DECONSTRUCTING INDEPENDENT EVOLUTION: A DEVELOPMENTAL APPROACH TO THE EVOLUTION OF ANIMAL CARTILAGE AND APPENDAGES By OSCAR ALEJANDRO TARAZONA REY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2015 © 2015 Oscar Alejandro Tarazona Rey To Francisca ACKNOWLEDGMENTS I thank Francisca Leal for her support and understanding during the development of this project. I want to thanks to my advisor Martin J. Cohn for everything he did to make me a better scientist, for his constant support, constructive criticism and for giving me the confidence to become a critical scientist. I also thank Barbara Battelle and the H. J. Brockmann and D. Julian labs (University of Florida) for Limulus eggs, Nadean Brown (Cincinnati Children's Research Foundation) for sharing protocols and reagents, GuangJun Zhang (Purdue University Vet School) for sharing data, and Monica Welten for assisting me with OPT scanning and 3D reconstructions. I thank HHMI International Student Research Fellow for giving me the independence to develop this project. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 2 MATERIALS AND METHODS ................................................................................ 16 Embryo Collection and Preparation ........................................................................ 16 Alcian Blue and Masson’s Trichrome Stain ............................................................. 16 Gene Cloning and RACE-PCR ............................................................................... 17 In situ Hybridization and Immunohistochemistry ..................................................... 18 In situ Hybridization and Phalloidin Staining ........................................................... 18 Optical Projection Tomography (OPT) of Collagen in situ Hybridization. ................ 19 Molecular Phylogenetic Analysis of Collagen and Sox Genes. ............................... 19 Treatments With Small Molecule Inhibitors ............................................................. 19 BrdU Labeling and BrdU Pulse Chase .................................................................... 20 Luciferase Assay .................................................................................................... 21 3 CARTILAGE DEVELOPMENT IN TWO PROTOSTOME LINEAGES .................... 26 Cartilage in Metazoans ........................................................................................... 26 Homology of Cartilage in Metazoans ...................................................................... 29 Origin of New Cell Types ........................................................................................ 29 Cell Identity and Gene Regulatory Networks .......................................................... 30 Histology and Molecular Nature of the ECM ........................................................... 31 Invertebrate Cartilage is Collagen Based and Contains Hyaluronan ...................... 32 Expression of Developmental Genes in Sepia chondrogenesis .............................. 34 Expression of Developmental Genes in Limulus chondrogenesis .......................... 35 Invertebrate SoxE proteins can activate vertebrate Col2a1 cartilage enhancer ...... 36 Late Chondrogenesis and Cartilage Appositional Growth....................................... 37 Hedgehog and β-catenin signaling pathways have antagonistic functions in Sepia chondrogenesis ......................................................................................... 38 Invertebrate and Vertebrate Chondrogenesis Share a Gene Regulatory Network . 39 Origin of Cartilage an the Chondrocyte in Bilateria ................................................. 40 Hypothesis 1 ........................................................................................................... 41 5 Hypothesis 2 ........................................................................................................... 41 Hypothesis 3 ........................................................................................................... 42 4 EVOLUTION OF CEPHALOPOD APPENDAGES .................................................. 61 Cephalopod Arms and Tentacles as Molluscan Evolutionary Novelties ................. 61 Deep Homology of Appendage Gene Regulatory Network ..................................... 62 Description of Limb Morphogenesis in Cuttlefishes ................................................ 64 Gene Expression and the Control of Axis of Growth in Cuttlefish Limb .................. 65 Anteroposterior Axis ......................................................................................... 65 Proximodistal Axis ............................................................................................ 66 Dorsoventral Axis ............................................................................................. 68 Developmental Basis of a Morphological Novelty ................................................... 69 A Conserved Appendage Developmental Program ................................................ 70 5 LIMB RE-EVOLUTION IN SNAKE-LIKE LIZARDS ................................................. 84 Phylogenetic Patterns of Limb Loss in Squamate Lizards ...................................... 84 Limb Loss and Re-evolution in Bachia .................................................................... 85 Limb Morphology of Bachia bicolor ......................................................................... 88 Embryonic Development of B. bicolor Hindlimbs .................................................... 89 B. bicolor Has an Autopod ...................................................................................... 89 Phylogenetic Patterns Hide Ontogenetic Processes ............................................... 91 6 CONCLUSION ...................................................................................................... 102 LIST OF REFERENCES ............................................................................................. 104 BIOGRAPHICAL SKETCH .......................................................................................... 114 6 LIST OF TABLES Table page 2-1 Sequence identifiers for the collagen phylogenetic analysis. .............................. 22 2-2 Sequence identifiers for the phylogenetic analysis of Sox genes. ...................... 23 2-3 Sequence identifiers for the phylogenetic analysis of HAS genes. ..................... 24 2-4 S. officinalis embryos used for each drug treatment and DMSO controls. .......... 25 7 LIST OF FIGURES Figure page 3-1 Animal phylogeny depicting the independent evolution of cartilage in the three major lineages of Bilateria ......................................................................... 44 3-2 Developmental series of chondrogenesis in Sepia and Limulus. ........................ 45 3-3 Molecular phylogenetic analysis of fibrillar collagen, Sox transcription factors (SoxC, SoxD, SoxE and SoxF) and Hyaluronan synthases in bilateria .............. 46 3-4 Protostome invertebrate cartilage is structurally similar to vertebrate cartilage, is ColA-based, and contains hyaluronan. ............................................ 48 3-5 ColAa and ColAb show the same pattern of gene expression in Sepia embryos. ............................................................................................................. 49 3-6 Deep conservation of gene expression, from induction of chondrogenesis to transcriptional regulation and secretion of a cartilage ECM, during protostome cartilage development ...................................................................... 50 3-7 Chondrogenesis of multiple cartilages occurs near Hedgehog-expressing tissues in Sepia .................................................................................................. 51 3-8 Patterns of gene expression in developing funnel cartilage of Sepia at stage 25 ....................................................................................................................... 53 3-9 Cuttlefish chondrogenesis is regulated positively by Hh signaling and negatively by β-catenin ....................................................................................... 54 3-10 Gill cartilage in Limulus is collagen-based and expresses SoxE during chondrogenesis .................................................................................................. 56 3-11 Luciferase reporter assay testing the human Col2a1 cartilage specific enhancer with vertebrate and invertebrate SoxE proteins .................................. 57 3-12 Cell proliferation during late chondrogenesis in Sepia ........................................ 58 3-13 Bright field micrographs