Butterfly wing color patterns result from the arrangement of monochromatic scales containing chemical pigments and a delicate architecture that can cause interference or diffraction of light, generating iridescent colors. The latter mechanism is known as structural coloration and, despite its ecological importance, little is known about the molecular mechanisms underlying the development of this trait. The Southern Dogface butterfly, Zerene cesonia, exhibits sexually dimorphic development of ultraviolet wing reflectance. Males possess a UV-reflective patch on the forewing that results from nano-structures on the wing scales, which are absent in females. This dimorphism offers an excellent opportunity to explore the genetic mechanisms involved in pattern formation and cyto-structural variation. We used RNA-seq data from imaginal wing discs through late larval and pupal development to identify genes involved in the regulation of color pattern and scale structure formation. We identified candidate genes for the regulation of wing color pre-patterning and sexually-dimorphic development of wing scales. Our results provide a genomic resource for the identification and characterization of genes that participate in the regulation of wing development in pierid butterflies.

La mise en place de la lignée germinale au cours du développement constitue une des étapes fondamentales conditionnant la fertilité de l’individu adulte. Au cours des dernières décennies, le nombre croissant de couples consultant pour une aide médicale à la procréation a fait émerger l’hypothèse d’une altération des fonctions de reproduction chez l’Homme qui pourrait trouver son origine dans la perturbation du développement précoce. Dans l’ovaire fœtal, les cellules germinales s’orienteront vers la voie de l’ovogénèse, caractérisée entre autres par l’entrée en méiose de ces cellules. La majorité des données actuelles relatives à ces évènements sont issues du modèle murin alors que le développement de l’ovaire humain est significativement diffèrent de celui de la souris. Il est donc nécessaire d’approfondir nos connaissances du développement ovarien humain et d’identifier ses éventuelles perturbations. L’objectif de mon travail a été de mettre au point un outil d’étude du développement ovarien et d’identifier de nouvelles voies impliquées dans la régulation de l’entrée en méiose des cellules germinales fœtales humaines et leurs perturbations éventuelles.Nous avons mis au point un nouveau modèle de xénogreffe d’ovaires fœtaux humains du premier trimestre de gestation (au moment de l’apparition des premières cellules méiotiques). Ce modèle nous a permis d’observer un développement de l’organe et une différenciation des cellules germinales similaires à ceux observés in vivo. Ce modèle permettra des travaux à des âges auxquels le matériel d’étude est peu accessible. En couplant ce modèle de xénogreffe à une stratégie d’ARN-interférence, il nous a été possible d’inhiber l’expression d’un gène spécifiquement exprimé dans les cellules germinales ovariennes, DMRTA2, et de mettre en évidence un potentiel rôle de ce gène dans leur différenciation pré-méiotique. Nous avons observé une diminution du nombre de cellules ayant initié la méiose après inhibition de l’expression de ce gène. Par ailleurs, nous avons également identifié la présence dans l’ovaire fœtal de nombreux marqueurs décrits comme testiculaires chez la souris (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). L’expression de ces marqueurs pourrait expliquer la présence de cellules mitotiques tardives dans l’ovaire fœtal humain que nous avons pu observer jusqu’à 30 semaines de gestation. En parallèle de ces travaux, nous avons testé la sensibilité des cellules germinales à la dexaméthasone, glucocorticoïde pouvant être administré au cours de la grossesse. Il a été observé une augmentation de l’expression de PLZF, gène cible de l’activation des récepteurs aux glucocorticoïdes, pouvant expliquer la diminution du nombre de cellules germinales.En conclusion, ce travail de thèse a permis d’identifier un nouveau gène potentiellement régulateur de la transition mitose/méiose dans l’ovaire humain, et d’affiner nos connaissances sur le développement de l’ovaire humain et l’entrée en méiose des cellules germinales. Toutefois, de nombreuses questions restent posées ainsi de futures études devront clarifier si les cellules germinales mitotiques observées à des stades tardifs sont capables de se différencier en ovocytes compétents. / Woman fertility is partially dictated by the set up of the human female germ line. During the last ten years, which saw an increased number of couples consulting for assisted reproductive cares, the hypothesis of an early alteration in reproduction functions has emerged.In the fetal ovary, germ cells enter the path of oogenesis differentiation characterized by meiotic initiation. On this subject, vast majority of the scientific data are obtained from the mouse model, even if differences with human ovarian physiology are widely acknowledged. Therefore it is necessary to extend our knowledge on human ovarian development and identify its perturbations. The objective of my work was to assess a new model to study ovarian growth, studying regulation of meiotic entry and perturbation of germ line differentiation.We sat up a new xenograft model of early human fetal ovaries, when very early meiotic germ cells appear. Organ growth and germ cells differentiation were comparable with in vivo observations. Using this model with an RNA-interference strategy, we inhibited the expression of an oogonia germ cell gene, DMRTA2. This inhibition conducted to a significantly reduced number of germ cells gene that initiated meiosis and DMRTA2 seemed to be required for mitotic-meiotic transition. In another hand, we identified, in the ovary, the expression of germ cells markers described as specifically male in rodent (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). The expression of these markers in the human ovary could explain the observation of mitotic germ cells in late fetal ovaries (30 wpf).In parallel, we tested germ cells sensibility to a synthetic glucocorticoid, dexamethasone, administrated during pregnancy in some justified pathologies. We observed an increased expression of PLZF that could explain the decreased number of germ cells observed in treated ovaries.In conclusion, we identified a new gene expressed in human fetal ovaries, potentially involved in the meiotic entry, and we extended our knowledge to characterized human germ line development. However, many points have to be clarified, as the possible competence of late mitotic germ cells to form oocytes.

