INSIGHTS INTO THE ROLE OF TGF-BETA SUPERFAMILY SIGNALING IN ANNELID DORSAL-VENTRAL AXIS FORMATION By ALEXIS R. LANZA 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 2019 © 2019 Alexis R. Lanza To Guillermina ‘Elena’ Wong ACKNOWLEDGMENTS I would first like to thank my PhD supervisor, Dr. Elaine Seaver, whose expertise and patience has helped me grow into a more confident and capable scientist. I thank all the members of my graduate committee for their helpful suggestions and support. I acknowledge the many inspirational post doctorial women at the Whitney Laboratory for Marine Bioscience for regularly providing technical advice and being strong rolemodels. I would particularly like to single out Dr. Danielle de Jong for her unwavering support in all things molecular-biology. I would like to thank my post-bachelor advisor, Dr. David Julian for giving me the opportunity to learn about academic research in his lab. Dave, without your guidance and the many wonderful people in your lab, I would not have embarked on this academic journey. I thank my mom, dad, and family at home in Belize for believing in me. I thank Helle and Binx for being there at the end of every long day. I thank, my best friend and partner, Sondre Skarsten, for being my emotional rock whether near or far. I give special thanks to my dear friend Andrea Murillo for cheering me on during the writing of my dissertation. Finally, I would like to thank my friends who have been an excellent support network, and contributed to making my entire grad school experience enjoyable. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 INTRODUCTION .................................................................................................... 14 Background ............................................................................................................. 14 Discovery of The Organizer .............................................................................. 14 Spiralia ............................................................................................................. 17 Standard Nomenclature of Unequal Spiral Cleavage ....................................... 17 A Brief History of Annelid Phylogenies ............................................................. 20 Investigation of Organizer Activity in Annelids .................................................. 21 Dissertation Objectives ........................................................................................... 22 2 AN ORGANIZING ROLE FOR THE TGF-Β SIGNALING PATHWAY IN AXES FORMATION OF THE ANNELID Capitella teleta ................................................... 25 Introduction ............................................................................................................. 25 Materials and Methods............................................................................................ 30 Animal Care ...................................................................................................... 30 Drug Treatments .............................................................................................. 30 Immunolabeling ................................................................................................ 31 Cloning of Ct-ADMP and Whole Mount In-situ Hybridization ............................ 32 Whole Mount In situ Hybridization .................................................................... 33 Lineage Tracing ................................................................................................ 34 Lucifer Yellow Microinjections .......................................................................... 34 Microscopy and Imaging................................................................................... 35 Statistical Analyses .......................................................................................... 35 Results .................................................................................................................... 36 Inhibition Experimental Design ......................................................................... 37 Activin/Nodal Inhibition ..................................................................................... 37 Axial Properties of C. teleta Larvae .................................................................. 38 Ct-ADMP Expression Is Predominantly Ventral ................................................ 43 Inhibition of TGF-beta Signaling In Early Embryos Leads to Abnormal Axial Markers ......................................................................................................... 44 Birth of Cell 2d and Its Daughter Cells 2d1 and 2d2 Are Not Affected by SB431542, Though Its Descendants Contribute to A Reduced Larval Trunk ............................................................................................................. 48 5 BMP Inhibition .................................................................................................. 50 Discussion .............................................................................................................. 55 ADMP as A Ventral Marker .............................................................................. 55 The Activin/Nodal Pathway Is Essential for Establishing the Dorsal-ventral Axis and Specification of Trunk Identity ........................................................ 55 Role of BMP in Patterning the Dorsal-ventral Axis in Other Spiralians ............. 60 BMP Signaling Is Not implicated in Organizing Activiy In C. teleta ................... 61 3 ACTIVIN/NODAL SIGNALING IS REQUIRED FOR ESTABLISHING THE DORSAL-VENTRAL AXIS IN Capitella teleta ......................................................... 72 Introduction ............................................................................................................. 72 Materials and Methods............................................................................................ 75 Animal Care and Embryo Acquisition ............................................................... 75 RNA-Seq Developmental Time-Course ............................................................ 75 Gene Knockdown with Morpholino Oligonucleotides ........................................ 76 cDNA and mRNA Synthesis ............................................................................. 77 Microinjections .................................................................................................. 78 Immunolabeling ................................................................................................ 79 Microscopy and Imaging................................................................................... 80 Statistical Analysis ............................................................................................ 80 Results .................................................................................................................... 80 Differential Gene Expression ............................................................................ 80 Morpholino Knockdown of Smad2/3 and Smad1/5/8 ........................................ 84 Standard-Control Morpholino ........................................................................... 86 Smad2/3 Knockdown ....................................................................................... 87 Rescue of Morpholino Phenotypes Using Smad2/3 mRNA .............................. 90 Recombinant Smad2/3 Detection .............................................................. 90 Phenotypic Rescue .................................................................................... 91 Statistical Analyses of Smad2/3 MO Phenotypes ............................................. 91 Smad1/5/8 Knockdown .................................................................................... 92 Statistical Analyses of Smad1/5/8 MO Phenotypes .......................................... 95 Discussion .............................................................................................................. 96 Differential Expression Data Hints at Promiscuous Ligand-Receptor Function ........................................................................................................ 96 Smads are Functional without an MH1 Domain ............................................... 98 Knockdown of Smad2/3 Confirms the Activin/Nodal Pathway is Essential in Dorsal-ventral Axis Patterning ....................................................................... 99 Smad1/5/8 is Not Essential for Dorsal-ventral Axis Patterning but Plays A Role in Early Development .......................................................................... 101 BMP Signaling in Spiralian Axis Patterning .................................................... 102 4 ACTIVIN/NODAL SIGNALING MEDIATES DORSAL-VENTRAL AXIS FORMATION IN Chaetopterus pergamentaceous DURING EARLY DEVELOPMENT. .................................................................................................