UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ Expression Microarray Analysis of Renal Development and Human Renal Disease A dissertation submitted to the Division of Graduate Studies and Research of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Program in Molecular and Developmental Biology of the College of Medicine 2006 by Kristopher Robert Schwab B.A., Blackburn College, 2001 Committee Chair: S. Steven Potter, Ph.D. Tom Doetschman, Ph.D. Chia-Yi Kuan, M.D., Ph.D. Dan Wiginton, Ph.D. James Wells, Ph.D. Abstract Renal morphogenesis involves the reciprocal inductive interactions between the ureteric bud and metanephric mesenchyme forming the collecting ducts and nephrons within adult kidney. We applied microarray technology to the study of renal morphogenesis in order to better understand the molecular mechanisms underlying development. Additionally, the techniques employed in the expression analysis of the embryonic kidney were extended to the study of renal disease. Embryonic kidneys representing different stages of renal development were analyzed using expression microarrays. Renal developmental analysis revealed many novel genes and genetic pathways involved in renal development. In addition, the normal renal development data provides a baseline for the analysis of gene targeted mice possessing disruptions in renal morphogenesis. Microarray analysis was also performed on the Hoxa11/Hoxd11 compound null renal defect throughout renal development. In conclusion, these microarray studies greatly advance our knowledge of gene expression within the normal renal morphogenesis and identify possible downstream candidate genes regulated by the Hox11 genes. Wnt signaling is crucial for normal renal morphogenesis. In Drosophila, the pygopus gene encodes a transcriptional co-activator required for canonical Wnt signaling. The targeted deletion of the mammalian orthologs of pygopus, Pygo1 and Pygo2, in mice was investigated in renal development. A disruption ii in ureteric number tip and morphology was identified in Pygo1/Pygo2 compound null kidneys. Additionally, canonical Wnt signaling as measure by the Bat-gal transgene is reduced within the ureteric compartment in Pygo1/Pygo2 null kidneys. Overall, these experiments suggest that Pygo function is required for activation of canonical Wnt signaling in the ureteric compartment of the developing kidney. Focal segmental glomerulosclerosis (FSGS) is characterized by the segmental scarring of the glomerulus, ultimately resulting loss of nephron function. To understand the molecular pathogenesis of this disease, gene expression analysis was performed on FSGS patient kidney biopsies and compared to normal kidney tissue. Hundreds of genes were identified significantly changed with in the FSGS patient groups. Furthermore, gene expression changes were identified in subsets of patients possessing different clinical manifestations of FSGS. In conclusion, the molecular analysis of gene expression in the FSGS kidney provides a better understanding of expression changes during renal disease. iii iv Acknowledgements First and foremost, I would like to thank my advisor S. Steven Potter. I greatly appreciate his enthusiasm for developmental biology and excellent guidance he has given me over the last few years. Also, these studies were greatly aided by the technical expertise of both Larry Patterson and Heather Hartman. I would also like to thank the members of my committee, Tom Doetschman, Alex Kuan, Dan Wiginton, and James Wells, for their assistance and guidance. Additionally, I would like to thank the past and present members of the Potter, Patterson, and Michael Bates laboratories for their friendship and help given to me over the years. Many thanks to the faculty, students, technicians, and staff of the Molecular and Developmental Biology program for constructing such an excellent environment for the study for developmental biology. Finally, a very special thanks goes to my wife, Jennifer, and the rest of our families for their love during this significant endeavor. I am grateful for your support. v Table of contents Abstract ii Acknowledgements v Table of contents 1 Chapter 1. General Introduction Overview 5 Mammalian kidney development 7 Role of Homeoxbox (Hox) genes in kidney development 15 Wnt signaling in renal development 21 Fsgs (Focal segmental glomerulosclerosis) 24 Significance of microarray analysis in renal development and 26 disease References 30 Figures 44 Chapter 2. A catalogue of gene expression in the developing kidney Abstract 51 Introduction 