitive endoderm and then expressing a set of lineage specific pancreatic transcription factors to induce the formation of β-cells (Aguayo-Mazzucato and Bonner-Weir, 2010). However, safety issues, including tumor formation from undifferentiated hESC residing in hESC-derived tissue as well as ethical concerns are always debated in stem cell based therapies (Bongso et al., 2008).

An alternative strategy would be to generate insulin producing cells from other adult tissues. Due to the similar embryological origin between the liver and pancreas, liver cells may provide a source for transdifferentiation into functional

β-cells (Meivar-Levy and Ferber, 2003). Recent in vivo and in vitro studies have demonstrated that the induction of pancreatic transcription factors can convert hepatocytes to functional β-cells (Ferber et al., 2000; Kojima et al., 2003; Ber et al., 2003; Horb et al., 2003; Sapir et al., 2005). This transdifferentiation strategy can eliminate the need for donors and complications related to immunosuppressive regimens (Meivar-Levy and Ferber, 2003).

In order for these cell based therapies to work, it is important to elucidate the molecular regulatory mechanisms involved in pancreas development. By

13 understanding how individual pancreatic cell types are specified, this knowledge can be applied to improve the efficiency of the above therapeutics or develop novel strategies for treating diabetes.

1.2 Xenopus to Study Pancreas Organogenesis

The vast majority of research into understanding pancreas development has been performed on conventional model organisms such as mouse and chick.

Traditionally, Xenopus laevis has been used to study gene regulatory networks and intracellular signaling pathways involved in early development (fertilization, gastrulation and neurulation). However, recent research has made use of Xenopus to study “later” developmental processes including organogenesis (Blitz et al.,

2006). The reasons for doing so, stems from the fact that frogs and mammals have the same organs, they are morphologically and functionally identical and the same genetic pathways are involved in the patterning and specification of the different cell types (Ferber et al., 2000; Horb et al., 2003; Gierl et al., 2006; Horb et al.,

2009). In addition, frogs are inexpensive to work with, pancreas development occurs faster, pancreatic tissue can be easily extracted and a large number of embryos can be experimented on at once allowing for high throughput data collection. Overexpression studies by microinjecting mRNA, loss-of-function studies using antisense morpholino oligonucleotides and the generation of transgenic tadpoles can all be performed rather quickly (Blitz et al., 2006; Pearl and Horb, 2008; Pearl et al., 2009). Xenopus fate maps have also been well characterized, thus allowing mRNA and morpholino microinjection targeting to

14 specific regions of the developing endoderm (Lane and Sheets, 2006). Due to these advantages, Xenopus is used as an initial tool for gene discovery and study before more complex and expensive mice studies are performed. In fact, an important pancreatic gene, Pdx1, was initially discovered in Xenopus before the mouse homologue was identified (Wright et al., 1989; Ohlsson et al., 1993).

1.3 Xenopus Pancreas Morphogenesis

The pancreas is a complex organ derived from the endodermal germ layer.

Similar to other vertebrates, the mature Xenopus pancreas forms as two ventral and single dorsal buds. Developing underneath the notochord, the dorsal anlage is visible at NF (Nieuwkoop and Faber) stage 35/36. On the other hand, the ventral anlage appears adjacent to the liver at NF37/38. In Xenopus, the two ventral pancreatic buds initially fuse with each other before finally fusing with the dorsal bud to form a single organ at NF40, 3 days post-fertilization (Kelly and Melton,

2000). However, in mice, one of the ventral buds regresses as the other later fusses with the dorsal bud by E12.5 (Jorgensen et al., 2007). As the pancreas develops, its morphology changes from a thin and long structure to one that becomes shorter and wider. During these stages of development, the pancreas becomes separated from the intestine and displaced to the right of the tadpole, as intestinal coiling is carried out from NF42 to NF48 (Pearl et al., 2009).

15 1.4 The Mature Vertebrate Pancreas

The mature pancreatic organ consists of two main tissue types, exocrine and endocrine. The exocrine component consists of acinar cells that secrete digestive enzymes into the duodenum. These include amylase, lipase, elastase, chymotrypsinogen and trypsinogen. The enzymes are transported to the digestive tract by the ductal cells. Ducts also have an exocrine function as they secrete bicarbonates to neutralize the acidic content of food arriving to the duodenum

(Slack, 1995; Jorgenson et al., 2007).

Pancreatic protein disulfide isomerase (XPDIp) is the first exocrine marker expressed in Xenopus pancreas. Its expression is observed throughout the entire pancreas at NF39 (Afelik et al., 2004). On the other hand, the expression of amylase, elastase and trypsinogen is initially detected solely in the ventral pancreas at NF41. Later, their expression expands throughout the entire pancreas by NF45 (Horb and Slack, 2002).

Another cell type present in the exocrine pancreas is pancreatic stellate cells (PaSC). In normal pancreas, PaSC reside in a quiescent state in the periacinar space. Upon pancreatic injury, PaSC become “activated” and migrate to the site of tissue damage and promote repair. However, sustained PaSC activation is associated with chronic pancreatitis and pancreatic cancer (Omary et al., 2007).

PaSC has been studied using mammalian models; however its developmental origins have yet to be elucidated. This cell type has not yet been identified and studied in Xenopus.

16 Endocrine cells accounts for 4% of the total pancreatic volume and are grouped into islets of Langerhans. Embedded within the exocrine compartment, the islets are composed of five cell types, α, β, δ, ε and PP. These cells secrete peptide hormones including, glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide respectively, directly into the bloodstream (Slack, 1995;

Jorgenson et al., 2007).

In Xenopus, the formation of an islet as seen in mammals whereby α-cells surround β-cells is not observed. Instead, glucagon and insulin expressing cells form separate, non-overlapping groups. The first endocrine marker to be expressed is insulin at 2 days post-fertilization (Kelly and Melton, 2000; Horb and

Slack, 2002). Early Xenopus insulin expression is detected in the dorsal endoderm

(underneath the notochord) before the appearance of the dorsal pancreatic bud.

Initially, insulin expression is restricted to the dorsal pancreas despite fusion of the ventral and dorsal pancreatic buds. Eventually, insulin appears in the ventral pancreas as well by NF47 (Kelly and Melton, 2000; Horb and Slack, 2002).

Glucagon and somatostatin first appear in the Xenopus stomach and duodenum at NF41. The initial expression in the dorsal pancreas is only visible at

NF44/45. Similarly to β-cell expression, α and δ expressing cells appear throughout the entire pancreas by NF47 (Kelly and Melton, 2000; Horb and

Slack, 2002). PP and ε have yet to be identified and characterized in Xenopus

(Pearl et al., 2009). In mice, the expression of insulin and glucagon precedes that of somatostatin, ghrelin and pancreatic polypeptide in the developing pancreatic buds. Insulin and glucagon are first detected at E9.5, ghrelin and pancreatic

17 polypeptide are observed at E10.5 and somatostatin expression is detected at the

E13.5 (Herrera et al., 1991; Teitelman et al., 1993; Heller et al., 2005)

1.5 Pancreatic and Duodenal Homeobox 1

1.5.1 Pdx1 Temporal and Spatial Expression

One of the earliest pancreatic transcription factors to be identified was

Pdx1. Initially discovered in Xenopus laevis as a novel homeobox gene over 20 years ago, it was given the named XlHbox8 (Wright et al., 1989). It is expressed in the anterior endoderm beginning at NF27, later expressed in the developing pancreatic anlagen, duodenum and stomach. XlHbox8 expression in the pancreas continues into adulthood, with none being observed in the liver and small intestine

(Wright et al., 1989; Afelik et al., 2006). Low levels of XlHbox8 have been observed in vegetal cells of Xenopus embryos at the end of gastrulation (NF12.5).

However, it is unclear what role it plays in endodermal pattering at such early stages (Gamer and Wright, 1995).

The mammalian homolog of XlHbox8 was discovered due to its ability to bind to somatostatin and insulin genes (Ohlsson et al., 1993; Leonard et al., 1993;

Miller et al., 1994). Initially named as insulin promoter factor 1 (IPF1), somatostatin transactivation factor-1 (STF-1) and islet/duodenum homeobox-1

(IDX-1), the term pancreatic and duodenal homeobox 1 (PDX1) has now been commonly adopted (Offield et al., 1996; Ahlgren et al., 1996). The mammalian

Xlhbox8 has a similar expression pattern to that of frogs. Pdx1 is initially observed in the pre-pancreatic foregut at E8.5. By E11.5, it becomes expressed in

18 the posterior stomach, duodenum, common bile duct and throughout the epithelium of pancreatic buds (Offield et al., 1996; Guz et al., 1995; Jonsson et al., 1994). After birth, Pdx1 becomes restricted to islet cells and the enterocytes and enteroendocrine cells of the duodenum (Offield et al., 1996). As the pancreas continues to develop and cells commit to the endocrine, exocrine and ductal cell lineages, Pdx1 expression decreases. By adulthood, Pdx1 is expressed mostly by mature β-cells to regulate insulin gene function (Guz et al., 1995). However, low levels of expression have been observed in other endocrine cells as well as exocrine and ductal cells (Guz et al., 1995; Wu et al., 1997).

1.5.2 Pdx1 Loss-of-functional Analysis

Due to the early presence of Pdx1 in the pre-pancreatic foregut, researchers believed in its role in specifying the pancreas. The first mouse knockout study was carried out in 1994 by Jonsson and colleagues. Null mutant mice were found to lack pancreatic tissue and die a few days after birth. In addition, Pdx1 loss-of-function did not seem to affect organogenesis of the surrounding organs including the gastrointestinal tract (Jonsson et al., 1994;

Jonsson et al. 1995). Since then, follow up studies were carried out to better understand the role of Pdx1 in initiation of pancreas development. In 1996,

Ahlgren and colleagues generated null Pdx1 mutants showing dorsal bud formation and the expression of few insulin and glucagon cells. However, further proliferation of the dorsal bud was inhibited. The detection of a few insulin- expressing cells suggests a population of β-cell-positive, Pdx1-negative cells that may form distinct from mature Pdx1-positive β-cells during development

19 (Ahlgren et al., 1996). These Pdx1 loss-of-function results were also confirmed by

Offield and colleagues (1996). In this case, the ablation of Pdx1 resulted in the formation of rudimentary pancreatic buds. The dorsal bud underwent limited branching but later degenerated and only a few glucagon positive cells were observed. Initial pancreatic bud formation, independent of Pdx1, is the result of inductive signals coming from the notochord and cardiac mesoderm instead of the epithelium itself (Kim et al., 1997; Rossi et al., 2001). However, Pdx1 is required to provide instructive signals that promote further outgrowth, proliferation and differentiation into the pancreatic cell types (Ahlgren et al., 1996; Offield et al.,

1996). In fact, Pdx1-positive cells give rise to all pancreatic cell types including endocrine, exocrine and ductal (Gu et al., 2002). On the other hand, it is not known whether Pdx1-positive cells give rise to PaSC as well. In addition to pancreas agenesis observed in Pdx1 null mice, patterning defects of the rostral duodenum was noted with the reduction of enteroendocrine cells (Offield et al.,

1996).

In the adult, Pdx1 is also essential for maintaining mature β-cell function.

Selective conditional inactivation of Pdx1 in β-cells of 3 and 5 week old mice resulted in a reduction of insulin and glucose transporter 2 expression (Glut2).

The loss of Glut2 and the subsequent impaired glucose-stimulated insulin release contributed to the development of hyperglycemia. Thus, with aging, the mice also exhibited an adult onset, non-insulin resistant diabetes phenotype (Algren et al.,

1998). Similar results were obtained using doxycycline-induced Pdx1 inactivation in adult mice (Holland et al. 2002). In addition to the loss of insulin expression and increased fasting blood glucose levels, a modest reduction in amylase and

20 elastase expression was also observed. This suggests that the low levels of Pdx1 present in adult exocrine cells may help maintain acinar function as well. These deletion studies show the importance of early and late Pdx1 function. Early Pdx1 expression is required for pancreas organogenesis, whereas late expression maintains β-cell identity and acinar cell function.

The loss-of-function analysis in Xenopus did not occur until 2006.

Antisense morpholino oligonucleotides targeted against Pdx1 mRNA resulted in ablation of exocrine markers. However, insulin expression appeared to be normal

(Afelik et al., 2006). In some respects this is similar to what was seen in mice knockout by Ahlgern and colleagues (1996), whereby all exocrine cells were absent except for a few differentiated insulin and glucagon expressing cells.

However, the fact that insulin expression remained in Xenopus, may be simply due to the fact that a knockdown experiment was used instead of a null deletion

(Pearl and Horb, 2008).

1.5.3 Clinical Significance

The results obtained from the Pdx1 deletion studies performed in mice have also been observed in humans. Pdx1 frame shift mutations which lead to a nonfunctional protein, has been identified in newborns displaying pancreas agenesis (Stoffers et al., 1997a). In addition, humans heterozygous for an inactivating mutation of Pdx1 has been linked to type 2 diabetes known as maturity onset diabetes of the young 4 (Stoffers et al., 1997b). This is concurrent in mice heterozygous for a Pdx1 deficiency. These mice are glucose intolerant with decreased islet cell mass, increased islet apoptosis, and abnormal islet

21 architecture (Brissova et al., 2002; Johnson et al., 2003). These finding demonstrate that gene dosage of Pdx1 is essential for glucose homeostasis.

