Bioinformatic and functional analyses of muscle cell diversification in Drosophila melanogaster Preethi Poovathumkadavil To cite this version: Preethi Poovathumkadavil. Bioinformatic and functional analyses of muscle cell diversification in Drosophila melanogaster. Quantitative Methods [q-bio.QM]. Université Clermont Auvergne, 2021. English. tel-03279910 HAL Id: tel-03279910 https://tel.archives-ouvertes.fr/tel-03279910 Submitted on 6 Jul 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Year 2021 Doctoral School of Life, Health, Agronomy, Environmental Sciences (SVSAE) University of Clermont Auvergne Thesis INSERM U1103, CNRS UMR6293, University of Clermont Auvergne Genetics, Reproduction and Development institute (iGReD) Preethi Poovathumkadavil Bioinformatic and functional analyses of muscle cell diversification in Drosophila melanogaster Specialty Bioinformatics and Biology - Health Thesis publicly defended on April 23, 2021 Jury members Dr. Athanassia Sotiropoulos President of the jury Dr. Michael V. Taylor Reviewer Dr. Maria Spletter Reviewer Dr. Laurence Dubois Examiner Dr. Maria Eugenia Gallego Examiner Dr. Krzysztof Jagla PhD supervisor A PhD brings out the best in you. It brings out the worst in you. It is so important to know both sides of oneself. Having been associated with the corporate IT world for a long time, this was another glimpse into the human psyche in an academic setting. This level of education is aptly titled ‘Doctorate of Philosophy’. I would like to thank Krzysztof Jagla, iGReD and the UCA for this opportunity. As someone who is interested in multiple domains, my PhD opened the door to the whole new world of biological experimentation. It reinforced my belief in the importance of cross domain interactions in any field. It again helped me recognize and appreciate the contribution of each domain, and how each of them is indispensable to a project, be it in IT or the Biological Sciences. It was great to have my autonomy during these three years. I learnt Science as well as some more life lessons, which is what education is really about for me. I would also like to thank everyone who came into my life, even for a period as short as a few seconds, because every interaction taught me something. “Anything worth doing in life has risks… In my case, I decided a long time ago that exploring the universe was worth taking a risk for.” - Chris Hadfield, astronaut, former commander of the International Space Station. The biggest blunder of our complex times might be our tendency to underestimate the power of simplicity. - Personal thought. Globalization is the new dynamite. It can give us people like Malala Yousafzai, Bill Gates and Greta Thunberg who are trying to find global solutions to global problems while staying true to their identities. It can also cause the global spread of COVID-19. I hope I learn from these exceptional people to think globally because a problem anywhere causes ripples everywhere. And because Science is for humanity. - Personal thought. ABSTRACT The voluntary skeletal muscles in vertebrates are the main effectors of locomotion. Processes and genes implicated in human myogenesis are of immense interest to better understand the deregulations caused in muscular and neuromuscular disorders and to find therapeutic targets. The body wall or somatic muscles of the fruit fly, Drosophila melanogaster, are similar to vertebrate skeletal muscles. As is the case for vertebrate skeletal muscles, each Drosophila embryonic somatic muscle possesses its specific identity that clearly distinguishes from its immediate neighbors. In Drosophila, some muscle identity transcription factors (iTFs) have been identified, but others remain elusive. In order to dissect mechanisms regulating the diversification of committed muscle cells to attain their final identity, the team had previously generated transcriptomics data for mRNA under translation in the Lms+ lateral transverse (LT) and Slou+ muscle subsets as well as the Duf+ global muscle set over three time windows of development. My analyses of this data helped identify the evolutionarily conserved gene that is part of the conserved Wnt enhanceosome, Sequence-specific single-stranded DNA-binding protein (Ssdp) as a determinant of final muscle identity. Its vertebrate homologue Single stranded DNA binding protein 3 (SSBP3) is downregulated and mis-spliced in human myotonic dystrophies, but its role in myogenesis has not been studied. My study reveals a role for Ssdp in embryonic myogenesis for the first time. A temporally regulated, isoform-specific expression of Ssdp was identified. Further analyses showed that the initial muscle identity program proceeds normally for the most part in the absence of zygotic Ssdp, but muscles fail to establish their final identity due to the deregulation of iTFs and identity processes that establish muscle morphology, innervation and attachment. Comparative analyses revealed that specific Ssdp mutant phenotypes overlap subsets of phenotypes observed in the context of loss of function of a Drosophila Wnt, Wg and dTCF, an effector of the canonical Wnt pathway, suggesting specific interactions between these factors. Potential genetic interactions between the LT iTF, Ap (a Lhx2 orthologue) with Mid and the Ssdp partner, Chi (a LDB1 orthologue, also part of the Wnt enhanceosome) were unveiled. In addition, my in silico analysis identified other potential candidates implicated in muscle identity such as the TFs D, Sox14 and Sox21b for LT muscles and Stat92E for Slou+ muscles, with Nf-YB acting as a potential upstream regulator. Muscle subset specific enrichments of CT-rich motifs in the LT subset and GATA motifs in the Slou subset were also identified. TABLE OF CONTENTS CHAPTER 1 – INTRODUCTION ......................................................................................... 1 1.1. STAYING GROUNDED AND STANDING UP TO GRAVITY ....................................................... 1 1.2. VERTEBRATE MUSCLE DEVELOPMENT – HOW DO THESE INCREDIBLE STRUCTURES FORM? ................................................................................................................................................ 4 1.2.1. Types of vertebrate muscles ..................................................................................... 4 1.2.2. Development of vertebrate skeletal muscles ............................................................ 4 1.2.2.1. Gastrulation and the specification of the mesoderm ......................................... 4 1.2.2.2. Mesoderm diversification and somitogenesis ................................................... 6 1.2.2.3. Somite specification ........................................................................................ 10 1.2.2.4. Vertebrate myogenesis .................................................................................... 11 1.2.2.4.1. Prenatal myogenesis ................................................................................. 13 1.2.2.4.1.1. Development of trunk muscles .......................................................... 13 1.2.2.4.1.2. Development of limb muscles ........................................................... 18 1.2.2.4.1.3. Development of craniofacial muscles ............................................... 19 1.2.2.4.2. Postnatal and adult myogenesis ................................................................ 20 1.2.2.4.3. Muscle attachment and innervation .......................................................... 20 1.3. THE NEED FOR SIMPLER MODEL SYSTEMS ....................................................................... 23 1.4. MUSCLE DEVELOPMENT IN DROSOPHILA MELANOGASTER ............................................. 25 1.4.1. Gastrulation and the formation of the mesoderm .................................................. 25 1.4.2. The development of embryonic, larval and adult somatic muscles ........................ 27 1.4.3. Muscle diversification and the mystery and complexity of muscle identity transcription factors (iTFs) .............................................................................................. 28 1.4.3.1. The LT muscle iTF code ................................................................................. 28 1.4.3.1.1. Drop (Dr) .................................................................................................. 29 1.4.3.1.2. Lateral muscles scarcer (Lms) .................................................................. 29 1.4.3.1.3. Apterous (Ap) ........................................................................................... 29 1.4.3.1.4. Krüppel (Kr) ............................................................................................. 30 1.4.3.1.5. Midline (Mid) ........................................................................................... 31 1.4.3.1.6. Araucan/Caupolican (Ara/Caup) .............................................................. 31 1.4.3.2. The Slou positive somatic muscle iTF code ...................................................