Le but de toute étude en biologie évolutive du développement est l’étude des mécanismes développementaux à l’origine des diversifications morphologiques. Dans ce contexte, j’ai décidé de me focaliser sur l’émergence des Vertébrés au cours de l’évolution, par la mise en œuvre d’études comparatives entre différents modèles de Deutérostomiens. Le travail réalisé durant ma thèse est subdivisé en trois projets: (i) j’ai abordé le lien entre l’évolution du cerveau chez les Chordés et les modifications de la signalisation à l’acide rétinoïque (AR) au cours du développement. En particulier, j’ai examiné les rôles de l’AR au cours du développement du cerveau chez la lamproie Lampetra fluviatilis, et j’ai comparé les résultats obtenus chez cette espèce aux mécanismes développementaux agissant chez l’amphioxus, un Chordé invertébré, et chez les modèles gnathostomes classiques. Les données obtenues lors de ces analyses comparatives ont permis une meilleure compréhension de l’évolution de la régionalisation cérébrale chez les Vertébrés. (ii) j’ai étudié l’évolution des séquences régulatrices présentes au sein des clusters de gènes hox, connus pour agir dans la régionalisation du système nerveux des Chordés. L’identification d’éléments non-codants conservés ainsi que d’éléments de réponse à l’AR (RARE) potentiels dans des clusters hox de Chordés, combinée à la caractérisation de RAREs in vivo en cellules murines a permis une vision intégrée de l’évolution du contrôle des gènes hox par l’AR, chez les Chordés. (iii) j’ai analysé l’évolution des microARNs chez les Chordés en comparant les répertoires microARN chez plusieurs espèces de Deutérostomiens. Cette étude a permis d’émettre de nouvelles hypothèses quant à l’émergence des microARNs chez les animaux. Toutes ces analyses ont abordé différents aspects de l’évolution des Chordés avec pour objectif la proposition d’une vision intégrée des mécanismes moléculaires à l’origine de l’émergence des Vertébrés. / The aim of the evolutionary developmental biology is to study the developmental mechanisms at the base of morphological diversification. In this context, I decided to focus my attention on the emergence of vertebrates during evolution by carrying out comparative analyses in several deuterostome models. The work carried out during of my thesis can be subdivided into three major projects: (i) I addressed the link between brain evolution and modifications in retinoic acid (RA) signaling during chordate development. In particular, I investigated the roles of RA signaling in brain development in a jawless vertebrate, the lamprey Lampetra fluviatilis, and compared the results with developmental mechanisms in the invertebrate chordate amphioxus and classical developmental model systems in jawed vertebrates. Data obtained from these comparative studies provided insights into the evolution of brain patterning in vertebrate evolution. (ii) I investigated the evolution of the regulatory landscape of hox gene clusters that are known to be fundamental for the patterning of the chordate central nervous system. The identification of conserved non-coding elements and putative RA response elements (RAREs) in hox clusters of different chordate species combined with the in vivo characterization of functional RAREs in mouse F9 cells provided an integrated view of the evolution of RA-dependent hox cluster regulation in chordates. (iii) I studied the roles of microRNAs (miRNAs) in chordate evolution by comparing the miRNA complements of different deuterostome species. This analysis provided novel insights about the general mechanisms of miRNA emergence in animals and highlighted species-specific miRNA complement amplifications in different deuterostome lineages. In sum, these studies have tackled different aspects of chordate evolution from an evo-devo perspective, aiming at an integrated view of the molecular mechanisms underlying vertebrate diversification.

Chez les eucaryotes, les ARNm cellulaires subissent une traduction dépendante de la coiffe qui nécessite des facteurs appelés eIFs pour produire des protéines, mais les ARNm Hox sont traduits dans un mécanisme non canonique en raison de deux régulateurs d'ARN dans l'élément 5'UTR appelé Internal Ribosome Entry Site (IRES) qui recrute le ribosome sans le besoin de coiffe, et un élément inhibiteur de traduction (TIE) qui empêche la translation dépendant de coiffe. L'objectif de ma thèse est de déchiffrer le mécanisme de deux éléments TIE a3 et a11 dans les ARNm Hox. Pour cela, nous avons utilisé le système de traduction sans cellules RRL. Notre modèle pour TIE a3 suggère qu'il inhibe la traduction en uORF qui se traduit par un ARNm Hox a3 UTR 5'UTR de pleine longueur et produit un peptide de 9 KDa avec l'implication de eIF2D, un facteur d'initiation non canonique indépendant du GTP. Pour TIE a11, il séquestre le ribosome 80S sur une combinaison de codons start-stop à 19 nucléotides en amont d'une structure de boucle de tige riche en GC qui bloque le 80S au codon stop. / In eukaryotes, cellular mRNAs undergo cap-dependent translation which requires factors called eIFs to produce proteins.However, Hox mRNAs are translated in a non-canonical mechanism due to two RNA regulons in the 5’UTR called Internal Ribosome Entry Site (IRES) element which recruits the ribosome without the need of a cap, and a Translation Inhibitory Element (TIE) which inhibits cap-dependent translation. The objective of my PhD is to decipher the mechanism of two TIE elements a3 and a11 in Hox mRNAs. For that, we used RRL cell-free translation system. Our model for TIE a3 suggests that it inhibits translation to a uORF which translates through full length 5’UTR Hox a3 mRNA and produces a peptide of 9 KDa with the involvement of eIF2D, a non-canonical GTP-independent initiation factor. For TIE a11, it sequesters 80S ribosome on a start-stop codon combination at 19 nucleotides upstream of a GC-rich stem loop structure which blocks the 80S at the stop codon.

