Characterization of Two G-Protein Coupled Receptors and One Fox Transcription Factor in Drosophila Embryonic Development By Caitlin D. Hanlon A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July 2015 ABSTRACT Cell migration is an exquisitely intricate process common to many higher organisms. Variations in the signals driving cell movement, the distance cells travel, and whether cells migrate as individuals, clusters, or as intact epithelia are all possible. Cell migration can be beneficial, as in development or wound healing, or detrimental, as in cancer metastasis. To begin to unravel the complexities inherent to cell migration, the Andrew lab uses the Drosophila salivary gland as a relatively simple model system for learning the molecular/cellular events underlying cell movement. The salivary gland begins as a placode of polarized columnar epithelial cells on the surface of the embryo that invaginates and move dorsally until a turning point is reached. There, it reorients and begins posterior migration, which continues until the gland reaches its final position along the anterior-posterior axis of the embryo. The broad goal of my work is to identify and characterize other key players in salivary gland migration. I characterized two G-protein coupled receptors (GPCRs) – Tre1 and mthl5 – which are expressed dynamically in the embryo. By creating a null allele of Tre1, I found that Tre1 plays a key role in germ cell migration and affects microtubule organization in the migrating salivary gland. I created a mthl5 mutant allele using the CRISPR/Cas9 system. mthl5 plays a role in the cell shape changes that drive salivary gland invagination. I have identified a potential ligand of Mthl5, fog, which plays a known role in mediating cell shape changes. Mutant alleles of fog phenocopy mutant alleles of mthl5. In a separate project, I characterized the role of the Fox transcription factor FoxL1 in the Drosophila embryo. Mis-expression of FoxL1 causes severe defects in salivary gland migration and muscle organization. I found that FoxL1 is upstream of the signaling molecule sema2a and plan to identify more targets through microarray analysis. Together, these data provide important information for how tissues integrate signaling information to arrive at the correct final destination. ii ACKNOWLEDGEMENTS A special thanks to all of those who have supported me throughout my journey at Hopkins. Without the people mentioned below, none of my accomplishments would have occurred, and life certainly would not be as fun. Thanks to my parents for coming to visit and bringing the rest family – “therapy” dogs included – to visit regularly. While my parents would check in regarding my work, I especially appreciated being able to just sit quietly with them in front a campfire. Thanks to my sister for listening to all of my science complaints and miniature accomplishments. Despite being a plant biologist, she’s a great scientist and is going to do amazing things. She inspires me to try new things with my work and to think about life in new and interesting ways. The Djuranovic family has a very special place in my heart. Sergej, Slavica, Vas, and Mila are my second family. Their home was always open to me, and they’ve helped me through so many events. They’ve encouraged me in my passions and listened to my frustrations. They were always there with a coffee, a beer, or a delicious meal. Celebrating Slava with them is one of my happiest times of the entire year. When I started graduate school, one of the best pieces of advice that was given to me was to “join a lab where you can thrive.” The Andrew lab has truly been the best place for me and my growth as a scientist and a person. Debbie’s unwavering enthusiasm and patience are legendary. I was fairly untrained when I joined the Andrew lab and all of the credit for what I’ve learned goes to her. The other members of the lab – Afshan, Rebecca, SeYeon, Aria, Jessica, iii Kyla, Dorothy, Sangjoon, Yim, Bilal, Raj, Mike, and others – have been great coworkers and friends and have contributed many helpful suggestions over the years. In particular, the postdocs in our lab have given me superb advice about how to overcome challenges in graduate school. I’m forever thankful for their input! I would also like to thank members of the Matunis lab for being great neighbors, and especially to Maggie for her advice and friendship. My friends have been vital to the completion of my thesis work. I’ve made amazing friends in Baltimore. The epicenter of many of these friendships is a now-closed corner dive bar in Canton. Together with these friends, I’ve watched many games of football (Here we go, Steelers…) and won many games of trivia. I’d