1.5.4 Pdx1 Gain-of-function Analysis

Various studies have attempted to elucidate the sufficiency of Pdx1 in promoting ectopic pancreatic cell fates. The conversion of other tissue types in the body to a pancreatic nature may provide a therapeutic means to treat diabetes. It is believed that the liver and pancreas arise from a common bipotential precursor and that they are specified by a single developmental decision involving a number of transcription factors (Deutsch et al., 2001). Therefore, due to the similar embryological origin between these two tissues, hepatic cells can be converted into functional β-cells (Meivar-Levy and Ferber, 2003). This transdifferentiation process was demonstrated in 2003 by Horb and colleagues. Using transgenic

Xenopus tadpoles, a modified form of Pdx1 (Pdx1-VP16) when expressed in the liver, was capable of converting differentiated hepatocytes into pancreatic tissue.

This pancreatic tissue expressed both endocrine and exocrine markers. The same construct was also able to convert mammalian HepG2 cells into both acinar and endocrine cells in vitro (Horb et al., 2003; Li et al., 2005). It was also shown that this transdifferentiation process yielded function β-cells (Li et al., 2005). More recent studies confirmed the sufficiency of this construct in converting liver to pancreas. In particular, Pdx1-VP16 was able to selectively convert rat hepatocytes into insulin producing β-cells that restored euglycemia in diabetic mice (Cao et al., 2004; Tang et al., 2006). Interestingly, only the modified form of Pdx1 carrying the VP16 transactivation domain of herpes simplex virus was able to

22 cause liver to pancreas transdifferentiation. It is believed that the VP16 domain allows for the interaction with various binding proteins ensuring a positive environment allowing Pdx1 to activate downstream targets (Horb et al., 2003).

Other in vivo studies using mammalian models have found that Pdx1 alone is also capable of reproducing the liver to pancreas transdifferentiation event.

Ferber and colleagues (2000) demonstrated that streptozotocin induced diabetic mice when treated with Pdx1 recombinant adenoviruses, ameliorated blood glucose levels and partially reversed diabetes. The regulation of blood glucose was due to a three fold increase in serum insulin levels when mice were treated with Pdx1 adenoviruses. Their results showed the sufficiency of Pdx1 in inducing biologically active insulin expression in the liver. However, adenovirus mediated conversion is not very efficient. Only 30-60% of hepatocytes expressed Pdx1 and only 1% of those cells produced insulin (Ferber et al., 2000). This low efficiency maybe due to the small number of Pdx1 gene copies induced per cell or due to the lack of appropriate Pdx1 protein partners (Fodor et al., 2007). In fact, the adenovirus mediated overexpression of unmodified Pdx1 in combination with pancreatic transcription factors NeuroD and Ngn3, significantly increases insulin production in the liver and ameliorated blood glucose levels in streptozotocin induced diabetic mice (Kaneto et al., 2005). Therefore, these findings suggest that

Pdx1 requires the recruitment of cofactors to fully exert its function during the liver to pancreas transdifferentiation process.

Other studies have also tried to address the potency of Pdx1 as a reprogramming factor to induce ectopic pancreas formation in different tissues.

The in ovo electroporation of Pdx1 in chick endoderm after its endogenous

23 expression, resulted in the formation of ectopic buds from the gut epithelium.

However, these Pdx1 overexpressing cells were not capable of differentiation into either the endocrine or exocrine cell types (Grapin-Botton et al., 2001). A similar study was carried out in Xenopus using a Pdx1 inducible gene construct. As in the

Grapin-Botton study, Pdx1 was overexpressed in the frog endoderm shortly after endogenous Pdx1 expression. Similarly, Pdx1 was unable to induce the formation of ectopic pancreas (Afelik et al., 2006). However, both studies overexpressed

Pdx1 shortly after endogenous expression which coincided with stable regional specification. Earlier Pdx1 overexpression before endodermal patterning was not studied.

In 2002, Yoshida and colleagues exogenously expressed Pdx1 in intestinal epithelium-derived IEC-6 cells. The overexpression of Pdx1 resulted in insulin expression accompanied by an upregulation of β-cell specific genes (Yoshida et al., 2002). The intestinal epithelium and pancreas share similar developmental backgrounds. During embryogenesis, both the gastrointestinal tract and pancreatic buds arise from the primitive gut tube (Grapin-Botton and Melton, 2000). This was likely to contribute to the successful formation of β-cells from non-insulin producing cells. Interestingly, using a transgenic approach, the sustained overexpression of Pdx1 in all pancreatic cell lineages during pancreas development, resulted in the formation of a small pancreas. In addition, acinar to ductal metaplasia was also observed (Miyatsuka et al., 2006). These findings suggest that the usefulness of Pdx1 in driving β-cell differentiation from non-β- cells depends on the context in which it is overexpressed.

24 1.5.5 Pdx1 Direct Gene Targets

The localized expression of Pdx1 to adult β-cells is believed to regulate various β-cell genes. Due to the presence of a homeodomain within Pdx1, this transcription factor can activate gene expression by binding DNA upstream of

TAAT sequences (Peshavaria et al., 1997). Pdx1 has been shown to bind to the following promoters: Insulin (Ohneda et al., 2000); glucose transporter 2 (Waeber et al., 1996); islet amyloid polypeptide (Macfarlane et al., 2000); glucagon (Wang et al., 2001); paired box 4 (Smith et al., 2000); glucokinase (Watada et al., 1996) and Pdx1 itself (in an autoregulatory loop) (Gerrish et al., 2001). Pdx1 binding to these promoters was first preformed in vitro and later confirmed in vivo as well

(Chakrabarti et al., 2002).

1.6 The Pancreatic Stellate Cell

1.6.1 PaSC Properties

Star shaped cells, known as stellate cells, were initially identified to be part of the exocrine pancreas in 1998. These myofibroblast-like cells comprise 4% of all pancreatic cells and are easily identified by the presence of intracellular fat droplets. In the normal pancreas, PaSC reside in a quiescent state mainly in the periacinar space where they express intermediate filament proteins, desmin and glial fibrillary acid protein (GFAP), but lack α-smooth muscle actin (Apte et al.,

1998; Bachem et al., 1998). It is hypothesized that due to the periacinar localization of PaSC, they may play a role in exocrine cell function. PaSC have also been identified in periductal and perivascular regions of the pancreas,

25 suggesting once again a possible role in regulating ductal and vascular cell function (Masamune et al., 2009).

In response to pancreatic injury, quiescent PaSC become “activated” and undergo various morphological changes. Activated PaSC display an enlarged nucleus and expanded endoplasmic reticulum network. They also obtain a myofibroblast-like phenotype by expressing α-smooth muscle actin in addition to desmin and GFAP, express extracellular matrix proteins and lose the ability to store lipid droplets (Apte et al., 1998; Bachem et al., 1998).

1.6.2 Activated PaSC

The activation of PaSC has been studied mainly with in vitro cultures of primary PaSC. The activation of these cells is regulated by various factors including cytokines (IL-1, IL-6, IL-8, TNF-α), growth factors (PDGF, TGF-β), ethanol, oxidative stress and reactive oxygen species. All of which can be secreted in a paracrine manner by neighboring cells as a result of pancreatic injury (Apte et al., 1999; Apte et al., 2000; Mews et al., 2002; Masamune et al., 2002). Once activated, PaSC produce a variety of autocrine factors (PDGF, TGF-β, IL-1, IL-6,

IL-8, MCP-1, RANTES, endothelin-1) which potentiate PaSC activation (Andoh et al., 2000; Shek et al., 2002; Phillips et al., 2003; Masamune et al., 2005; Aoki et al., 2006a; Aoki et al., 2006b). In addition to these factors, the activated PaSC secrete extracellular matrix components (collagens and fibronectin) as well as extracellular matrix degradation enzymes (matrix metalloproteinases and their inhibitors) (Phillips et al., 2003). The resulting autocrine and paracrine factors play an important role in enhancing PaSC proliferation, migration, contraction,

26 phagocytosis and extracellular matrix remodeling for tissue repair (Omary et al.,

2007).

Once PaSC become activated and migrate to the site of injury, they can follow either of two paths. Persistent injury or inflammation will lead to the formation of pancreatic fibrosis (part of the wound healing process). The participation of PaSC in the repair process has been observed in rats and humans with acute pancreatitis (Yokota et al., 2002; Zimmermann et al., 2002). Injury or inflammation that is only transient causes the activated PaSC to undergo apoptosis thus terminating the tissue repair process (Klonowski-Stumpe et al., 2002).

1.6.3 Embryonic Origin of PaSC

The study of PaSC is a relatively new field based upon extensive work carried out on hepatic stellate cells (HSC). However, recent genome-wide studies have revealed the similarities between these two cell types. Cultured HSC and

PaSC differ by 0.1% at the mRNA level (Buchholz et al., 2005). Thus it is believed that they may in fact share a common origin. Due to the expression of various neural stem cell markers (GFAP, NCAM1, nestin), it is hypothesized that stellate cells may arise from the neural crest (Niki et al., 1999). However, this has been recently disproven (Cassiman et al., 2006). Studies using transgenic mice with enhanced green fluorescent protein, have suggested a bone marrow contribution to the PaSC and HSC populations (Baba et al., 2004; Watanabi et al.,

2009). Despite these studies, the embryonic origin of stellate cells remains controversial.

27 1.7 Hypothesis and Objectives

Despite the identification of gene targets in mature β-cells, very little is known about Pdx1 downstream targets during initial stages of pancreas development. By identifying those targets, we can better understand the regulatory network involved in specifying the various pancreatic cell types. To achieve this, we make use of overexpression analysis. Pdx1 is sufficient to induce ectopic pancreas formation in the liver. However, it remains to be elucidated whether Pdx1 is sufficient and necessary to promote ectopic pancreas when misexpressed in the naïve endoderm.

Objectives:

1. Determine the sufficiency of Pdx1 in promoting ectopic pancreatic

tissue in naïve endoderm.

2. Identify possible Pdx1 downstream targets.

28 Chapter 2: Materials and Methods

In Vitro Fertilization and Microinjection

Xenopus laevis females were primed 3-5 days before the induction of ovulation by injecting 50 units of PMSG into the dorsal lymph sac. Ovulation was induced with 500 units of hCG, 15 hours before the start of fertilization. After ovulation, the ventral posterior surface of the frog was massaged and the expelled eggs collected. Male frogs were sacrificed by immersing in 0.05% benzocaine at room temperature for 30 minutes. The testis were dissected and placed in 1X

MMR and stored at 4°C until fertilization. The eggs were fertilized in vitro with a small part of the testis crushed in 0.1X MMR with 0.05mg/ml gentamycin

(MMRG). Fertilization was observed once the eggs have rotated so that the animal hemisphere faces upwards. To prepare the embryos for microinjection, the protective jelly membrane surrounding the embryos was removed. This was achieved by swirling the embryos in 4% cysteine (Fisher) at pH 7.6 for 15 minutes. After dejellying, the cysteine was washed out by rinsing the embryos with 1X MMR and then kept in 0.1X MMRG until they reach eight-cell stage for microinjection.

For mRNA microinjections, 1000pg Pdx1 and 50pg Pdx1-VP16 mRNA was co-injected with 400pg GFP mRNA into the dorsal vegetal blastomeres of eight-cell stage embryos. For temporal regulation of Pdx1 overexpression, Pdx1 and Pdx1-VP16 were cloned into GR-CS2 vectors. The mRNA was synthesized in

29 vitro using SP6 transcription mMessage mMachine Kit (Ambion). 1500pg Pdx1-

GR and 150pg Pdx1-VP16-GR mRNA was co-injected with 400pg GFP mRNA into the dorsal vegetal blastomeres of eight-cell stage embryos. The embryos were placed in Noble agar to help healing and grown overnight at 18ºC in 0.1X MMR with 2% ficoll (Sigma). At NF12, the GR fusion proteins were activated using dexamethasone (Sigma). Dexamethasone was prepared as a 5mM stock solution in 100% ethanol. The mRNA injected embryos were incubated with a final concentration of 10μM dexamethasone in 0.1X MMRG (Afelik et al., 2006).

Those embryos not activated with dexamethasone were used as controls. Embryos were grown at 23ºC and kept in dexamethasone until NF44/45. At NF44/45, whole guts were isolated from tadpoles anesthetized in a 1:2000 dilution of MS-

222 (Sigma). Pdx1 and Pdx1-VP16 mRNA targeting to the anterior gut region was confirmed by examining GFP fluorescence. Well targeted guts were fixed in

MEMFA for 1h, washed with 100% ethanol for 15 minutes and stored in glass vials at -20ºC in 100% ethanol.

Probe Synthesis and Whole-mount In situ Hybridization

Whole-mount in situ hybridization was performed according to Harland

(1991), with dioxigenin-substituted ribonucleotide probes. Antisense dioxygenin- labeled RNA probes were synthesized and purified using Sephadex columns. The table below represents those antisense RNA probes generated (Table 1). Genes are based on published Xenopus laevis sequences.

