Investigating the roles of Hes and Sox genes during embryogenesis of the spider P. tepidariorum Christian Louis Bonatto Paese This thesis was submitted in partial fulfillment of the requirements of the award of Doctor of Philosophy June 2018 “Our greatest glory is not in never falling, but in rising every time we fall” Confucius Aquele uísque “Lembro-me daqueles dias, Em que assistíamos ao pica-pau E rachavamos o bico dando risada de algumas passadas. Lembro-me de que possuíamos aspirações e sonhos selvagens. Cada qual, à sua maneira, pôs-se a caçar seu horizonte. Sabíamos que nenhuma estrada se emenda no passado, Todas se amarram apenas no porvir, E, só com muita sorte, nos achamos novamente. Mas a memória, ah! Essa é uma cruel concubina, Pois nunca nos esquecemos daqueles dias. Eles voltam em espasmos mistos de tristeza e alegria. No saldo da vida, Parece que atingimos muito do que poderíamos, Mas sempre ficamos na dúvida De uma dose daquele uísque: Velho olho vermelho.” Pablo Jamilk Essa tese é o resultado de 3 anos que me trouxeram um incrivel auto-aprendizado. Todo esse esforço é dedicado a minha família – em especial aos meus sobrinhos Leonardo e Leticia. Table of Contents Abstract ...................................................................................................................................................... 6 List of Abbreviations .................................................................................................................................. 8 List of Figures ............................................................................................................................................. 9 List of Tables ............................................................................................................................................ 11 General Introduction.................................................................................................................................. 12 Introduction ............................................................................................................................................. 13 1 Spider studies in embryology and developmental biology .............................................................. 13 2 Segmentation .................................................................................................................................... 17 2.1 Segmentation in Vertebrates ......................................................................................... 20 2.2 Segmentation in Annelids .............................................................................................. 22 2.3 Segmentation in Arthropods ......................................................................................... 23 2.3.1 The GRN for formation of the SAZ and segment addition in P ............................... 30 2.3.2 The hairy/Hes genes in spiders ............................................................................... 31 3 Neurogenesis .................................................................................................................................... 34 3.1 Neurogenesis in Vertebrates ......................................................................................... 34 3.2 Neurogenesis in Arthropods .......................................................................................... 35 3.3 Sox genes and its involvement in neurogenesis ............................................................ 36 4 Aims of the thesis ............................................................................................................................. 39 Expression of bHLH-TF Encoding Genes in P. tepidariorum................................................................ 41 Introduction ............................................................................................................................................. 41 Identification and characterization of Hairy-Related orthologues .......................................................... 42 Expression of P. tepidariorum bHLH-containing genes ........................................................................... 46 Functional analysis of the bHLH genes .................................................................................................... 51 Discussion ................................................................................................................................................ 53 Conservation of bHLH-containing Hes-like genes in the common house spider ................................ 53 hairy is expressed in a segmental pattern ........................................................................................... 54 deadpan, hey and side are expressed in clusters of neurogenic cells ................................................. 56 Expression of Sox Genes in P. tepidariorum .......................................................................................... 60 Introduction ............................................................................................................................................. 60 Isolation of Sox genes in spiders .............................................................................................................. 61 Sox Gene Expression during P. tepidariorum embryogenesis ................................................................. 65 SoxB group ........................................................................................................................................... 65 SoxN ..................................................................................................................................................... 66 Sox21b-1 .............................................................................................................................................. 67 SoxC group ............................................................................................................................................... 70 SoxD, E and F groups ............................................................................................................................... 71 Discussion ................................................................................................................................................ 74 Conservation and divergence of Sox Gene Expression ........................................................................ 74 A SoxB1 orthologue has segmentation expression pattern ................................................................ 75 SoxC to F groups have divergent expression patterns ........................................................................ 77 A SoxB Gene Regulates Segmentation in the Spider ............................................................................ 82 Introduction ............................................................................................................................................. 83 Sox21b-1 regulates prosomal and opisthosomal segmentation ............................................................. 83 Sox21B-1 is involved in cell maintenance during spider embryogenesis ................................................ 88 Effects of Sox21b-1 knockdown on the germ disc and mesoderm formation ........................................ 90 Effects of Sox21b-1 knockdown on segmentation .................................................................................. 94 Discussion ................................................................................................................................................ 99 A SoxB gene is required for two different mechanisms of spider segmentation .............................. 100 Sox21b-1 exhibits highly pleiotropic phenotypes during early spider embryogenesis ..................... 102 The evolution of Sox21b-1 ................................................................................................................. 103 Identification of Putative Regulatory Regions for Spider Manipulation ............................................ 106 Introduction ........................................................................................................................................... 106 Establishing tools for genome editing and other applications in P. tepidariorum ................................ 110 Characterization and isolation of RNApol II-associated promoters ...................................................... 110 Beta-actin........................................................................................................................................... 111 Elongation Factor-1 ........................................................................................................................... 113 Heat-shock protein 70 ........................................................................................................................ 114 PolyUbiquitin ...................................................................................................................................... 115 Characterization and isolation of RNApol III-associated promoters ..................................................... 116 Micro-injection of spider embryos ........................................................................................................ 118 Discussion .............................................................................................................................................