ABSTRACT CHARACTERIZATION OF THE MLR19 TRANSGENIC MOUSE LINE AND THE ROLE OF MYOCARDIN IN THE BLADDER By Kevin David Wright Obstructive uropathy is the leading cause of the end-stage renal failure in children, yet there remain few genetic animal models to study this disorder. The MLR19 megabladder mice are a transgene-induced insertional mutant mouse line that fails to develop the smooth muscle surrounding the urinary bladder, creating an obstructive uropathy. The aim of this project is to further characterize the MLR19 mutants to determine what gene is being affected to cause the phenotype. Complementation studies suggest that myocardin is the relevant gene in the MLR19 megabladder phenotype, even though the transgene insertion is 335kb upstream from myocardin. Morphometric analysis of MLR19 heterozygotes and myocardin heterozygotes suggests that even mild reductions in myocardin have a significant effect on thickness of the bladder smooth muscle. Together, these data support the conclusion that myocardin is a critical gene involved in the development of the urinary bladder smooth muscle. CHARACTERIZATION OF THE MLR19 TRANSGENIC MOUSE LINE AND THE ROLE OF MYOCARDIN IN THE BLADDER A Thesis Submitted to the Faculty of Miami University In partial fulfillment of The requirements for the degree of Master of Science Department of Zoology by Kevin David Wright Miami University Oxford, Ohio 2009 Advisor ________________________________ Dr. Michael L. Robinson Reader _________________________________ Dr. Joyce Fernandes Reader _________________________________ Dr. Paul Schaeffer Reader _________________________________ Dr. Yoshinori Tomoyasu TABLE OF CONTENTS TITLE PAGE i TABLE OF CONTENTS ii LIST OF FIGURES AND TABLES iii ACKNOWLEDGEMENTS iiii CHAPTERS 1. Introduction 1 2. Targeted knock-out Construct for 24kb region on chromosome 11 14 2.1. Introduction 14 2.2. Materials and Methods 15 2.3. Results and Discussion 18 3. Complementation study to determine myocardin’s role in MLR19 phenotype 32 3.1. Introduction 32 3.2. Materials and Methods 32 3.3. Results and Discussion 34 4. Morphometric analysis of urinary bladder smooth muscle 43 4.1. Introduction 43 4.2. Materials and Methods 43 4.3. Results and Discussion 44 6. Conclusions 50 APPENDIX 53 REFERENCES 55 ii LIST OF TABLES Tables page 2.1 Primers to amplify the 5’ and 3’ homology arm for knockout construct 22 3.1 Primers used to genotype the MLR19 and Myocardin mutants 37 3.2 Ureter lumen area of MLR19-/myocd- and wild type animals 38 4.1 Mean thickness of the bladder smooth muscle in WT, MLR19+/-, and Myocd+/- 47 iii LIST OF FIGURES Figures page 1.1 Urinary bladder development 11 1.2 WT and MLR19-/- bladders and map of MLR19 mutation 12 1.3 Map of myocardin cardiac and smooth muscle isoforms 13 2.1 Diagram showing location of the homology arm primers 23 2.2 Maps of plasmids PL451 and PL253 24 2.3 Restriction digests showing successful ligation of 5’ homology arm into pl451 25 2.4 Restriction digests showing successful ligation of 3’ homology arm into pl451+5’ arm 26 2.5 Restriction digests showing successful ligation of TK gene into pl451+homology arms 27 2.6 Sequences of ligation junctions in the completed knockout construct 29 2.7 Diagram summarizing Southern blot strategy for screening ES cells 31 3.1 Diagram of MLR19-/myocd- breeding plan with chromosome 11 39 3.2 PCR and gross histology of MLR19-/myocd- compound heterozygotes 40 3.3 Hemotoxylin and Eosin staining of MLR19-/Myocd- compound heterozygotes 41 3.4 Immuhistochemistry of smooth muscle actin in newborn urinary bladders 42 4.1 Comparison of wild type, MLR19+/-, Myocd+/- bladder smooth muscle thickness 48 4.2 Comparison in lumen area between wild type, MLR19+/-, and Myocd+/- animals 49 Appendix A. Myocardin western blot analysis 56 iv ACKNOWLEDGEMENTS I thank Dr. Michael Robinson for his guidance, patience and support throughout my work in his lab. I thank Dr. Joyce Fernandes, Dr. Paul Schaeffer, and Dr. Yoshinori Tomoyasu for sitting on my committee as well as for their valuable input on the project. I thank Brad Wagner for all his help in the lab, without him our lab would not function as well as it does. I would also like to thank other members of the Robinson lab for their companionship and insight into the project. Thanks for making the lab a great place to work. I thank Kristen Lucia and Dr. Robert Schaefer for their help running the statistics, and Dr. John Hawes for his help and insight with the protein analysis aspect of the project. I would like to thank everyone in the Department of Zoology, for creating a great work environment and providing assistance when needed. I would especially like to thank my parents, family and friends, without them I would not be the person I am today. Your constant support and love are truly appreciated. v CHAPTER 1. Introduction Obstructive uropathy Obstructive uropathy (OU) is any urinary tract abnormality caused by an obstruction at any level of the urinary tract (Roth et al. 