30 Gene Vector Linearization Transcription Enzyme Enzyme Somatostatin pCR Script EcoRI T7 Glucagon pCR Script EcoRI T7 Insulin pCR Script EcoRI T3 Transthyretin (TTR) pCR Script EcoRI T3 Elastase pCMV EcoRI T7 Pancreatic protein pBK-CMV BamHI T7 disulfide isomerase (XPDIp) α-1 pCR Script EcoRI T3 microglobulin/bikunin (AMBP) Insulinoma associated PCRII HindIII T7 protein 1 (Insm1) Paired box 4 (Pax4) PCRII XhoI SP6 Collagen type II α1 PCRII HindIII SP6 (Col2A1) Connective tissue growth PCRII NotI T7 factor (CTGF) Cytochrome b-245, alpha PCRII NotI T7 polypeptide (CYBA) Transformation growth PCRII NotI T7 factor β2 (TGFB2) Tissue inhibitor of PCRII NotI T7 metalloproteinase 1 (TIMP1) Collagen type IX α1 PCRII NotI SP6 (Col9A1) Cellular retinoic acid PCRII NotI SP6 binding protein 2 (CRABP2) Fatty acid binding protein PCRII NotI SP6 6 (FABP6) Iroquois homeobox 1 PCRII NotI SP6 (IRX1) Frizzed related protein 5 PCRII NotI SP6 (FRP5) Neural cell adhesion PCRII NotI SP6 molecule 1 (NCAM1) Hyaluronan synthase 2 PCRII BamHI T7 (HAS2)

Table 1: Generated antisense RNA probes for in situ hybridization.

31 The following Xenopus genes for whole-mount in situ hybridization were cloned by PCR from Xenopus laevis NF35 cDNA. Primers (Invitrogen) were designed based on published Xenopus laevis sequences (Table 2).

Gene Forward Primer Reverse Primer TIMP1 5’-ATGTTGTACCTTGTGGTTGTGC-3’ 5’-TTATTGTGCTGTGGCAGCAGA-3’ CYBA 5’-TCCTCGACAAAGACTATTCG-3’ 5’-CCGCAGAGGAAACACCGAC-3’ Col2A1 5’-TGGTCTACCAGGTCAGCGTG-3’ 5’-CCGCTCTTCCATTCAGGGTC-3’ TGFB2 5’-AGTGCCCTCGACATGGATCA-3’ 5’-GCTCCACACAGATACGGAC-3’ CTGF 5’-ATGTCTGCAGGAAAAGTGAC-3’ 5’-GGCCTCAAAGATGTCGTTGT-3’ FABP6 5’-ATGGCTTTACCGGAAAGTACG-3’ 5’-TGCCAACCTCTTACTGATTC-3’ Col9A1 5’-GGATTAGCTGGTTTGCCTGG-3’ 5’-TGGTTCAGTCAGAGATGCTG-3’ HAS2 5’-GATCCGGCGTCATCAGTGGAG-3’ 5’-GCGTAAAGTATTGCACCAAC-3’ CRABP2 5’-CAACTTCTCAGGACACTGGA-3’ 5’-AGTCCCTGATGTAAATCCGT-3’ IRX1 5’-CCGACAGGAGCTGAGTTGG-3’ 5’-ACACCCAACAAGGACCTCAT-3’ NCAM1 5’-GGATGATGGTGGCTCTCCT-3’ 5’-CTGCTTTGGCTCAGGAGTAG-3 Pax4 5’-ATGGAAGACTGTATAATGGA-3’ 5’-TTAATAATGGTAGACAGGATG-3’

Table 2: Designed PCR primers for cloning genes for in situ hybridization.

For whole-mount in situ hybridization, fixed samples stored in glass vials were rehydrated in 5 minute washes of:

• 100% methanol • 75% methanol 25% water • 50% methanol 50% water • 25% methanol 75% PTw (1X PBS, 0.1% Tween20) • four washes of 100% PTw

- Samples were incubated for 15 minutes in 1ml of 10μg/ml proteinase K.

- Samples rinsed twice with 0.1M trisethanolamine pH 7-8 for 5 minutes.

- 12.5μl of acetic anhydride (Sigma) was added and incubated for 5 minutes.

- The samples were washed twice with PTw for 5 minutes and then incubated with 4% paraformaldehyde (PFA) for 20 minutes.

32 - PFA was removed with five, 5 minute washes of PTw.

- Samples incubated in 1 ml hybridization buffer for 10 minutes at 60ºC.

- Prehybridization was then performed for 2-3 hours at 60ºC with fresh hybridization buffer.

- 1μg of desired probe was added to prehybridization buffer and allowed to incubate for 18 hours at 60ºC.

- The samples were washed for 10 minutes at 60ºC in hybridization buffer.

- Two washes of 2X SSC for 20 minutes and then two washes of 0.2X SSC for 30 minutes at 60ºC was carried out.

- Samples were washed twice with maleic acid buffer (MAB) for 15 minutes at room temperature.

- Samples incubated in 1ml of MAB containing 2% BMB blocking solution for 1 hour.

- 1 hour incubation was performed in MAB containing 2% BMB and 20% goat serum.

- 1:2000 dilution of α-dioxygenin antibody (Roche) was added to MAB containing 2% BMB, 20% goat serum.

- Incubation with antibody was carried out on a nutator at 4ºC overnight.

- Four washes of MAB at room temperature and a fifth wash with MAB overnight at 4ºC was performed.

- Samples incubated twice for 5 minutes with 1ml alkaline phosphatase buffer.

- Color reaction commenced with the addition of 0.5ml BM purple AP substrate

(Roche) containing 5 mM levamisol.

33 - Color reaction stopped with MEMFA once staining became apparent. Pictures were taken with a Leica DFC480 camera mounted onto a Leica MZ-16FA microscope.

Pax4 Cloning with RACE

Partial cloning of Xenopus laevis Pax4 was obtained through nested PCR of partial Xenopus tropicalis Pax4 sequence published in JGI XT Genome 4.1

(e_gw1.480.46.1). The following nested primers (Invitrogen) based on tropicalis

Pax4 sequence were designed:

Forward (fwd) 1: 5’-GGTGTTACCAGTGTCAATCA-3’

Forward 2: 5’-AATCAGTTGGGAGGGGTGTT-3’

Forward 3: 5’-GTGTTTGTAAATGGGCGTCC-3’

Reverse (rev) 1: 5’-ACTGATGTCACCTGAAATCT-3’

Reverse 2: 5’-AATCTCCTTGTCTTTCTTCA-3’

Reverse 3: 5’-CTTCAGCTTCTCTTCACGCC-3’

PCR was carried out with primers fwd1 and rev1, NF22 Xenopus laevis cDNA, 40 cycles at 50ºC annealing temperature. 1μl PCR product was then used as template for the following PCR reaction using nested fwd2 and rev2 primers,

25 cycles at 52ºC annealing temperature. 1μl PCR product was then used as a template for the following PCR reaction using nested fwd3 and rev3 primers, 25 cycles at 54ºC annealing temperature. Entire PCR product was run on 1% agarose gel (Invitrogen) and extracted using Qiax II Gel Extraction Kit (Qiagen). Product was then ligated in PCRII using TOPO TA Cloning Kit (Invitrogen).

34 To obtain the full length sequence of Xenopus laevis Pax4, BD SMART

RACE cDNA Amplification Kit (BD Biosciences) was performed according to manufacturer’s specifications. The 5’ sequence was obtained through PCR with gene specific primer (GSP): 5’-TGTCTGCTGCCAGTTTCTCCCTTCT-3’ and universal primers mix (UPM), 5’ RACE ready cDNA, 25 cycles at 55ºC annealing temperature. A nested PCR was then carried out using 2μl of PCR product, nested gene specific primer (NGSP): 5’-GTTGCGTTGCTGGCAGTTTGGAGG-3’ and nested universal primer (NUP), 25 cycles at 55ºC annealing temperature. Entire

PCR product was run on 1% agarose gel (Invitrogen) and extracted using Qiax II

Gel Extraction Kit (Qiagen). Product was then ligated in PCRII using TOPO TA

Cloning Kit (Invitrogen). The 3’ sequence was obtained through PCR with primers GSP: 5’-ATCCGTCGCTGTTTGCTTGGGAGAT-3’ and UPM, 3’

RACE ready cDNA, 25 cycles at 55ºC annealing temperature. A nested PCR was then carried out using 2μl of PCR product, nested primers NGSP: 5’-

GTCATCTATCAATCGTGTCCTAGG-3’ and NUP, 25 cycles at 55ºC annealing temperature. Entire PCR product was run on 1% agarose gel (Invitrogen) and extracted using Qiax II Gel Extraction Kit (Qiagen). Product was then ligated in

PCRII using TOPO TA Cloning Kit (Invitrogen). The Xenopus laevis Pax4 coding sequence was then obtained by analyzing the 5’ and 3’ RACE sequences.

Cloning Pdx1-GR Fusion Constructs

Pdx1-VP16-GR-CS2 construct was obtained as a kind gift from T. Hayata.

Pdx1-GR fusion construct was generated by fusing the coding region of Pdx1 with

35 the ligand binding domain of the human glucocorticoid receptor (GR) (Hollenberg et al., 1993). Xenopus laevis Pdx1 PCR isolated from Pdx1-CS2 (kind gift from C.

Wright) using SP6 primer and 3’ primer containing ClaI site, 5’-

ATCTCTATCGATTTTCTGCCTGC-3’. It was cut with ClaI and subcloned into the ClaI site of GR-CS2 vector. RNA for microinjection was generated by linearizing both Pdx1-GR-CS2 and Pdx1-VP16-GR-CS2 with NotI and transcribed with SP6 using mMessage mMachine Kit (Ambion).

RNA Extraction, Purification and Microarray Analysis

1500pg Pdx1-GR and 150pg Pdx1-VP16-GR mRNA was microinjected into the dorsal vegetal blastomeres of eight-cell stage embryos. To ensure proper mRNA targeting to the anterior endoderm, Pdx1-GR and Pdx1-VP16-GR mRNA was co-injected with 400pg GFP mRNA. Dexamethasone was added at NF12 to activate the GR fusion constructs. Those embryos not activated by dexamethasone were treated as controls. At NF44/45, the foregut region from targeted embryos was isolated from mRNA injected-embryos activated by dexamethasone as well as those not activated by dexamethasone. Ten foreguts from each condition was collected and placed in 1ml eppendorf tubes with 0.5ml of RNAlater (Ambion).

RNA extraction was performed using Trizol Reagent (Invitrogen) as specified by the manufacturer. The extracted total RNA was then purified using RNAeasy

Micro Kit (Qiagen). Three different replicates of extracted RNA from different fertilizations were obtained for the microarray analysis. RNA bioanalysis using

RNA 6000 Nano Kit (Agilent), cDNA synthesis and hybridization to Xenopus

36 laevis 2.0 GeneChip 3’ IVT (Affymetrix) was performed by McGill University and Genome Quebec Innovation Centre. The results were analyzed with

FlexArray 1.4.1 software (http://genomequebec.mcgill.ca/FlexArray). The data was normalized using RMA algorithm. The EB (Wright & Simon) statistical algorithm was used to calculate the log base 2 (fold change) between treatment and control groups. Those genes having a fold change greater than 1.5 and a P- value less than 0.05 were studied further. Gene annotation as well as functional clustering was performed with DAVID Bioinformatics Resources 6.7 (Dennis et al., 2003; Huang et al., 2009) and GeneCodis 2.0 (Carmona-Saez et al., 2007;

Nogales-Cadenas et al., 2009).

Microarray Validation by RT-PCR

Reverse transcription reactions were performed on cDNA generated from extracted RNA replicates sent for microarray analysis. 1μg of total RNA was incubated in the presence of 5mM dNTPs and oligodT primers for 5 minutes at

65ºC. Solution was then incubated with 5X first strand buffer and 0.1M DTT for 2 minutes at 42ºC. cDNA was transcribed using SuperScript II Reverse

Transcriptase (Invitrogen) by incubating for 50 minutes at 42ºC. Enzymatic reaction was inactivated by heating mixture at 70ºC for 15 minutes. PCR was performed on the following genes of interest. Primers (Invitrogen) were designed based on published Xenopus laevis sequences (Table 3). The RT-PCR was normalized to elongation factor 1 alpha (EF1A).

