UNIVERSITY OF CALIFORNIA, IRVINE Regulation of Regenerative Responses by Factors in the Extracellular Matrix during Axolotl (Ambystoma mexicanum) Limb Regeneration DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences by Anne Quy Phan Dissertation Committee: Professor David M. Gardiner, Chair Professor Susan V. Bryant Professor Marian L. Waterman Professor Thomas F. Schilling Associate Professor Rahul Warrior 2014 © 2014 Anne Quy Phan DEDICATION This dissertation is dedicated to my family. To my mother, Nu Iris Dinh, who fought vehemently against the notion that educating a female is not equivalent to pouring good wine into your best shoes. To my father, Bao Quy Phan who has instilled a value of intelligence, and plotted and worked to ensure I had the highest probability of developing intellect. To my sister, April Ai Han Phan, who learned at a very early age how to aspirate cancer cells, while other kids got to play outside. To my brothers, Andy Khai Phan and Dat Hy Phan, who always made it a challenge to keep it up academically. I am always proud to call you family. To the Phan clan who always accepted and supported their ‘mad scientist’ cousin. To the Dinh family, it is an honor. To the Frys and Hamils, thank you for welcoming me and being my family away from home. To Alexander Hamil, I would not be anywhere without you. ii TABLE OF CONTENTS LIST OF FIGURES .......................................................................................................... v LIST OF TABLES ........................................................................................................... vii ACKNOWLEDGEMENTS ............................................................................................. viii CURRICULUM VITAE .....................................................................................................ix ABSTRACT OF THE DISSERTATION ........................................................................... xii CHAPTER 1: Introduction ............................................................................................... 1 Regeneration ............................................................................................................... 2 Axolotl Limb Regeneration ........................................................................................... 5 Polar Coordinate Model of Positional Information ........................................................ 8 Accessory Limb Model ............................................................................................... 13 Heparan Sulfate Glycosaminoglycans ....................................................................... 15 FGF Signaling in Limb Regeneration ......................................................................... 19 Regenerative Capacity of Mammals .......................................................................... 21 Summary of Dissertation Aims .................................................................................. 23 CHAPTER 2: ................................................................................................................. 24 Heparan Sulfates Mediate Anterior/Posterior Positional Information by Position-Specific Growth Factor Regulation during Axolotl Limb Regeneration ........................................ 24 Abstract ..................................................................................................................... 25 Introduction ................................................................................................................ 26 Results ....................................................................................................................... 29 The Accessory Limb Model as an in vivo Gain-of-function Assay for Limb Regeneration ............................................................................................................. 29 Cell-free Extracellular Matrix (ECM) Derived from Different Positions around the Limb Circumference can both Inhibit and Enhance Ectopic Limb Regeneration ................ 33 Position-specific Information in the ECM is Dependent on the Presence of Heparan Sulfates ...................................................................................................................... 34 Heparan sulfate (HS) is Sufficient to Induce Pattern Formation in an Ectopic Blastema .................................................................................................................................. 35 The ECM can Function both as a Source and Sink of Growth Factors for Regulating Regeneration ............................................................................................................. 38 Position-specific Mediation of FGF Signaling Regulates Blastema Formation and Pattern Formation ...................................................................................................... 40 Heparan Sulfate Sulfotransferases are Differentially Expressed in Anterior and Posterior Blastema Cells during Regeneration .......................................................... 47 Differential Levels of Glycosaminoglycan Sulfation across the Anterior/Posterior Limb Axis ............................................................................................................................ 51 Discussion ................................................................................................................. 52 Materials and Methods .............................................................................................. 61 CHAPTER 3: ................................................................................................................. 68 Positional Information in Mammalian Extracellular Matrix ............................................. 68 Abstract ..................................................................................................................... 69 Introduction ................................................................................................................ 71 Results ....................................................................................................................... 73 iii Mouse ECM Induces Heparan Sulfate-dependent Pattern Formation in Axolotl Blastemas .................................................................................................................. 73 The Ability of Mouse Limb ECM to Affect Ectopic Axolotl Limb Regeneration is both Position-specific and Developmental Stage Dependent ............................................ 77 The Expression of Heparan Sulfate Sulfotransferases is Differentially Regulated in both Time and Space ................................................................................................ 79 Discussion ................................................................................................................. 81 Materials and Methods .............................................................................................. 85 CHAPTER 4: ................................................................................................................. 88 Extracellular Factors in Axolotl Limb Regeneration ....................................................... 88 Introduction ................................................................................................................ 89 Results and Discussion ............................................................................................. 90 A Limb from Scratch: Synthetic Induction of Axolotl Limb Regeneration by Defined Mammalian Factors ................................................................................................... 90 Expression of Cell-surface Heparan Sulfate Proteoglycans ...................................... 95 Chondroitin Sulfate Glycosaminoglycans ................................................................ 100 Materials and Methods ............................................................................................ 104 CHAPTER 5: Conclusions and Outlook ...................................................................... 108 Summary ................................................................................................................. 109 Novel Mechanism for Positional Information and Pattern Formation in Regeneration ................................................................................................................................ 110 Glycans in the Extracellular Matrix as Candidates for Mediating Positional Information ................................................................................................................................ 110 Positional Information is a Blueprint of Position-specific Growth Factor Regulation Embedded in the Extracellular Matrix ...................................................................... 112 Future Directions ..................................................................................................... 113 Why do Mammals have a Limited Ability to Regenerate? ........................................ 115 Implications for Cancer ............................................................................................ 117 Implications for Aging .............................................................................................. 118 REFERENCES ............................................................................................................ 121 ABBREVIATIONS