Temporal Regulation of the Muscle Gene Cascade by Macho1 and Tbx6 Transcription Factors in Ciona Intestinalis

For over a century, muscle formation in the ascidian embryo has been representative of \u27mosaic\u27 development. The molecular basis of muscle-fate predetermination has been partly elucidated with the discovery of Macho1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c. However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear. By combining misexpression assays with CRM analyses, we have identified the mechanisms through which Ciona Macho1 (Ci-Macho1) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and we have unveiled the cross-regulatory interactions between the latter transcription factors. Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1). We use these representative examples to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos

The role of highly conserved non-coding DNA sequences in vertebrate development and evolution

PhDComparisons between vertebrate genome sequences, from mammals to fishes, have revealed thousands of conserved non-coding elements (CNEs) that are associated with developmental genes. Interestingly, the vast majority of these CNEs cannot be found in invertebrate genomes by sequence homology. As many CNEs have been demonstrated to act as enhancers in-vivo, it has been postulated that CNEs represent gene regulatory elements with crucial roles in aspects of development that are shared between vertebrates. To trace the evolution of CNE sequences in vertebrates, a preliminary search for CNEs in the lamprey genome was conducted using the draft lamprey genome sequence. This thesis documents how the CNEs identified in lamprey have been used as a guide to ask questions about the function and evolution of CNEs in the vertebrate lineage. Through the combined use of comparative genomics and developmental biology techniques, including a newly developed reporter assay for sea lamprey embryos, crucial first steps have been taken toward systematically de-coding these ancient gene regulatory elements. Special attention is paid toward utilising the low sequence identity of lamprey CNEs for „phylogenetic footprinting‟, an approach which uncovers striking enrichment of CNEs for a set of motifs that are characteristic of Hox-regulated elements. These findings help to establish CNEs within a developmental and evolutionary context.School of Biological and Chemical Sciences Queen Mary University of London

The role of vertebrate conserved non-coding elements in hindbrain development and evolution

Vertebrate conserved non-coding elements (CNEs) act as cis-regulatory modules of developmental genes. To assess their roles in coordinating gene expression during embryogenesis, CNEs were subjected to motif searches. Using reporter gene assays in zebrafish (Danio rerio) embryos, Pbx-Hox (TGATNNAT) motifs are demonstrated to be poor predictors of hindbrain enhancer activity. Hindbrain enhancer CNEs are distinguished from hindbrain negative CNEs accurately by virtue of co-occurring Pbx-Hox (TGA TDDA TKD) and Meis/Pknox (CTGTCA) motifs. The grammar of these motifs was investigated using a bioinformatic pipeline for the detection of multiple conserved motifs, revealing no patterns in their relative organisation aside from spatial co-occurrence. These motifs were then used to identify additional conserved hindbrain enhancers with high efficacy (89%). Substitutions targeted to either motif abrogate expression by the enhancer or generate ectopic reporter gene expression, suggesting that motif co-occurrence is required for efficient and segment-specific hindbrain activation. Pbx-Hox and Meis/Pknox motifs are enriched in gnathostome CNE sets but are not detected in invertebrate chordate CNEs. Furthermore the presence (or absence) of the hindbrain syntax correlates with the conservation (or lack thereof) of segment-restricted enhancer activity in orthologous CNEs from the sea lamprey. A library of zebrafish hindbrain regulatory elements is made available. The heterogeneity of function and the loose grammar of motifs are consistent with combinatorial factor binding; a model of CNEs as exceptionally well- conserved billboard enhancers is presented (inflexible billboard model). The implications of these data for models of the evolution of the vertebrate hindbrain are discussed. Several components of the hindbrain gene regulatory network are shared- derived characters of gnathostomes, suggesting the establishment and elaboration of the conserved regulatory code controlling hindbrain development on the vertebrate and gnathostome stems, respectively

Gut Patterning In Development And Evolution: A Comparative Differential Transcriptomics Approach