37 Gene Forward primer Reverse primer Tissue inhibitor of 5’-GTAACTTCATTGTTCCTTGG-3’ 5’-TTATTGTGCTGTGGCAGCAGA-3’ metalloproteinase 1 (TIMP1) Cytochrome b-245, 5’-CAATGACGCGTCAACCCTCA-3’ 5’-CCGCAGAGGAAACACCGAC-3’ alpha polypeptide (CYBA) Collagen type II α1 5’-GTGGTGAAACTGGCCCCTCT-3’ 5’-CCGCTCTTCCATTCAGGGTC-3’ (Col2A1)

Transformation 5’-CCAGGTTTGCAGGTATTGAT-3’ 5’- growth factor β2 GCTCCACACAGATACGGACAAG-3’ (TGFB2) Connective tissue 5’-ACTCTGCATGGTCAGGCCCT-3’ 5’-GGCCTCAAAGATGTCGTTGT-3’ growth factor (CTGF) Insulin growth 5’-CAGGAGCCTATGTCAGCTGC-3’ 5’-CGCTGGGTATGTGCTCGGCG-3’ factor binding protein 2 (IGFBP2) Fatty acid binding 5’-GGCATCCCTGCTGAGACCAT-3’ 5’-TGCCAACCTCTTACTGATTC-3’ protein 6 (FABP6) Collagen type IX 5’-GTCCACGTGGTATTCCAGGC-3’ 5’-TGGTTCAGTCAGAGATGCTG-3’ α1 (COL9A1) Hyaluronan 5’-CAACTTGTAGGCCTTATCAA-3’ 5’-GCGTAAAGTATTGCACCAAC-3’ synthase 2 (HAS2) Cellular retinoic 5’-GCTGCATCTAAACCAGCAGT-3’ 5’-AGTCCCTGATGTAAATCCGT-3’ acid binding protein 2 (CRABP2) Tetraspanin-5 5’-CAACTGCACCGACTCTAACG-3’ 5’-TCTGGCAAGCACTACAAGGG-3’ (TSPAN5) Iroquois homeobox 5’-TCAATGTCGCTGGGTAAGGA-3’ 5’-ACACCCAACAAGGACCTCAT-3’ 1 (IRX1) Silver (SILV) 5’-GTTCAAGTAGCTCCAGTTGC-3’ 5’-CCAACTAGGACAATGCCACC-3’ Paired box 9 5’-ATGTCCTCAACGGTTTAGAG-3’ 5’-GGACTTGCGTGTTTGGATAC-3’ (PAX9) Collagen type II α 5’-GCCGTGACCTGAAGATGTGC-3’ 5’-TCTGAGAGGCTTCAGTGGCC-3’ 1 (COL2A1) α smooth muscle 5’-CTGCTGAGCGTGAAATTGTG-3’ 5’-AATGCCAGGGTACATGGTGG-3’ actin (SMA) Desmin (DES) 5’-CTCAACCAGGCAGCTAAGAA-3’ 5’-AGCAGTTTACGGTATGTGGC-3’ Synaptophysin 5’-CAGCCATGGCCGTGTACATG-3’ 5’-CACACGTTTCCGAGCCAGAG-3’ (SYP) Vimentin (VIM) 5’-CACCTGCGAGATTGATGCAA-3’ 5’-GTTTCTCTCAGGCTCATTGT-3’

38 Endothelin 5’-GACTGGTGGCTGTTTGGCTT-3’ 5’-GAGTTCAGCGCTGCTAGGTT-3’ receptor A (EDNRA) Neural cell 5’-GAGACCACCACTTTAACTTC-3’ 5’-CTGCTTTGGCTCAGGAGTAG-3’ adhesion molecule 1 (NCAM1) Nestin (NES) 5’-CAACCAGACGTTCATGAAGA-3’ 5’-GCGAGACCATAAAGTCAACT-3’ Fibronectin (FN) 5’-GCCATCCTATTGACCATGAG-3’ 5’-TAACCCAGGCACTTGCACCA-3’ Platelet derived 5’-CCATGAAGTGTACGACATCA-3’ 5’-CTGAGACGGGGTCAATATCA-3’ growth factor receptor A (PDGFRA) Elongation factor 1 5’-ATGCACCATGAAGCCCTTAC-3’ 5’-GCAAGCAATGTGAGCAGTGT-3’ α (EF1A)

Table 3: Designed primers for RT-PCR.

39 Chapter 3: Results

Early Pdx1 Overexpression Caused Gastrulation Defects

It has been previously shown using Xenopus transgenics that a modified form of Pdx1 (Pdx1-VP16) when expressed in the liver, is capable of converting differentiated hepatocytes into pancreatic tissue (Horb et al., 2003). However, it remains to be elucidated whether an ectopic pancreatic fate may arise when Pdx1 is overexpressed in the early (naïve) endoderm. To achieve this, Pdx1 and Pdx1-

VP16 mRNA was co-injected with GFP mRNA into the dorsal vegetal blastomeres of Xenopus laevis eight-cell stage embryos. The co-injection with

GFP mRNA ensured the visualization of proper targeting of the RNA to the anterior endoderm where pancreas organogenesis takes place.

The overexpression of Pdx1 at such early stages in development resulted in a high mortality rate. This was largely due to gastrulation defects (Figure 1).

Pdx1 injected embryos did not form a blastopore by NF (Nieuwkoop and Faber) stage 12 compared to GFP only injected controls (Figure 1A,C). Those few embryos that survived past gastrulation and neurulation showed a shortening of the anterior-posterior body axis by tadpole stages (Figure 1B,D). Similar results were observed with Pdx1-VP16 injected embryos. However, the rate of mortality and the severity of the gastrulation defects were much more pronounced (data not shown).

Figure 1: Early Pdx1 overexpression resulted in gastrulation defects. (A) Blastopore developed normally in GFP only injected controls. (B) GFP injected control tadpoles were elongated by NF33. (C) NF12 embryos did not form a blastopore when Pdx1 was overexpressed. (D) Tadpoles at NF33 showed shortening of anterior-posterior axis when Pdx1 was overexpressed. BP, blastopore.

Established Temporal Regulation of Pdx1 Overexpression

Due to the high morality rate and defects in gastrulation, it became necessary to temporally regulate Pdx1 and Pdx1-VP16 overexpression. A hormone inducible variant of Pdx1 was generated by fusing the human glucocorticoid receptor (GR) ligand binding domain to the C-terminus of Pdx1 and Pdx1-VP16. To perform the overexpression study, Pdx1-GR and Pdx1-VP16-

GR mRNA was co-injected with GFP mRNA into the dorsal vegetal blastomeres of eight-cell stage embryos. The translated Pdx1 proteins remain in an inactive state bound to heat shock proteins until activation with dexamethasone (dex). The proteins were activated by treating the embryos with dex at stage 12 (twelve hours post-fertilization), immediately after gastrulation, but well before endogenous

41 Pdx1 expression is observed. The embryos were kept in this activation media from NF12-NF44/45. Pdx1-GR and Pdx1-VP16-GR mRNA injected embryos not incubated in dex caused protein function inhibition and were considered as controls.

Pdx1 Overexpression Resulted in Ectopic Tissue Formation

NF44/45 tadpoles showing well targeted GFP fluorescence to the anterior endoderm were kept for further analysis (Figure 2A,E,I,M). Pdx1-GR and Pdx1-

VP16-GR injected tadpoles following incubation in the presence of dex showed a similar morphological phenotype (Figure 2F,N). The tadpoles no longer had difficulties completing gastrulation and did not show shortening of the anterior- posterior axis. Guts of NF44/45 tadpoles that expressed GFP fluorescence uniformly throughout the foregut were isolated (Figure 2C,G,K,O). It was observed that the isolated guts of Pdx1-GR and Pdx1-VP16-GR activated tadpoles were morphologically similar (Figure 2H,P). Both displayed the presence of ectopic tissue in the anterior foregut region with undefined organ domains. In addition, intestinal coiling was not observed. The RNA injected embryos not incubated in the presence of dex were wild-type in nature, with well defined organs and proper gut coiling (Figure 2B,D,J,L). This demonstrated that the generated GR fusion constructs were working correctly.

Figure 2: Pdx1-GR overexpression caused ectopic tissue formation. (A) GFP fluorescence showing Pdx1-GR mRNA localization to the anterior endoderm in no dex control tadpoles. (B) Tadpole controls were elongated and wild-type. (C) GFP fluorescence showing Pdx1-GR mRNA localization to foregut region in no dex control guts. (D) Without the addition of dex, Pdx1-GR injected guts appeared normal with defined organs and gut coiling. (E) GFP fluorescence showing Pdx1-GR mRNA localization to the anterior endoderm in dex activated tadpoles. (F) Activated Pdx1-GR injected tadpoles were elongated showing ectopic tissue in the foregut. (G) GFP fluorescence showing Pdx1-GR mRNA localization to the foregut region in activated tadpoles. (H) Activated Pdx1-GR injected tadpoles showed ectopic tissue formation in the anterior foregut region. (I) GFP fluorescence showing Pdx1-VP16-GR mRNA localization to the anterior endoderm in no dex control tadpoles. (J) Tadpole controls were elongated and wild-type. (K) GFP fluorescence showing Pdx1-VP16-GR mRNA localization to foregut region in no dex control guts. (L) Without the addition of dex, Pdx1- VP16-GR injected guts appeared normal with defined organs and gut coiling. (M) GFP fluorescence showing Pdx1-VP16-GR mRNA localization to the anterior endoderm in dex activated tadpoles. (N) Activated Pdx1-VP16-GR injected tadpoles were elongated showing ectopic tissue in the foregut (O) GFP fluorescence showing Pdx1-VP16-GR mRNA localization to the foregut region in activated tadpoles. (P) Activated Pdx1-VP16-GR injected tadpoles showed ectopic tissue formation in the anterior foregut region. P, pancreas; L, liver; St/d, stomach and duodenum; ET, ectopic tissue.

43 Ectopic Tissue Displayed Reduced Exocrine Differentiation

Since the overexpression of Pdx1-GR and Pdx1-VP16-GR in the naïve endoderm resulted in formation of ectopic tissue in the foregut that was morphologically ill defined, we determined which anterior markers were affected.

Due to the known effects of Pdx1 in promoting an ectopic pancreas in the liver, we first aimed to determine whether this tissue formation expressed pancreatic markers. Elastase and pancreatic protein disulfide isomerase (XPDIp) are two acinar markers present throughout the pancreas (Figure 3A,C,E,G). The expression of elastase and XPDIp were severely reduced in Pdx1-GR and Pdx1-

VP16-GR injected embryos treated with dex (Figure 3B,D,F,H).

Figure 3: Ectopic tissue displayed reduced exocrine markers. (A,C,E,G) Elastase and XPDIp are expressed throughout the pancreatic tissue in isolated NF44/45 whole gut controls. (B) Activation of Pdx1-GR injected embryos displayed reduced expression of elastase (n=5). (D) Decrease in elastase expression was observed in Pdx1-VP16-GR injections (n=13). (F) XPDIp expression was reduced in the ectopic tissue with the activation of Pdx1-GR (n=6). (H) Reduction of XPDIp was also observed with the activation of Pdx1- VP16-GR (n=18).

Ectopic Tissue Displayed Reduced Endocrine Differentiation

We next determined whether the ectopic tissue has committed to the endocrine cell lineage. By NF44/45, the gastrointestinal and pancreatic endocrine cells, glucagon and somatostatin, are expressed in the stomach, duodenum and throughout the dorsal pancreas (Figure 4A,C,E,G). Both these markers were absent in the ectopic tissue with the overexpression of Pdx1-GR and Pdx1-VP16-

GR (Figure 4B,D,F,H). In controls, insulin expression is solely visible in the dorsal pancreas (Figure 4I,K). However, the activation with dex has lead to an absence of insulin expression in the ectopic tissue (Figure 4J,L).

Figure 4: Ectopic tissue displayed reduced endocrine differentiation. (A,C,E,G) Glucagon and somatostatin are expressed throughout the stomach, duodenum and dorsal pancreas in isolated NF44/45 control guts. (B) Glucagon was not present within the ectopic tissue with the overexpression of Pdx1-GR (n=4). (D) Absence in glucagon expression was seen in Pdx1-VP16-GR overexpression (n=5). (F) Activation of Pdx1-GR showed absence of somatostatin expression in the ectopic tissue (n=5). (H) Similar absence was noted with the addition of dex in Pdx1-VP16-GR injections (n=5). (I,K) Insulin is present only in the dorsal pancreas in non activated gut controls. (J) Insulin expression was absent in the ectopic tissue when Pdx1-GR was overexpressed (n=9). (L) Absence in insulin expression was observed in Pdx1-VP16-GR overexpression (n=17).

To better understand the reasons for the absence in endocrine expression, we investigated whether initial differentiation of endocrine cells occurred. Pax4 plays an important role in insulin and somatostatin producing cell maturation

(Sosa-Pineda et al., 1997). However, Xenopus laevis Pax4 has never been cloned.

The use of Rapid Amplification of cDNA Ends (RACE) permitted the cloning of the Pax4 coding sequence (data not shown). The Pax4 expression pattern is

46 similar to that of somatostatin and glucagon. In no dex controls, Pax4 is expressed in a punctate manner throughout the stomach, duodenum and dorsal pancreas (Figure 5A,C). Pax4 is moderately reduced in our overexpression model with the activation of dex (Figure 5B,D). We also decided to look further upstream in the endocrine differentiation pathway to determine whether endocrine progenitors were specified. Insulinoma associated protein 1 (Insm1) is expressed in all cells that give rise to the various endocrine cell types (Gierl et al., 2006). It is localized to the stomach, duodenum and dorsal pancreatic region (Figure 5E,G).

Insm1 expression in the ectopic tissue was significantly reduced (Figure 5F,H).

These data suggest that the ectopic tissue formation resulting from Pdx1-GR and

Pdx1-VP16-GR overexpression was not of an exocrine or endocrine character.

Figure 5: Reduction in initial endocrine cell differentiation. (A,C) Pax4 is present in punctate form throughout the stomach, duodenum and dorsal pancreas in NF44/45 control guts. (B) Reduction of Pax4 was seen in Pdx1-GR overexpression (n=5). (D) Reduction in Pax4 expression was also observed in Pdx1-VP16-GR overexpression (n=7). (E,G) Insm1 is expressed throughout the stomach, duodenum and dorsal pancreatic regions in control guts. (F) Activated Pdx1-GR injections, the ectopic tissue showed significant decrease in Insm1expression (n=5). (H) Similar Insm1decrease was noted in Pdx1-VP16-GR overexpression (n=7).

Ectopic Tissue Disrupted Foregut Organ Development

We next examined whether development of stomach, duodenum and liver was normal. The stomach and duodenum marker frizzled related protein 5 (FRP5) is expressed in a gradient fashion from the posterior stomach to the duodenum

(Figure 6A,C). We found FRP5 to be significantly reduced in Pdx1-GR and Pdx1-

VP16-GR injected embryos (Figure 6B,D). To assess whether liver organogenesis was normal in our overexpression study, we examined whether the expression of transthyretin (TTR) and α-1 microglobulin/bikunin (AMBP) was affected. In controls, TTR is expressed throughout the intestinal epithelium and in the liver

48 (Figure 6E,G). Whereas AMBP is expressed exclusively in the liver (Figure 6I,K).