2002, Becker and Baum 2006). There are many different disorders that cause obstructions, including: kidney stones, ureterocele, urethral valve blockages, and prune belly syndrome. If left untreated, these disorders often result in mild to severe kidney damage from hydronephrosis. Hydronephrosis is a condition caused by the buildup of hydrostatic pressure proximal to the obstruction. The urinary back pressure causes dilation of the ureters and ultimately dilation of the kidneys (hydronephrosis) (Peters 1995, Roth et al. 2002). In many of the severe conditions, the urinary tract is obstructed for long periods of time causing irreversible damage to the kidneys leading to end-stage renal disease (ESRD) a condition where the kidneys are unable to maintain organismal homeostasis. This leads many patients with ESRD to a lifelong dependency on dialysis or the need for renal transplant. The most severe cases can even lead to death. In children needing renal transplants, obstructive uropathies are the leading cause accounting for approximately 22% of children with renal insufficiency and leading to 1,538 transplants from 1987 to 2008 in North America alone (Benfield et al. 2003, Warady and Chadha 2006, Morris and Kilby 2008). For children with urinary tract obstructions, without intervention, surgical or otherwise, the condition can be lethal shortly after birth. Congenital uropathies are often due to structural deformities that lead to blockage during the development of the urinary system in the fetus. While the blockage can be surgically removed and normal urinary system function restored many children still develop ESRD. Because of this, there remains a need to understand what is happening to the renal and urinary systems at the cellular and biochemical level during and post obstruction to better understand kidney function and the potential long term implications of the blockage. Even at the state-of-the-art strategic planning workshop hosted by the American Society of Pediatric Nephrology and NIH, this need was listed as the first major aim for future research goals (Chevalier and Peters 2003). Animal Models to Study the Cause and Pathology of Obstructive Uropathies To date there are few spontaneous animal models where obstructive uropathies, that occur during renal development, can be studied (Peters 1997, Peters 2001, Bascands and 1 Shanstra 2005). One such model is the Congenital progressive hydronephrosis (cph) mouse strain that develops a progressive hydronephrosis (Horton et al. 1988) from overproduction of urine. However, the cph mice do not have an obstruction prior to the urine accumulation; it is the inability to void the large amounts of urine that produces the obstruction (McDill et al. 2006). Since, many of the cases of OU in children are not due to overproduction of urine, but by obstruction, the cph mice are an insufficient model to study the progression to ESRD due to obstruction. As an alternative to spontaneous (genetic) models of obstructive uropathy, researchers have created surgical models in which different portions of the urinary tract are artificially ligated to form an obstruction. This method has been performed in a wide array of animal models including sheep, pig, canine, and opossum (reviewed in Peters 2001). This method of creating urinary obstructions has several advantages, such as having the option to choose which level of the urinary tract to obstruct and the predictability in which the specimens develop pathological symptoms. Yet, with these advantages, there are also drawbacks - the surgeries are extremely invasive and some models, such as rabbits, are sensitive to the anesthesia and have a high mortality rate with surgery (Peters 2001). Also, larger animals that are more amendable to surgical obstruction are often extremely costly to keep and maintain. Sheep have been the favored animal model for induced obstructive uropathies for many years, as the size of the sheep, both adult and fetal, are convenient for surgery. This model has provided great insight into the effects of obstruction on the kidneys (reviewed in Peters 1997). However, one of the drawbacks of this model is that females only have one or two offspring each pregnancy, after a relatively lengthy gestation period, making sheep an inconvenient model for large scale studies using multiple fetal specimens. Disadvantages like this, and the time cost of performing the surgeries have led to a desire for genetic models of functional obstructive uropathies in smaller animal models such as mice. One of the other main advantages of using mice as a model system includes the wealth of genetic information and the ease of manipulating the mouse genome to create mutants that will help give a better understanding the genes/molecules required for normal urinary system function. Currently, there are very few genetic mouse models where the animals develop obstructive uropathies without having severe developmental defects that affect multiple organ systems.