also like to acknowledge the members of my intramural football team, who are intelligent, athletic, and fun people. I would especially like to thank Lisa, Nina, Jess, Gayle, Natalie, and Jon, who have been the greatest friends a pre-doctoral fellow could ask for. A special part of my heart goes to thanking Alex, who is always quick with a smile, who knows how to make me smile, and who would brag about my work to anyone who could listen. His enthusiasm has brought me out of many work-induced funks, and together we’ve had many adventures with hopefully many, many more to come. My friends from college and home have always been supportive of my endeavors. So to Lauren, Evan, HD, Lauren Huff, Jonelle, KTP, Ali, Jen, and Maureen – thank you! iv TABLE OF CONTENTS Abstract ii Acknowledgements iii Table of Contents v List of tables viii List of figures ix Introduction 1 References 7 CHAPTER 1: Building and specializing epithelial tubular organs: the Drosophila salivary gland as a model system for revealing how epithelial organs are specified, form and specialize 8 Abstract 9 Introduction 10 Specification of Salivary glands 13 Construction of the Secretory tubes 25 Elongation of the secretory tubes 29 Positioning the salivary gland 33 Formation of the salivary duct 38 Salivary gland function: secretion and production of tissue specific gene products 40 The larval pupal salivary gland 44 Conclusions 52 Acknowledgements 55 References 56 CHAPTER 2: Outside-In Signaling: A review of the Drosophila GPCR Family 65 Abstract 66 Introduction 67 GPCR Stucture-Function 71 Signal Propogation and the GPCR cycle 73 GPCR families 76 Ligand Identification 78 Drosophila GPCRs 79 Conclusions 92 References 93 CHAPTER 3: The Role of the GPCR Tre1 in Germ Cell and Salivary Gland Migration 98 Abstract 99 Introduction 100 Materials and Methods 103 Fly Strains 103 Tre1 antibody generation 103 Immunohistochemistry and in situ hybridization 103 Result 105 Existing alleles of Tre1 are not completely null 105 Creation of a Tre1 null allele 109 v Germ cell migration is severely impaired in the Tre1KO flies 109 Salivary gland migration is not affected with loss of Tre1 112 Other tissues are not affected in Tre1KO flies 116 Over- and mis- expression of Tre1 causes a range of defects in multiple tissues 116 Searching for a Tre1 ligand 124 Discussion 128 References 132 CHAPTER 4: The Role of the GPCR mthl5 in Salivary Gland Invagination 135 Abstract 136 Introduction 137 Materials and Methods 140 Fly Strains 140 Mthl5 antibody generation 140 Immunohistochemistry and in situ hybridization 141 Results 142 Mthl5 is transiently expressed in the early salivary gland 142 Creation of a mthl5 null allele 145 Loss of mthl5 causes salivary gland defects 148 Localization and over-expression of Mthl5 in salivary gland cells 149 Fog as a ligand for both Mist and Mthl5 155 Discussion 162 References 166 Chapter 5: Characterization of the Fox Family Transcription Factor FoxL1 in Drosophila Embyrogenesis 169 Abstract 170 Introduction 171 Materials and Methods 174 Fly Strains 174 FoxL1 antibody generation 174 Immunohistochemistry and in situ hybridization 175 Results 177 FoxL1 is expressed in a subset of somatic muscles that contact the migrating salivary gland 177 Loss of FoxL1 does not overtly affect salivary gland placement 177 Hindgut morphology is normal in FoxL1 mutants 183 FoxL1 is expressed in muscle VIS5/muscle 33 186 Creating a new, tagged, allele of FoxL1 to assay for muscle phentotypes 190 FoxL1 over-expression disrupts the morphology of multiple tissues 194 Sema2a functions downstream of FoxL1 200 Discussion 204 References 208 Chapter 6: The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity 211 Abstract 212 vi Introduction 213 Materials and Methods 216 Fly Strains 216 Electrophoretic mobility shift assays 216 Site directed mutagenesis of SPCG enhancer lacZ reporters 217 Chromatin immunoprecipitation and quantitative PCR 217 Transmission electron microscopy 217 Immunohistochemistry and in situ hybridization 218 Generation ofCrebA maternal zygotic mutants 218 Microarray experiments to identify CrebA target genes in Drosophila 219 HeLa cell culture, transfection, and immunofluorescence 219 Cell sorting, RNA extraction, and microarray analysis from HeLa cells 220 Accession numbers 220 Results 221 CrebA binds directly to SPCG enhancers in vitro and in vivo 221 CrebA directly activates SPCG expression 226 CrebA is sufficient to induce SPCG expression 229 CrebA regulates additional secretory pathway genes as well as secreted cargo 229 Loss of CrebA leads to defects consistent with secretory dysfunction 235 CrebA is related to the mammalian proteins Creb3L1 and Creb3L2 238 Creb3L1 and Creb3L2