The observed ectopic tissue displayed a liver domain which was significantly smaller than controls (Figure 6F,H,J,L). These results demonstrated that the development of almost the entire anterior endoderm was disrupted by overexpression of Pdx1-GR and Pdx1-VP16-GR.

Figure 6: Ectopic tissue perturbed stomach, duodenum and liver development. (A,C) FRP5 is expressed throughout the stomach and duodenum regions of isolated NF44/45 control guts. (B) The ectopic tissue formation resulting from Pdx1-GR overexpression caused significant reduction in FRP5 expression (n=6). (D) Similar reduction in FRP5 was observed in Pdx1-VP16-GR overexpression (n=14). (E,G,I,K) TTR and AMBP are expressed in the liver of isolated NF44/45 control guts. (F) Significant reduction in the amount of liver TTR when Pdx1-GR was overexpressed (n=4). (H) Liver TTR was reduced in the overexpression of Pdx1-VP16-GR (n=8). (J) Small liver had been observed when staining for AMBP in activated Pdx1-GR injections (n=5). (L) Small liver in the ectopic tissue was also observed when stained for AMBP in Pdx1-VP16-GR overexpression (n=5). Arrows point to hepatic tissue.

49 We studied the possibility that the maintained overexpression of Pdx1 has

“trapped” the ectopic tissue in an undifferentiated state. In our overexpression model, Pdx1-GR and Pdx1-VP16-GR expression was maintained for 3 days throughout the development of the embryo (NF12-44/45). We decided to activate the GR constructs for only 4 hours (NF12-15) with dex and then remove dex, effectively shutting down Pdx1 function for the remainder of the embryo’s development. This would assess whether the maintained Pdx1 overexpression was preventing the ectopic tissue from further differentiating. However, we obtained a similar phenotype as previously observed. The ectopic tissue formation displayed reduced expression of endocrine and exocrine markers (Figure 7). Normal stomach, duodenum and liver organogenesis were also severely disrupted (data not shown). The ability of Pdx1 overexpression to form ectopic tissue early in endoderm development, before formation of foregut organs, suggests that this ectopic tissue develops directly from the endoderm and is not the result of transdifferentiation from mature stomach, duodenum and pancreatic tissue.

Figure 7: Transient activation did not result in differentiation of ectopic tissue. Pdx1-GR and Pdx1-VP16-GR injected embryos were incubated in dex media for four hours (NF12-15) as apposed to continuous incubation (NF12- 44/45). (A,C,E,G) Glucagon and somatostatin are expressed throughout the stomach, duodenum and dorsal pancreas in isolated gut controls. (B) Glucagon was not present in the ectopic tissue with the overexpression of Pdx1-GR (n=4). (D) Absence in glucagon was seen in Pdx1-VP16-GR overexpression (n=6). (F) Somatostatin expression was not observed in the ectopic tissue (n=4). (H) Absence in somatostatin was noted with the addition of dex in Pdx1-VP16-GR injections (n=8). (I,K) Insulin expression is restricted to the dorsal pancreas in controls. (J) Insulin was absent when Pdx1-GR was overexpressed (n=4). (L) Absence in insulin expression was seen in Pdx1-VP16-GR overexpression (n=7).

Identifying Ectopic Tissue Formation by Microarray

To define the character of the ectopic tissue, we performed a microarray analysis comparing ectopic tissue to control foreguts (Figure 8). We hypothesized that by uncovering those genes that are upregulated within the ectopic tissue, we

51 would obtain a better understanding of the cell type formed when Pdx1-GR and

Pdx1-VP16-GR are overexpressed in the naïve endoderm. Three different replicates of extracted RNA from different fertilizations were obtained for the microarray analysis. The RNA was reverse transcribed, labeled and hybridized to probes on the Affymetrix Xenopus laevis 2.0 GeneChip 3’ IVT (McGill

University, Genome Quebec Innovation Center). The results were analyzed with

FlexArray 1.4.1 software. The data was normalized using RMA algorithm. The

EB (Wright & Simon) statistical algorithm was used to calculate the log base 2

(fold change) between treatment and control groups. Those genes having a fold change greater than 1.5 and P-value less than 0.05 were studied further.

Redundantly represented genes in the Affymetrix GeneChip were removed and the human orthologs for the differentially expressed genes were obtained using

BLAST. Thus for simplicity of the analysis, each Xenopus gene was assigned an official gene symbol and name from the human counterpart.

Figure 8: Experimental design of microarray analysis. Foreguts of NF44/45 tadpoles were isolated from treatment (+Dex) and control (-Dex) groups. Microarray analysis on extracted RNA was performed with Affymetrix Xenopus laevis 2.0 GeneChip and analyzed using FlexArray software.

52 Reduction in Pancreas, Liver and Intestinal Markers Confirmed by Microarray

We began the analysis by looking at the expression of genes that were significantly downregulated (P-value<0.05) by a factor of -1.5 or greater (Figure

9). 151 genes were found to be decreased in the ectopic tissue in the Pdx1-GR group, while 246 genes were downregulated in the Pdx1-VP16-GR group.

Interestingly, the majority of these downregulated genes were common between both treatment groups. 92% of genes downregulated in Pdx1-GR were also downregulated in Pdx1-VP16-GR. 57% of genes downregulated in Pdx1-VP16-

GR were also downregulated in Pdx1-GR. The large number of commonly downregulated genes is due to the similarity of the overexpression phenotype present between both treatment groups. Therefore, we decided to focus specifically on those common downregulated genes.

Pdx1-GR 12 139 107 Pdx1-VP16-GR

Figure 9: Venn diagram displaying downregulated microarray genes. Genes having a P-value less than 0.05 and a fold change greater than -1.5 were considered. 12 genes were specifically downregulated in Pdx1-GR overexpression. 107 genes were specifically downregulated in Pdx1-VP16-GR overexpression. 139 genes were commonly downregulated between both groups.

Our preliminary findings using whole-mount in situ hybridization revealed a significant decrease in the expression of pancreatic, liver, stomach and

53 duodenum markers. Thus, we would expect that the majority of the downregulated genes would be classified within these tissue categories. In fact,

74% of the 139 common downregulated genes were classified as being expressed in pancreas, liver or intestine (Tables 4, 5 and 6). This categorization was achieved based on results obtained from the Hayata and colleagues (2009) microarray. Using Affymetrix Xenopus GeneChip arrays, they were able to obtain the differential gene expression profiles of pancreas, liver and intestine from stage

42 tadpoles. Many of the genes identified by these researchers were verified extensively by others. Those genes not able to be categorized based on the Hayata microarrays were categorized using DAVID Bioinformatics Resources 6.7

(Dennis et al., 2003; Huang et al., 2009).

In Table 4, we obtained a list of pancreatic downregulated genes present in the overexpression microarray. Many of the genes have enzymatic activity and play a role in the exocrine function of the pancreas. These included CELA1,

AMY2A, CTRB1, CPA1, PNLIP, PRSS3, DNASE1, PRSS2, CPB1, CEL,

CELA2A, PRSS1 and CA2. Some endocrine genes have also been identified including INS and SST. Surprisingly, very few transcription factors have been identified: PTF1A necessary for endocrine and exocrine development, while

PAX6 is important for endocrine formation. Nonetheless, the majority of the pancreatic genes significantly downregulated in the ectopic tissue are expressed by acinar and endocrine cells.

54 Gene Name and Symbol Pdx1-GR* Pdx1-VP16-GR* chymotrypsin-like elastase family, member 1 -5.46 -5.43 (CELA1) amylase, alpha 2A (AMY2A) -5.40 -6.37 chymotrypsinogen B1 (CTRB1) -5.17 -5.30 carboxypeptidase A1 (CPA1) -5.10 -6.11 pancreatic lipase (PNLIP) -4.81 -5.54 protease, serine, 3 (PRSS3) -4.68 -6.34 deoxyribonuclease I (DNASE1) -4.13 -5.14 insulin (INS) -3.63 -3.38 lectin, mannose-binding, 1 (LMAN1) -3.50 -4.82 protease, serine, 2 (PRSS2) -3.44 -4.82 carboxypeptidase B1 (CPB1) -3.35 -4.60 carboxyl ester lipase (CEL) -3.28 -4.29 chymotrypsin-like elastase family, member 2A -3.27 -4.25 (CELA2A) protease, serine, 1 (PRSS1) -3.21 -3.43 serpin peptidase inhibitor, member 2 (SERPINI2) -3.11 -4.20 branched chain aminotransferase 1 (BCAT1) -2.80 -3.20 carnosine dipeptidase 1 (CNDP1) -2.71 -2.37 keratin 18 (KRT18) -2.54 -2.73 activating transcription factor 5 (ATF5) -2.39 -2.69 prolyl 4-hydroxylase, beta polypeptide (P4HB) -2.39 -3.46 pancreas specific transcription factor 1a (PTF1A) -2.30 -3.16 sideroflexin 2 (SFXN2) -2.16 -2.58 phosphoserine aminotransferase 1 (PSAT1) -2.11 -1.77 carbonic anhydrase II (CA2) -2.10 -2.62 neurensin 1 (NRSN1) -1.90 -2.56 aquaporin 1 (AQP1) -1.77 -2.22 tripartite motif-containing 2 (TRIM2) -1.76 -1.54 SEC11 homolog C (SEC11C) -1.67 -2.07 nuclear receptor subfamily 5, group A, member -1.62 -1.73 2 (NR5A2) methionine adenosyltransferase I, alpha -1.61 -2.05 (MAT1A) cytochrome P450, family 1, subfamily A, -1.57 -2.19 polypeptide 1 (CYP1A1) paired box 6 (PAX6) -1.56 -1.67 somatostatin (SST) -1.54 -2.20 * log base 2 fold change relative to controls

Table 4: Downregulated microarray genes present in pancreas. Fold changes of Pdx1-GR and Pdx1-VP16-GR were calculated relative to their respective controls.

55 Gene Name and Symbol Pdx1-GR* Pdx1-VP16-GR* lectin, mannose-binding, 1 (LMAN1) -3.50 -4.82 transmembrane 4 L six family member 4 -3.50 -3.87 (TM4SF4) proprotein convertase subtilisin/kexin type 9 -3.41 -2.97 (PCSK9) hydroxyacid oxidase 1 (HAO1) -3.03 -3.40 glucokinase (GCK) -2.58 -2.67 solute carrier family 12 member 3 (SLC12A3) -2.57 -3.12 aldo-keto reductase family 1, member D1 -2.48 -2.66 (AKR1D1) activating transcription factor 5 -2.39 -2.69 (ATF5) prolyl 4-hydroxylase, beta polypeptide (P4HB) -2.39 -3.46 cytochrome P450, family 51, subfamily A, -2.36 -1.67 polypeptide 1 (CYP51A1) adenylate kinase 3 (AK3) -2.21 -2.48 aminocarboxymuconate semialdehyde -2.17 -2.80 decarboxylase (ACMSD) angiotensin I converting enzyme 2 (ACE2) -2.16 -2.19 sideroflexin 2 (SFXN2) -2.16 -2.58 cytochrome P450, family 8, subfamily B, -2.15 -2.16 polypeptide 1 (CYP8B1) arylsulfatase A (ARSA) -2.13 -2.33 lipase, hepatic (LIPC) -2.13 -2.40 3-hydroxy-3-methylglutaryl-Coenzyme A -2.10 -1.59 reductase (HMGCR) hydroxy-delta-5-steroid dehydrogenase, 3 beta- -2.05 -2.18 and steroid delta-isomerase 7 (HSD3B7) pyruvate carboxylase (PC) -2.04 -2.15 aminoadipate aminotransferase (AADAT) -1.95 -1.98 aldo-keto reductase family 1, member C1 -1.89 -2.17 (AKR1C1) butyrylcholinesterase (BCHE) -1.82 -1.70 emopamil binding protein-like (EBPL) -1.76 -1.51 glucan (GBE1) -1.76 -1.60 deiodinase, iodothyronine, type I (DIO1) -1.75 -2.24 sulfotransferase family, cytosolic, 2A, member -1.75 -2.52 1 (SULT2A1) glyoxylate reductase/hydroxypyruvate -1.74 -2.83 reductase (GRHPR) xanthine dehydrogenase (XDH) -1.69 -2.01 3-hydroxy-3-methylglutaryl-Coenzyme A -1.66 -1.54 synthase 1 (HMGCS1) sulfotransferase family 1E, estrogen-preferring, -1.66 -1.99 member 1 (SULT1E1)

56 ferredoxin reductase (FDXR) -1.62 -1.84 cytochrome P450, family 27, subfamily A, -1.61 -1.66 polypeptide 1 (CYP27A1) dynein, axonemal, heavy chain 10 (DNAH10) -1.59 -2.20 cytochrome P450, family 1, subfamily A, -1.57 -2.19 polypeptide 1 (CYP1A1) superoxide dismutase 2, mitochondrial (SOD2) -1.57 -1.80 cytochrome P450, family 4, subfamily V, -1.56 -1.85 polypeptide 2 (CYP4V2) ectonucleoside triphosphate -1.54 -1.74 diphosphohydrolase 5 (ENTPD5) acyl-CoA synthetase medium-chain family -1.53 -1.89 member 3 (ACSM3) radial spoke 3 homolog (RSPH3) -1.52 -2.26 * log base 2 fold change relative to controls

Table 5: Downregulated microarray genes present in liver. Fold changes of Pdx1-GR and Pdx1-VP16-GR were calculated relative to their respective controls.