No description available.

Univ Arizona, Dept Mol & Cellular Biol

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Adipose tissues have long been considered to derive from a common origin. Even the functionally different brown and white adipose tissues were generalized to share a common origin. Brown adipose tissue is a highly innervated and vascularised tissue containing multilocular and multimitochondrial brown adipocytes. Brown adipose tissue expends energy through sympathetic nervous system-mediated non-shivering thermogenesis, where uncoupling protein 1 (UCP1) is the key player. In contrast, white adipose tissue consists of unilocular white adipocytes with a main role to store energy in the form of the lipid droplet. We know today that this generalisation is exaggerated since adipocytes can derive from more than one origin and not only be brown or white. We and others have demonstrated that the brown adipocyte has a dermomyotomal origin and derives from the adipomyocyte, the precursor cell that can also become a myocyte, whereas white adipocytes are suggested to derive from pericytes, cells that are embedded within the vascular vessel walls. For a long time there has been evidence that energy-expending adipocytes reside within certain white adipose tissues, based on the fact that cold exposure, by switching on the sympathetic nervous system, leads to levels of UCP1 that are not detectable in mice housed at thermoneutrality. We demonstrated that these cells have a molecular signature that is distinct from brown and white adipocytes. Since these energy-expending cells reside within certain white adipose tissues, we chose to name them brite (brown in white) adipocytes. Moreover, we also identified regulatory factors that were specifically expressed in each adipocyte type, thus, facilitating the possibility to identify the three adipocytes: the brown, the white and the brite. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

La presente tesis comprende una revisión del desarrollo de Rhodnius prolixus (Stähl, 1859); anotación de genes del desarrollo temprano y estudios de expresión y funcionales de dos de esos genes. El objetivo general ha sido aportar datos que contribuyan a establecer redes génicas como blanco de la evolución de la forma en los insectos. Los objetivos específicos fueron: Identificar y anotar en el genoma de R.prolixus genes ortólogos a los genes HOX de Drosophila melanogaster (Meigen, 1830); caracterizar el cluster HOX y determinar la función de genes HOX mediante genómica funcional. Se identificaron 70 genes, la mayoría de ellos correspondientes al grupo de TF con homeobox. Se analizaron, curaron y anotaron 26 secuencias; incluyendo a los ocho HOX canónicos. Se logró demostrar que los genes HOX de R.prolixus están agrupados en un cluster y se plantean cinco agrupamientos probables. Los ensayos funcionales se realizaron usando un gen HOX ‒scr‒ y un activador HOX ‒caudal‒ involucrado en el establecimiento del eje anteroposterior. Para ello, se pusieron a punto las técnicas de hibridación in situ de embriones completos ‒WMISH‒ y de ARNi parental. La expresión de scr mostró un patrón acorde a lo esperado en relación a las observaciones hechas en otros insectos. La ARNi mostró variantes en comparación con especies relacionadas, pero se ajusta muy bien a lo esperado. La expresión de caudal muestra las siguientes similitudes con respecto a otras especies estudiadas: (1) actúa tempranamente como gen de efecto materno, (2) se expresa en la región posterior del huevo en estado de blastodermo, (3) los efectos de la ARNi son semejantes a los encontrados en otros insectos de banda germinal corta. Sin embargo, en estados de banda germinal, los resultados de expresión difieren respecto a otras observaciones, esto puede estar en relación con mecanismos de sañalización aún no descriptos para este gen. / This thesis deals with the embryology of Rhodnius prolixus (Stähl, 1859), the establishment of functional techniques in this new developmental model (whole mount in situ hybridization ‒WMISH‒ and parental RNAi), the annotation of early developmental genes, and the functional assay for two of those genes, Sex combs-reduced (Rp-Scr) and caudal (Rp-cad). The aim of the investigation is to contribute to define and understand the basic components of developmental genetic networks that are target of evolutionary processes that shape insect morphology. The goals are to indentify and annotate orthologs to the HOX genes of Drosophila melanogaster (Meigen, 1830) in the R .prolixus genome, characterize the HOX cluster and determine the HOX function in R. prolixus by means of functional genomics. Seventy genes were identified, mostly homeobox-containing transcription factors. Twentysix sequences were analysed in detail, cured and annotated, including the eight canonical HOX. The results showed that the HOX genes of R. prolixus are clustered and five plausible organizations were proposed. The functional assays were performed by using the HOX gene Rp-Scr and the activator Rp-cad , involved in the anteroposterior axis patterning. Rp-Scr expression was similar to other insects. Rp-Scr RNAi showed some variants compared to related species, but it was in agreement with its known function, which was relevant to validate the techniques developed. Rp-cad expression showed that (1) it function early as a maternal gene, (2) it is expressed in the posterior of the egg in blastoderm stage, (3) the RNAi effects shows a posterior-to-anterio effect. In the germband stage, the expression suggests a long distance signalling mechanism at work.

No description available.