All bilaterians share a common kit of transcription factors and cis-regulatory elements that compose the Gene Regulatory Network (GRN) essential for the development of the body plan. Modifications within the elements of the GRN determine the evolution of animal forms. In this study, the GRN for embryonic gut specification in two echinoderm species, the sea urchin Strongylocentrotus purpuratus and the sea star Patiria miniata, has been studied with the purpose of acquiring further knowledge on the process of gut patterning in the sea urchin larva and to compare it with the sea star embryo, focusing on the role that Xlox and Cdx transcription factors have in this process in both echinoderm species. Taking advantage of genome-wide approaches and modern high-throughput technologies, a partial reconstruction of the sea urchin GRN around Xlox and Cdx transcription factors, known for their key role to start the gut specification process, has been achieved leading to describe the putative interactions of the TFs in the network. An important novel node of the sea urchin GRN featuring the interaction between Sp-Meis, an homeobox gene, and Sp-Lox protein has been revealed and the occupancy of Sp-Lox protein on Sp-Meis regulatory region has been demonstrated by ChIP-PCR. The comparison of the differentially expressed genes after Xlox and Cdx perturbation in both sea urchin and sea star embryos has revealed that, although the absence of these two proteins affects some digestive functions and developmental processes related to its specification, however many of the regulatory genes involved in these mechanisms are not the same in the two species. This result suggests a phenomenon of rewiring of the gut GRN in S. purpuratus and P. miniata that, although belonging to the same phylum Echinodermata, are distant million years in term of evolutionary time, as recorded by fossil records

Analyse bioinformatique des événements de transferts horizontaux entre espèces de drosophiles et lien avec la régulation des éléments transposables

Transposable elements (TEs) are repeated DNA sequences that are able to move (transpose) within their host genome. To counteract the negative effects of their TEs, regulation mechanisms of the TE transposition are present in the host genome. Once a TE is regulated, the progressive accumulation of mutations in its sequence will inevitably lead to the definitive loss of its transposition capacity. My work during this thesis is was to better understand the succss and the maintaining of these peculiar repeated sequencest, with the study of horizontal transfers (HTs) of TEs enabling them to escape host regulation mechanisms, and the study of this regulation. The first part of my thesis concerns the study of HTs between two closely related drosophila species. I have developed a new bioinformatic method for the detection of HTs between two eukaryotic genomes. The development of this method brought me to work on the unilateral multiple testing problematic for which I have developed a new procedure to control the expected false discovery rate (FDR). The second part of my thesis focuses on the regulation of TEs by the small RNA pathway, an RNA interference mechanism. For this study, I have analyzed sequencing data of small RNAs and total RNAs. For this work, I have developed an analysis pipeline, to study differences of expression between repeated sequences. Some features of the small RNA dataset required the development of a new procedure to parse them. This procedure was extended and implemented in a software to be used for the quality control of next generation sequencing dataLes éléments transposable (ET) sont des séquences d'ADN qui ont la capacité de se déplacer au sein des génomes. Pour contrebalancer les effets négatifs liés à l'activité des ET, il existe chez leurs hôtes des mécanismes régulant l'activité de transposition. Une fois qu'un ET est régulé, l'accumulation progressive de mutations dans sa séquence conduit fatalement à la perte définitive de son activité de transposition. J'ai cherché au cours de cette thèse à mieux comprendre le succès et le maintien de ces séquences répétées, avec d'une part l'étude des transferts horizontaux (TH) d'ET, un moyen d'échapper aux mécanismes de régulation , et d'autre part l'étude de leur régulation. Dans la première partie de ma thèse, je me suis intéressé à l'étude des TH entre deux espèces proches de drosophiles. Dans cette étude, j'ai développé une nouvelle méthode bioinformatique permettant la détection de séquences transférées horizontalement entre deux génomes eucaryotes qui m'a permis détecter de nombreux TH d'ET. Ce travail m'a aussi conduit à développé une nouvelle méthode de contrôle du taux de faux positifs moyen applicable aux tests multiples unilatéraux. Dans la deuxième partie de ma thèse, j'ai étudié la régulation des ET par la voie des petits ARN, un mécanisme de l'ARN interférence. Dans cette étude, j'ai analysé des données de séquençage de petits ARN, ainsi que d'ARN totaux issues de différentes populations de D. simulans. Ce travail a conduit au développement d'un pipeline d'analyse permettant d'étudier des différences d'expression entre des séquences répétées ainsi que d'une nouvelle procédure de contrôle qualité de ce type de donné