Gene Name and Symbol Pdx1-GR* Pdx1-VP16-GR* solute carrier family 5, member 5 (SLC5A5) -5.00 -5.92 transmembrane 4 L six family member 1 -3.62 -3.97 (TM4SF1) ATPase, H+/K+ exchanging, beta polypeptide -3.55 -4.62 (ATP4B) aquaporin 8 (AQP8) -3.22 -3.85 meprin A, beta (MEP1B) -3.04 -2.70 ATPase, H+/K+ exchanging, alpha polypeptide -3.03 -4.26 (ATP4A) keratin 8 (KRT8) -2.86 -2.89 carnosine dipeptidase 1 (CNDP1) -2.71 -2.37 keratin 18 (KRT18) -2.54 -2.73 N-acetylated alpha-linked acidic dipeptidase- -2.54 -1.88 like 1 (NAALADL1) carbonic anhydrase II (CA2) -2.46 -2.68 lectin, galactoside-binding, soluble, 4 (LGALS4) -2.37 -2.92 cytochrome P450, family 51, subfamily A, -2.36 -1.67 polypeptide 1 (CYP51A1) tetraspanin 8 (TSPAN8) -2.36 -2.62 meprin A, alpha (MEP1A) -2.20 -2.52 chromosome 9 open reading frame 9 (C9orf9) -2.18 -3.29 tektin 2 (TEKT2) -2.15 -2.63 arylsulfatase A (ARSA) -2.13 -2.33

57 lactase (LCT) -2.13 -2.25 family with sequence similarity 154, member -2.06 -2.71 B (FAM154B) solute carrier family 7, member 9 (SLC7A9) -2.04 -1.97 secreted frizzled-related protein 2 (SFRP2) -2.01 -2.20 peroxisomal D3,D2-enoyl-CoA isomerase -1.93 -2.14 (PECI) tubulin polymerization-promoting protein -1.93 -2.64 family member 3 (TPPP3) EF-hand domain containing 1 (EFHC1) -1.87 -2.92 chromosome 14 open reading frame 45 -1.84 -2.19 (C14orf45) outer dense fiber of sperm tails 3B (ODF3B) -1.82 -2.77 3-oxoacid CoA transferase 1 (OXCT1) -1.81 -1.91 WD repeat domain 16 (WDR16) -1.78 -2.37 UDP-GlcNAc:betaGal beta-1,3-N- -1.70 -1.83 acetylglucosaminyltransferase 7 (B3GNT7) chromosome 15 open reading frame 26 -1.69 -2.15 (C15orf26) xanthine dehydrogenase (XDH) -1.69 -2.01 solute carrier family 22, member 2 (SLC22A2) -1.67 -1.65 mucin 2 (MUC2) -1.66 -1.82 tescalcin (TESC) -1.63 -2.05 interleukin 17B (IL17B) -1.59 -1.99 superoxide dismutase 2 (SOD2) -1.57 -1.80 paired box 6 (PAX6) -1.56 -1.67 somatostatin (SST) -1.54 -2.20 dynein, axonemal, light intermediate chain 1 -1.53 -2.24 (DNALI1)

KIAA0664 -1.53 -1.52 solute carrier family 5 member 1 (SLC5A1) -1.53 -1.76 meiosis expressed gene 1 (MEIG1) -1.52 -2.37 radial spoke 3 homolog (RSPH3) -1.52 -2.13 progestin and adipoQ receptor family member -1.50 -1.70 V (PAQR5) * log base 2 fold change relative to controls

Table 6: Downregulated microarray genes present in stomach/duodenum. Fold changes of Pdx1-GR and Pdx1-VP16-GR were calculated relative to their respective controls.

58 Upregulated Genes were Common between Treatment Groups

The analysis of upregulated microarray genes would help better identify the nature of the ectopic tissue. As was observed with the downregulated genes, the majority of the upregulated genes were common between Pdx1-GR and Pdx1-

VP16-GR (Figure 10). 89% of genes upregulated in Pdx1-GR were also upregulated in Pdx1-VP16-GR. 82% of genes upregulated in Pdx1-VP16-GR were also upregulated in Pdx1-GR. Once again it is not surprising as the phenotype between these two groups is very similar. Therefore, we focused our study specifically on upregulated genes common between both treatment groups.

Pdx1-GR 43 355 77 Pdx1-VP16-GR

Figure 10: Venn diagram displaying upregulated microarray genes. Genes having a P-value less than 0.05 and a fold change greater than 1.5 were considered. 43 genes were specifically upregulated in Pdx1-GR overexpression. 77 genes were specifically upregulated in Pdx1-VP16-GR overexpression. 355 genes were commonly upregulated between both groups.

Validation of Microarray by RT-PCR and In Situ Hybridization

To validate the data from the microarray, we assessed the expression of genes that were commonly upregulated. We verified the expression of 17 genes by RT-PCR that were shown to be differentially expressed by the microarray analysis. As shown in Figure 11, 14 genes showed significant expressional differences between treatment and controls for both Pdx1-GR and Pdx1-VP16-GR

59 (COL2A1, MMP13, IGFBP2, COL9A1, HAS2, IRX1, PAX9, SILV, EDNRA,

TSPAN5, TGFB2, TIMP1, CTGF, and FABP6). Two genes were differentially expressed only in the Pdx1-VP16-GR group (NCAM1 and CRABP2). Only

CYBA was not observed to be upregulated in either group by RT-PCR. The results obtained demonstrate the soundness of the microarray analysis. The validation of downregulated genes by RT-PCR was not performed as some genes were previously confirmed to be reduced in the ectopic tissue by in situ hybridization.

Gene Name and Symbol Pdx1-GR* Pdx1-VP16-GR* collagen, type II, alpha 1 (COL2A1) 5.52 6.23 matrix metallopeptidase 13 (MMP13) 3.04 3.24 insulin-like growth factor binding protein 2 3.58 3.44 (IGFBP2) collagen, type IX, alpha 1 (COL9A1) 3.87 4.54 hyaluronan synthase 2 (HAS2) 3.69 4.33 iroquois related homeobox (IRX1) 3.00 3.48 paired box 9 (PAX9) 3.72 4.36 silver homolog (SILV) 2.96 2.79 endothelin receptor type A (EDNRA) 1.85 2.08 tetraspanin 5 (TSPAN5) 3.08 2.67 transforming growth factor, beta 2 (TGFB2) 2.57 3.07 tissue inhibitor of metalloproteinase 1 (TIMP1) 3.86 3.95 connective tissue growth factor (CTGF) 2.07 2.20 fatty acid binding protein 6 (FABP6) 5.10 5.21 neural cell adhesion molecule 1 (NCAM1) 2.27 3.24 cellular retinoic acid binding protein 2 2.62 3.39 (CRABP2) cytochrome b-245, alpha polypeptide (CYBA) 2.82 2.02 * log base 2 fold change relative to controls

Figure 11: Validation of microarray results by RT-PCR. (A) Table showing fold changes of upregulated microarray genes. Fold changes of Pdx1-GR and Pdx1-VP16-GR were calculated relative to their respective controls. (B) RT-PCR of upregulated microarray genes. Majority of the genes showed differential expression between treatment and controls. The RT-PCR was normalized to elongation factor 1 alpha (EF1A).

To further validate the microarray analysis, we verified the spatial expression of some differentially upregulated genes confirmed by RT-PCR. This was achieved by performing whole-mount in situ hybridization on isolated wild- type whole guts (Figure 12). COL2A1 was expressed throughout the duodenum

61 and stomach regions as well as in the gall bladder and common bile duct (Figure

12A). No expression was visible in the pancreas. A similar expression pattern was also observed with COL9A1 (Figure 12B). CRABP2 was expressed solely in pancreatic tissue (Figure 12C). A punctate expression pattern was visible for

CTGF in the stomach and duodenum (Figure 12D). CYBA was expressed throughout the foregut organs (Figure 12E). Its expression was observed in the liver, stomach, duodenum and pancreas. FABP6 was seen in the liver, posterior duodenum and distal intestine (Figure 12F). HAS2 was localized throughout the stomach, duodenum and pancreas (Figure 12G). IRX1 was strongly observed in the posterior intestine, however weak staining was seen in the stomach, duodenum and pancreatic regions (Figure 12H). Staining of TGFB2 was observed mainly throughout the distal duodenum and intestinal regions (Figure 12I). A punctate pattern of TIMP1 was noted throughout the stomach, duodenum, pancreas and liver (Figure 12J). A punctate NCAM1 pattern was also observed throughout the stomach and duodenum (Figure 12K). From both validation experiments, we confirmed that the upregulated genes were in fact differentially expressed in the microarray analysis and localized to the foregut region as expected.

Figure 12: Validation of microarray results by in situ hybridization. (A) COL2A1 expressed in stomach, duodenum, gall bladder and bile duct. (B) COL9A1 has similar expression pattern as COL2A1. (C) CRABP2 expressed in pancreas. (D) CTGF punctate expression in stomach and duodenum. (E) CYBA punctate expression in stomach, duodenum, liver and pancreas. (F) FABP6 expressed in the liver, duodenum and distal intestine. (G) HAS2 expressed throughout stomach, duodenum and pancreas. (H) IRX1 expressed in stomach, duodenum, pancreas and distal intestine. (I) TGFB2 expressed in distal duodenum and intestine. (J) TIMP1 punctate expression in stomach, duodenum, liver and pancreas. (K) NCAM1 punctate expression in stomach and duodenum.

Functional Categories of Differentially Expressed Genes

To obtain an understanding of gene function and thus a perspective into the nature of the ectopic tissue, we utilized GeneCodis 2.0 to analyze gene annotations for the 355 commonly upregulated genes present in the microarray analysis (Carmona-Saez et al., 2007; Nogales-Cadenas et al., 2009). Gene

Ontology (GO) terms were assigned to genes with statistically significant

63 representation (P-value<0.05). P-values comparing the upregulated genes to all genes present in the microarray were calculated using hypergeometric analysis corrected by false discovery rate. The significantly over-represented GO categories were obtained and organized into Table 7.

Many upregulated genes were seen to have myogenic and contractile properties. These included myogenic transcription factor 6 (MYF6) and the various myosin light and heavy chains (MYLs and MYHs). Various factors have been identified to promote angiogenesis including endothelin receptor A

(EDNRA). Response to tissue damage had also been assigned to a number of differentially expressed genes. The expression of toll-like receptor 5 (TLR5), interleukin 1β (IL1B) and interleukin 8 (IL8) all play a role in immune function.

Other genes displayed properties of ion transport and cell migration. Interestingly, many genes were observed to play a role in extracellular matrix organization.

Extracellular matrix proteins including matrix metalloproteinases (MMPs) and collagens (COLs) were highly expressed in the microarray analysis. Various genes were characterized to play a role in cellular proliferation, cell death and cell communication. Cell-cell adhesion was also observed to be a functional GO category. Claudins (CLDNs) and neural cell adhesion molecule 1 (NCAM1) were all categorized to this GO term. The expression of various growth factor binding genes has been observed including insulin-like growth factor binding protein 2

(IGFBP2) and connective tissue growth factor (CTGF). Forty transcription factors were seen to be overexpressed in the microarray; all of which exhibit various developmental roles.

64 With GeneCodis 2.0, we were able to identify key molecular interaction networks using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The molecular pathways that were significantly over-represented included the peroxisome proliferator-activated receptor (PPAR), transforming growth factor-beta (TGF-beta) and Wnt signaling pathways. Interestingly, genes that play a role in cancer were also categorized.