53 Materials and methods 57 Results 60 Discussion 77 Acknowledgements 79 References 80 Figures and tables 91 1 Chapter 3. Comprehensive microarray analysis of Hoxa11/Hoxd11 mutant kidney development Abstract 106 Introduction 107 Materials and methods 110 Results 114 Discussion 124 Appendix 134 Acknowledgements 135 References 136 Figures and tables 148 Chapter 4. Pygo1 and Pygo2 roles in Wnt signaling in mammalian kidney development Abstract 159 Introduction 160 Materials and methods 163 Results 169 Discussion 179 Acknowledgements 185 References 186 Figures and tables 193 2 Chapter 5. Microarray analysis of focal segmental glomerulosclerosis (FSGS) Abstract 210 Introduction 211 Materials and methods 213 Results 216 Discussion 229 Appendix 231 Acknowledgements 232 References 233 Figures and tables 239 Chapter 6. General Discussion Microarray expression analysis of kidney development 247 Microarray expression analysis of Hoxa11/Hoxd11 null mutants 249 Analysis of Pygo1/Pygo2 null mutants 252 Focal segmental glomerulosclerosis 254 References 257 3 Chapter 1 General Introduction 4 Overview How a single fertilized egg differentiates into the many diverse tissues and organ systems of an organism is a fundamental question of developmental biology. Embryogenesis relies on many different processes, such as cell proliferation, apoptosis, polarization, migration, and differentiation to occur within a specific spatio-temporal manner generating the basic body plan, limbs, and organs of the organism. A common theme in organogenesis is the requirement of specific instructive interactions between different tissue types; one example is the interaction of the epithelial tube with the surrounding unorganized mesenchyme in the developing metanephric kidney. The ablation of either the mesenchyme or epithelial component in this system results in a remarkable loss of developmental potential. Likewise, the disruption of specific molecular signals and cell-cell interactions between these tissues result in abnormal development. The formation of the developing kidney relies on many complex genetic and signaling pathways between the ureteric bud, an epithelial outgrowth from nephric duct which extends throughout the intermediate mesoderm, and the surrounding metanephric mesenchyme (Dressler, 2006). The ureteric bud must undergo many rounds of branching while inducing the surrounding metanephric mesenchyme to undergo a mesenchymal to epithelial transformation forming renal vesicles. These vesicles undergo nephrogenesis, a complex process of elongation and differentiation forming the nephron, the functional unit of the kidney. During these processes, the metanephric mesenchyme is also actively 5 regulating ureteric branching, while the ureteric bud tips regulate nephron induction. These reciprocal inductive interactions generate the 800,000 to 1,200,000 nephrons present in the adult human kidney functioning in the removal of metabolic wastes from the bloodstream. These developmental interactions of the metanephric kidney can easily be studied in metanephric kidney organ culture, recapitulating in vivo development and providing an excellent experimental system for further analysis (Grobstein, 1955; Grobstein, 1956). After development has completed successfully, the organ must fulfill its physiological role within the organism while properly responding to insults and stresses from the environment or intrinsic to organism, such as genetic and autoimmune diseases. The adult kidney filters nearly 200 liters of fluid a day from the bloodstream, concentrating waste products into a significantly smaller volume while reclaiming electrolytes, metabolites, and water. Many human renal diseases result from stresses, insults, or genetic defects within the glomeruli, the site of blood filtration. The glomerulus consists of podocytes with complex cellular processes surrounding a specialized endothelium allowing the selective passage of small molecules. Focal segmental glomerulosclerosis (FSGS) is an example of such a devastating condition, resulting in the ablation of podocytes and segmental scarring of the glomeruli causing loss of nephron function which eventually leads to renal failure. Although the etiology of a small percentage of FSGS cases is due to alleles containing mutations in podocyte-specific genes, the majority of FSGS cases are idiopathic. Due to the heterogeneity of FSGS cases, many clinicians have suggested that FSGS is a pathological diagnosis of 6 different, uncharacterized glomerular diseases resulting in similar lesions.