Gene P-Value GO Term Gene Symbols Number 42 1.74476e- GO:0003012 ACTA1,TNNT3,MYOM1,PGAM2,IL1B,MYBPC 34 :muscle 3,CYBA,CHRNG,MYL2,MYL7,MYBPH,CHRN system process A1,TNNI1,MYH8,ACTN2,TPM1,PTGS2,TPM2, CACNG1,TTN,CASQ2,TRIM72,CHRND,TNNI2, TNNC2,TNNI3,TCAP,EDNRA,SMPX,MYL6,M YL1,ATP2A1,ARG2,TNNT2,MYH7,MYOM2,M YH6,GJC1,KBTBD10,TNNC1,MYL4, MYF6

26 9.38875e- GO:0001568 LECT1,IL1B,FN1,TGFB2,HOXA3,NPPB,SERPI 14 :blood vessel NE1,SHH,TDGF1,LAMA4,MMP2,CDH2,LOX,T development NNI3,TNFRSF12A,FOXC2,FOXC1,EDNRA,IL8, CSRP3,MEOX2,JUNB,CTGF,NOX1,GJC1,RUN X1

32 2.03777e- GO:0009611 AOAH,IGFBP1,TLR5,IL1B,MYF6,CYBA,FN1,G 11 :response to RHL3,TGFB2,FCN2,PTAFR,SERPINE1,TPM1,P wounding TGS2,ADIPOQ,SHH,FABP4,VCAN,A2M,TNFA IP6,TRIM72,LOX,PTX3,EDNRA,IL8,SCNN1B,B MP2,CTGF,SLC1A3,CHIA,ANXA1,NOX1

36 4.06918e- GO:0006811 UCP2,KCNE3,CHRNG,STEAP4,ATP6V1A,ATP 10 :ion transport 6V1G3,SLC26A6,CHRNA1,KCTD15,SLC26A4, RHCG,RHBG,PTGS2,ATP6V0A4,CACNG1,CLI C3,SLC22A8,SLC16A3,CHRND,SLCO2B1,NIP AL4,SLC5A8,SLC25A12,EDNRA,SCNN1B,ATP 1B2,SLC25A4,SLC34A2,ATP2A1,ATP6V1B1,A TP6V0D1,SLC5A2,SLC1A3,NOX1,ATP12A,SL C12A1

24 3.05706e- GO:0016477 IL1B,FOXE1,LAMA2,FN1,TGFB2,SERPINE1,T 09 :cell migration PM1,SHH,TDGF1,LAMA4,TBX5,VCAN,NKX2. 1,CDH2,TNFRSF12A,MMP9,FOXC1,IL8,SCNN 1B,SERPINE2,CTGF,NOX1,SLIT3,TWIST1

65 28 3.47634e- GO:0030198 EGFL6,COMP,CCDC80, COL9A1, COL9A2, 08 :extracellular MMP7,COL9A3, COL11A1, COL21A1, matrix CTGF,VCAN, DCN, EPYC, FN1, FBLN1, organization HAPLN3, TIMP1,LAMA2, LAMA4, MMP13, MMP2, COL2A1, MMP3, MMP9, POSTN, UCMA, WNT7B, MMP1

29 9.40203e- GO:0042127 IL1B,TIMP1,MMP7,TGFB2,HOXA3,EMP3,PTN, 07 :regulation of BMP7,SERPINE1,MSX2,PTGS2,SHH,TDGF1,F cell ABP4,DLX6,TBX5,FABP6,PRTN3,MYCN,EDN proliferation RA,IL8,NFKBIA,BMP2,MAB21L1,ANXA1,NO X1,ODC1,SOX9,RARG

33 7.54843e- GO:0012501 IL1B,MPO,TGFB2,SOCS3,BMP7,MSX2,ACTN2 06 :programmed ,PTGS2,ADIPOQ,SHH,TDGF1,CAT,TBX5,TNF cell death RSF11B,DDIT4,PIM1,TNFAIP3,TNFRSF12A,SL C5A8,FOXC2,COL2A1,MMP9,FOXC1,NFKBIA, DNASE1L3,COMP,ATP2A1,TNFAIP8,ANXA1, TWIST1,SOX9,HSPB1,RARG

14 4.70848e- GO:0016337 CLDN6,IL1B,CLDN1,ADIPOQ,CLDN5,CDH2,C 05 :cell-cell OL2A1,CLDN4,FAT4,NCAM1,CDH11,CTGF,A adhesion LX1,SOX9

29 0.0003043 GO:0010646 LECT1,FSTL3,TLR5,IL1B,TGFB2,SOCS3,BMP7 94 :regulation of ,RGS10,IGFBP2,PTGS2,ADIPOQ,TDGF1,TTN,C cell AT,NKX2.1,DKK1,CALB1,CDH2,DDIT4,TNFA communicatio IP3,DKK2,NFKBIA,GSC,NCAM1,BMP2,HTRA n 1,SLC1A3,SOCS1,SLIT3

8 0.0004937 GO:0019838 IGFBP1,IGFBP2,NTRK2,A2M,COL2A1,CTGF,H 46 :growth factor TRA1,LTBP3 binding

40 0.043354 GO:0003677 GCM1,MYOG,SMYD1,IRX2,DLX2,MYF6,PAX :DNA binding 9,FOXE1,GRHL3,HOXA3,HOXD4,IRX1,TFAP2 A,MSX2,FOXN2,FOXI1,DLX6,TBX5,NKX2.1,T NFAIP3,MYCN,FOXC2,FOXC1,SOX3,CEBPD, DNASE1L3,TBX1,GSC,MEOX2,LHX8,JUNB,L MO4,ALX1,VENTX,RUNX1,TRIM29, TWIST1,SOX9,HEY1,RARG

16 4.43849e- (KEGG) PTGS2,BMP2,NFKBIA,FN1,MMP2,TGFB2,FZD 05 05200 7,RUNX1,SHH,LAMA4,MMP1,WNT7B,SLC2A :Pathways in 1,IL8,LAMA2,MMP9 cancer 7 0.0002066 (KEGG) ADIPOQ,SCD,MMP1,FABP6,PCK1,ACSL6,FAB 43 03320 :PPAR P4 signaling pathway 6 0.0039941 (KEGG) BMP2,DCN,TGFB2,BMP7,THBS3,COMP 5 04350 :TGF- beta signaling pathway

66 6 0.0330452 (KEGG) MMP7,FZD7,WNT7B,DKK1,DKK2,WIF1 04310 :Wnt signaling pathway

Table 7: Functional categories of upregulated microarray genes. The commonly upregulated Pdx1-GR and Pdx1-VP16-GR microarray genes were categorized into GO categories using GeneCodis 2.0. P-values were calculated using hypergeometric analysis.

Microarray Suggests Stellate Cell Formation

The analysis using GeneCodis 2.0 permitted the functional categorization of the upregulated microarray genes. The above GO categories are properties highly expressed by stellate cells. This cell type resides in the acinar pancreas and plays an important role in the tissue repair process. Based on this categorization, we hypothesized that the ectopic tissue obtained in our overexpression model, may in fact be pancreatic stellate cells. We decided to look at a few known stellate cell markers to see if they are expressed in the ectopic tissue. Using RT-PCR, we observed a differential increase in endothelin receptor A (ENDRA) and platelet derived growth factor receptor (PDGFR) in both Pdx1 overexpression groups

(Figure 13). Nestin (NES), neural cell adhesion molecule 1 (NCAM1) and synaptophysin (SYP) were differentially upregulated only in Pdx1-VP16-GR. No differences were observed with alpha smooth muscle actin (SMA), collagen type

1 alpha 1 (COL1A1), desmin (DES), vimentin (VIM) and fibronectin (FN).

Figure 13: Ectopic tissue expressed stellate cell markers. RT-PCR was performed on various stellate cell markers. ENDRA, PDGFR, NES, NCAM1 and SYP show differential gene expression between treatment and controls. RT-PCR was normalized to elongation factor 1 alpha (EF1A).

We decided to test whether a stellate cell marker would stain the ectopic tissue using whole-mount in situ hybridization. Using NCAM1, we observed a punctate pattern present throughout the stomach and duodenum in the no dex control groups (Figure 14A,C). In the overexpression study, we clearly observed a significant increase in NCAM1 throughout the ectopic tissue (Figure 14B,D).

Based on our results, we believe that Pdx1 overexpression in the early endoderm results in ectopic pancreatic tissue displaying a stellate cell character.

Figure 14: Ectopic tissue formation marked for stellate cells. (A,C) In control groups, NCAM1 is expressed in a punctate expression pattern throughout the stomach and duodenum. (B) NCAM1 is expressed throughout the ectopic tissue when Pdx1-GR is overexpressed (n=5). (D) NCAM1 is also expressed throughout the ectopic tissue in Pdx1-VP16-GR (n=8).

69 Chapter 4: Discussion

Numerous studies have addressed the sufficiency of Pdx1 in promoting ectopic pancreas formation in the liver. However, it is unclear whether ectopic pancreatic tissue formation can arise from the early (naïve) endodermal overexpression of Pdx1. In this study, we report that the overexpression of Pdx1 has resulted in ectopic tissue formation displaying a reduction in pancreatic exocrine and endocrine differentiation. Stomach, duodenum and liver organogenesis was also severely perturbed in our overexpression model. To obtain a better understanding of the identity of the ectopic tissue, we carried out a microarray analysis which confirmed the reduction in pancreatic endocrine, exocrine cells as well as the reduction of stomach, duodenum and liver tissue.

The analysis of upregulated ectopic tissue genes displayed characteristics to those of pancreatic stellate cells. Therefore, the results obtained in our analysis, suggest that the early endodermal overexpression of Pdx1 is sufficient to induce the formation of pancreatic stellate cells. The lack of exocrine and endocrine formation suggests that Pdx1 alone is not adequate to induce further cytodifferentiation.

Early Pdx1 Overexpression Caused Gastrulation Defects

To ascertain the sufficiency of Pdx1 in promoting ectopic pancreatic fates in the naïve endoderm, we injected Pdx1 and Pdx1-VP16 mRNA into the dorsal vegetal blastomeres of eight-cell stage Xenopus embryos. The early

70 overexpression resulted in gastrulation defects. The blastopore would not develop normally and tadpoles appeared to have a shortening of the anterior-posterior axis.

Gastrulation and elongation of the body axis is the result of morphogenetic processes known as convergent extension. This is the result of the involution of marginal zone cells into the blastopore lip. These cells then converge along the mediolateral axis and extend along the anterior-posterior axis of the embryo.

Elongation of the body axis continues until tadpole stages. The non-canonical

Wnt/planar cell polarity pathway is the cellular signal required for convergent extension processes to take place (Keller et al., 2008; Tada and Kai, 2009). We hypothesize that this signaling pathway which controls cell polarity and movement is disrupted with early misexpression of Pdx1. Since Pdx1 is a homeobox protein, it will bind to target gene promoters as complexes with other cofactors and coactivators. At such early stages in development, Pdx1 may be interacting inappropriately with certain partners and causing the observed gastrulation defects.

Reduced Foregut Differentiation Markers

To overcome the gastrulation defects, we generated a hormonally derived variant of Pdx1 by fusing Pdx1 and Pdx1-VP16 to the ligand binding domain of the human glucocorticoid receptor. This hormone inducible system provided a useful means to temporally regulate Pdx1 overexpression in the naïve endoderm.

The fusion constructs were posttranslationally activated towards the end of gastrulation, by incubating the embryos in media containing dex. RNA injected embryos not incubated with dex were wild-type in nature and used as controls.

71 The inactivity of Pdx1 in absence of dex is due to the association of the ligand binding domain to heatshock proteins (Pratt and Toft, 2003). Using this conditional experiment, we obtained ectopic tissue arising in the foregut region that displayed a reduction in anterior markers.

The absence of endocrine and exocrine differentiation within the ectopic tissue is likely due to the limited amount of appropriate Pdx1 binding partners present at such early stages in endoderm development. Pdx1 requires the cooperation of protein partners in order to magnify its transcriptional activity. The trimeric complex Pdx1-Pbx1b-Meis2b with the cooperation of Ptf1a, synergistically activate the elastase gene (Liu et al., 2001). Moreover, the binding of Pdx1 to the insulin enhancer, results in the recruitment of E47/Pan1,

BETA2/NeuroD1 and high-mobility group protein I. This cooperative binding is essential for transcription of downstream Pdx1 targets (Ohneda et al., 2000).

Therefore, it is hypothesized that the function of Pdx1 in our overexpression is restricted due to the large quantities of Pdx1 present and insufficient amounts of cofactors needed for further endocrine and exocrine cytodifferentiation. This can be tested by co-expressing Pdx-GR1 mRNA with known binding partners.

The transdifferentiation process observed by overexpressing Pdx1 in the liver is very different from what is described in our study. In our report, Pdx1 was overexpressed in multipotent embryonic progenitors while liver to pancreas conversion experiments were carried out on already differentiated hepatocytes.

The fact that ectopic Pdx1 expression in the liver leads to the production of exocrine and endocrine cells but no differentiation in the naïve endoderm is observed, demonstrates the context dependency of Pdx1 function. By

72 overexpressing Pdx1 in the early endoderm, well before its endogenous expression, we are uncertain which binding partners aid in the formation of the ectopic tissue and can be very different from those present in the liver. In addition, at early stages in endodermal development, there is cross-talk between the various germ layers allowing for the regional specification of the endoderm

(Horb and Slack, 2001). Therefore, ectopic Pdx1 expression is affecting normal signaling pathways which are essential for the development of the endoderm and the foregut organs. On the other hand, the liver can be considered as an isolated system. The in vivo overexpression of Pdx1 in the liver would not affect surrounding organs and any interacting signaling pathways. These reasons may explain why Pdx1 can promote exocrine and endocrine differentiation in the liver but not in the naïve endoderm.

The loss of mature cellular differentiation accompanied by the decreased expression of lineage specific pancreatic transcription factors raises the possibility that the ectopic tissue is being maintained in a progenitor or undifferentiated state.

This is supported by the fact that Pdx1 itself is a progenitor marker giving rise to all pancreatic cell types (Gu et al., 2002). However, the transient activation of

Pdx1 from NF12-15 reproduced the ectopic phenotype similarly to the continual dex activation from NF12-44/45. The ability of ectopic Pdx1 to form this phenotype early in endoderm development, suggests that this ectopic tissue develops directly from the endoderm. In addition, the establishment of this tissue at the expense of acinar, endocrine, liver, stomach and duodenum formation demonstrates the change in endodermal competency brought by Pdx1. In other words, Pdx1 is changing the fate of the endoderm, such that cells programmed to

73 commit to pancreatic, liver, stomach and duodenum, are instead becoming this ectopic tissue. This also raises the question of the stage in which Pdx1 overexpression is causing this phenotype. The in ovo electroporation of Pdx1 in chick endoderm shortly after its endogenous expression, simply caused gut epithelium budding, but no exocrine and endocrine differentiation was observed

(Grapin-Botton et al., 2001). Similarly, Pdx1 was unable to induce the formation of ectopic tissue when overexpressed in the Xenopus endoderm at NF27 (Afelik et al., 2006). However, in both studies, Pdx1 was overexpressed in the endoderm shortly after endogenous expression. We also observed no ectopic tissue formation when Pdx1 was overexpressed past NF20 (data not shown). However, unlike the previous studies, we showed that by overexpressing Pdx1 earlier in development, it did result in ectopic tissue formation. Therefore, the effects of

Pdx1 overexpression are temporally regulated. More detailed analysis can reveal precisely the stage at which Pdx1 is having an effect.

Microarray Analysis on Ectopic Tissue Formation

To define the character of the ectopic tissue, we performed a microarray analysis comparing ectopic tissue to control foreguts. The microarray analysis revealed that the vast majority of downregulated genes were common between

Pdx1-GR and Pdx1-VP16-GR. This was due to the similarity in phenotype present between both treatment groups. In addition, we revealed that these genes were in fact present in the acinar and endocrine pancreas, as well as in the stomach, duodenum and liver. In these tissue categories, we recorded the fold change of

74 each gene’s expression level. We observed that in almost all cases, the fold change was greater in the Pdx1-VP16-GR group. When analyzing the isolated guts in our overexpression model, the quantity of ectopic tissue formation was noted to be greater with the addition of the VP16 domain to Pdx1. Thus, the phenotype observed was slightly more severe. As a result, it was not surprising to see a greater fold difference in expression level with the added VP16 domain. The larger difference in expression maybe due to the heightened transcriptional activity brought about by the interaction of the VP16 domain with RNA polymerase complexes (Hall and Struhl, 2002).

Similar to the analysis of downregulated genes we also observed that the majority of upregulated genes were common between both treatment groups. We verified the validity of the microarray analysis by RT-PCR and whole-mount in situ hybridization. The in situ on isolated wild-type tadpole guts showed the expression of those select genes to be in the foregut region. This is expected as we believed the ectopic tissue to be of foregut origin due to the known developmental function of Pdx1. However, the in situ results showing the localization of gene expression can be misleading. Certain genes, including COL2A1 and COL9A1 were found to be expressed solely in the stomach and duodenum. This infers that the ectopic tissue obtained is stomach and duodenum in nature. However, all our previous results proved otherwise. The reason for this discrepancy lies from the fact that the in situ validation experiments were performed on wild-type tissues. In wild-type conditions, one cell type in the stomach expresses COL2A1 while a pancreatic cell type may not. But once Pdx1 is overexpressed, the fate and

75 function of those cells will change. Therefore, the expression pattern of those genes will be affected as well.

GeneCodis 2.0 enabled us to better identify the ectopic tissue formation.

This was achieved by using Gene Ontology (GO) terms that are assigned to the commonly upregulated microarray genes. The grouping of genes with similar functions into the various GO categories enabled us to hypothesize that the tissue formation obtained in our overexpression model, mimics the characteristics of pancreatic stellate cells (PaSC).

In the normal pancreas, PaSC reside in a quiescent state in the periacinar space. In response to pancreatic injury, quiescent PaSC become “activated”, undergo proliferation, contraction and migrate to the site of tissue damage to help in the repair process. The GO terms that match these processes were significantly over-represented in the microarray. 29 genes were grouped in to the regulation of cell proliferation GO term. Bone morphogenic protein 7 (BMP7) has been shown to enhance stellate cell proliferation (Tacke et al., 2007). In the muscle system processes GO term, we observed upregulated genes that were shown to have myogenic and contractile properties. These included myogenic transcription factor

6 (MYF6) and the various myosin light and heavy chains (MYLs and MYHs).

These genes have been shown to have contractile properties in stellate cells

(Walker et al., 2001). The GO category of cell migration encompasses 24 genes.

N-Cadherin (CHD2) was shown to play an important role in enhancing invasion and migration of stellate cells into epithelial cancers (De Wever et al., 2004).

Response to tissue damage/ wounding has also been assigned a GO term to a number of differentially expressed genes and is a characteristic of PaSC. The

76 expression of toll-like receptor 5 (TLR5) has been shown to be expressed by pancreatic stellate cells (Masamune et al., 2008). In addition to their role in immune function, interleukin 1β (IL1B) and interleukin 8 (IL8) have been shown to activate PaSC. These stellate cells also secrete these factors in an autocrine manner to further potentiate their activity (Andoh et al., 2000; Aoki et al., 2006).

The GO category of extracellular matrix organization encompasses 28 genes from the microarray analysis. Activated PaSC have been shown to secrete extracellular matrix components (collagens and fibronectin) as well as extracellular matrix degradation enzymes: matrix metallopeptidases (MMP2,

MMP9 and MMP13) and their tissue inhibitor (TIMP1) (Phillips et al., 2003).

These genes facilitate extracellular matrix remodeling and fibrosis which are essential aspects of the repair process. Once the repair process is completed, PaSC undergo apoptosis (Klonowski-Stumpe et al., 2002). We observed a number of genes categorized into the programmed cell death GO term.

The expression of growth factors and their binding proteins has been well studied in stellate cells, specifically in the hepatic counterpart. However, due to the transcriptional similarities between pancreatic and hepatic stellate cells (HSC)

(99.9% similar), inferences can be made about genes expressed in PaSC based on hepatic studies (Buchholz et al., 2005). Upon activation, HSC secrete insulin growth factor 1 (IGF1) which potentiates proliferation and deposition of extracellular matrix proteins. In addition, IGF1 modulates the production of insulin growth factor binding proteins (IGFBP) which in turn affects local IGF1 secretion (Gentilini et al., 1998). In our study we revealed a number of growth factor binding proteins, most notably IGFBP1 and IGFBP2.

77 Forty genes upregulated have been characterized as having transcriptional activity according to the GO analysis. Some of these genes were discovered to be expressed in both quiescent and activated stellate cells. HSC express adipogenic factors CCAAT/enhancer binding proteins (CEBP). Expression levels decline when the HSC become activated. Overexpression of CEBP inhibits HSC activation (Huang et al., 2004). Similar results were observed in PaSC (Kim et al.,

2010). We observed an increase in CEBP-δ expression in our analysis. Activating protein 1 (AP1) is upregulated in activated stellate cells. This transcription factor controls the expression of fibrogenic factor TIMP1 (Bertrand-Philippe et al.,

2004). The ectopic tissue displayed an increase in an AP1 member protein JUNB.

Runt related transcription factor 1 (RUNX1) is also upregulated in the microarray and has been shown to regulate TIMP1 promoter activity through the cooperation with AP1 proteins (Bertrand-Philippe et al., 2004). The upregulation of sex determining region Y box 9 (SOX9) leads to overexpression in the ectopic tissue.

The SOX9 transcription factor is highly upregulated in activated stellate cells and is capable of regulated extracellular matrix deposition by affecting collagen gene expression (Bell et al., 1997; Hanley et al., 2008).

The transcription factors mentioned above are involved in mediating the activation of stellate cells from a quiescent to active state in vitro. Since we do not know the embryonic origins of PaSC, we are uncertain which factors are involved in the developmental process. Therefore, certain transcription factors present in our microarray may be essential for the normal development of this cell type. This can be demonstrated by understanding what role they may play in endoderm development. In addition, there is high probability that some of these transcription

78 factors are Pdx1 targets. This will help us elucidate the transcriptional regulatory network required for pancreatic tissue to form from the endoderm.

GeneCodis 2.0 permitted the identification of key molecular interaction networks using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The analysis revealed significantly over-represented KEGG categories that are highly expressed in stellate cells. The activation of the peroxisome proliferator activated receptor (PPAR) pathway maintains PaSC in a quiescent state by inhibiting proliferation and extracellular matrix deposition (Masamune et al., 2002). The TGFβ pathway plays an important role in PaSC function. The activation of TGFβ intracellular signaling increases PaSC activation, extracellular matrix synthesis, attenuates proliferation and reduces MMP3 and MMP9 expression (Shek et al., 2002). In addition to its role in various developmental processes including differentiation, cell polarity and proliferation, the Wnt signaling pathway has been implicated in regulating stellate cell function. The activation of this pathway maintains cells in an active, fibrogenic state (Jiang et al., 2006).

It was interesting to observe that the ectopic tissue displayed genes previously shown to be involved in various cancer related pathways. Ductal pancreatic adenocarcinoma is the most common form of pancreatic cancer with a

5 year survival rate of 5% (Jemal et al., 2010). There is accumulating evidence that a symbiotic relationship exists between PaSC and pancreatic adenocarcinoma cells. The interaction results in progression of the pancreatic cancer. Evidence for this relationship emerged when it was shown that conditioned media from pancreatic cancer cells stimulated the proliferation and extracellular matrix

79 production in PaSC. In addition, conditioned media from PaSC increased the rate of pancreatic tumor growth, migration, and its invasion (Bachem et al., 2005;

Hwang et al., 2008; Vonlaufen et al., 2008). These results were further supported by in vivo studies showing that the co-subcutaneous injections of PaSC and cancer cells in nude mice have a greater tumor size then if cancer cells we injected alone.

Thus, the PaSC are creating a microenvironment where the tumor cells may thrive

(Bachem et al., 2005).

Pdx1 has been implicated in pancreatic cancer. Human patients that underwent pancreaticoduodenectomy showed the re-expression of Pdx1 in pancreatic cancer cells (Liu et al., 2007). In addition, the exogenous overexpression of Pdx1 in pancreatic cancer cell lines resulted in an increase in tumor growth and invasion of these cancer cells (Liu et al., 2008). However, it is unclear whether Pdx1 overexpression directly results in pancreatic cancer or whether it is simply a secondary affect of the tumor itself.

The results obtained from the analysis of the ectopic tissue, suggests stellate cells form from the overexpression of Pdx1 in the naïve endoderm. If this is proven, then we may have provided an alternative model for the implication of

Pdx1 in pancreatic cancer cell development. In other words, the overexpression of

Pdx1 may lead to the proliferation of pancreatic stellate cells. In turn, the secreted factors from these cells potentiate cancer cell progression.

To confirm the analysis that the ectopic tissue displays stellate cell characteristics, we looked at a few known markers expressed by stellate cells.

Endothelin receptor A (ENDRA), platelet derived growth factor receptor

(PDGFR), nestin (NES), neural cell adhesion molecule 1 (NCAM1) and

80 synaptophysin (SYP), alpha smooth muscle actin (αSMA), collagen type 1 A1

(COL1A1), desmin (DES), vimentin (VIM) and fibronectin (FN) have all been shown to be expressed by stellate cells (Klonowski-Stumpe et al., 2003;

Jaskiewicz et al., 2003; Omary et al., 2007; Loo and Wu, 2008). Using RT-PCR, we observed differential expression in EDNRA, PDGFR, NES, NCAM1 and

SYP. We did not observe differential expression in the other markers.

Interestingly the markers that were not differentially expressed are known intermediate filament proteins. These intermediate proteins have limitations as they characterize a wide variety of cell types. In addition, stellate cells from different species express different intermediate filaments (Omary et al., 2007).

Using whole-mount in situ hybridization, we observed the expression of cell adhesion and communication protein NCAM1 throughout the ectopic tissue in both the overexpression of Pdx1-GR and Pdx1-VP16-GR. The staining of stellate cell marker NCAM1 within the ectopic tissue supports the possible stellate cell phenotype hypothesis. However, future histological experiments with various stellate cell markers will clearly confirm whether this is indeed the case.

If the ectopic PaSC hypothesis is disproven, then the alternative possibility to explain the resultant phenotype may lie in the manner at which the microarray dissections were originally performed. Since the overexpression of Pdx1 results in the formation of a large anterior ectopic tissue, during dissection, more anterior endodermal tissue is obtained compared to controls. By affecting normal anterior endoderm development, Pdx1 overexpression may also be affecting the development of more anterior organs including the lungs and heart. Therefore,

81 future studies need to address the effects of Pdx1 overexpression on more anterior endodermal tissues.

The question that becomes raised due to this study is why Pdx1 is promoting a single cell type when it is responsible for the formation of all pancreatic cells. The answer comes back to what was discussed earlier in the discussion: differences in the spatial and temporal overexpression of Pdx1 results in different outcomes. Misexpression in the liver causes endocrine and exocrine cell differentiation while in the early endoderm it does not. Pdx1 overexpression in the early endoderm results in ectopic tissue formation while later expression it does not. The timing and location also affects which binding partners are present.

This in turn will also affect the transcriptional activity of Pdx1 on target genes.

All of these factors affect Pdx1 function and therefore the cell type that is produced from the endoderm. The identification of different protein partners and

Pdx1 downstream targets will help elucidate how endodermal cells are being directed to this ectopic fate.

Conclusions and Future Direction

In conclusion, Pdx1 overexpression in the naïve endoderm is sufficient to induce the formation of ectopic tissue that is characteristic to that of pancreatic stellate cells. However, Pdx1 alone cannot cause the ectopic tissue to differentiate into the endocrine or exocrine cell lineages.

Future experiments confirming the stellate cell formation will be performed. Its confirmation will provide a better understanding of how PaSC

82 develop. This will also help elucidate the role of Pdx1 in pancreatic cancer progression. In addition, a small group of PaSC has been shown to display progenitor properties (Kordes et al., 2009). Therefore, there may also be novel therapeutic implication of our study to the treatment of diabetes.

The goal of our research is to understand how the pancreas develops from the endoderm. One way to achieve this is by elucidating the transcriptional regulatory network involved in pancreas organogenesis. This can be achieved by identifying Pdx1 targets in the naïve endoderm. Once we have ensured that the ectopic tissue formation is that of pancreatic stellate cells, we can apply this information to narrow our focus in selecting targets which play a role in PaSC formation. The information obtained from these future experiments will help us better understand pancreas organogenesis in hopes to improve the efficiency of current diabetic therapies and develop novel strategies.

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