In Drosophila, the functionally conserved transcription factor, doublesex (dsx), is pivotal to the specification of sexual identity in both males and females. One of its key dedicated roles involves regulating the development of a sexually dimorphic nervous system (NS) that underlies both male and female reproductive behaviours. Specific inhibition of the function of dsx-expressing neurons in males and females results in a global disruption of these sex-specific behavioural outputs. However, little is known about the functional organisation of this dsx circuit that encodes the potential to display these behaviours. Such investigations require the generation of a novel transgenic tool, capable of separating the function of dsx in the NS from that of the body. To achieve this, I generated a novel split-GAL4 dsx^GAL4-DBD hemidriver by ends in homologous recombination. Coupling the novel tool with the pan-neuronal elav^VP16-AD hemidriver, revealed spatial restriction of dsx^GAL4-DBD/elav^VP16-AD expression to dsx neurons only; enabling the realisation of novel patterns of dsx-expression in the peripheral NS. Next, the ability to elicit male-specific behavioural outputs upon activation of all dsx neurons formed the basis of a large behavioural screen aimed at parsing dsx circuitry into functionally distinct clusters. I utilised the novel dsx^GAL4-DBD hemidriver to screen a large collection of extant enhancer trap lines (ET^VP16-AD), for the elicitation of distinct sub-behaviours of male courtship. Here, I show that the activity of dsx-expressing clusters in: i) the brain (dsx-pC1, -pC2 and -pC3 collectively) regulate the early steps of male courtship (initiation, orientation and wing extension), ii) the pro- and mesothoracic ganglia (dsx-TN1 and -TN2) regulate the middle steps of male courtship (wing extension and possibly courtship song) and iii) the abdominal ganglia (dsx-Abg) regulate the late steps of male courtship (abdominal curling, attempted copulation and copulation). These data establish functional correlations between dsx clusters in distinct neuroanatomical foci and specific sub-behaviours of the courtship repertoire. Furthermore, the novel intersectional tool primed a collaborative study on female post-copulatory behaviours. We identified key sensory neurons in the female reproductive tract involved in initiating post-mating behaviours. Subsequent functional interrogations of dsx circuitry in the central NS revealed a subset of dsx-expressing neurons in the Abg that mediate changes in the female behavioural repertoire after mating. Characterisation of this relatively simple neural circuitry sheds light on the organisation of the fly brain. Ultimately, future studies will define principles of neural circuit operation, which may be similarly conserved in the nervous systems of higher animals.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20955号 / 理博第4407号 / 新制||理||1633(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 曽田 貞滋, 准教授 渡辺 勝敏, 教授 中川 尚史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Sex determination is a key developmental process in several species regulated by sexrelated transcription factors. In many species a gene called doublesex and mab-3 related transcription factor 1 (dmrt1), plays an important role in sexual differentiation. I used African clawed frogs (Xenopus) to examine function of dmrt1 in two species: a diploid species, X. tropicalis, and an allotetraploid species, X. laevis. In both species, dmrt1 is an autosomal gene; Xenopus tropicalis has one copy of dmrt1 and X. laevis has two homeologous copies that by definition are derived from whole genome duplication: dmrt1.L and dmrt1.S in X. laevis. We generated knockouts of each of these genes to further examine their function in sexual differentiation. Histological examination showed testicular dysgenesis in X. tropicalis dmrt1 and X. laevis dmrt1.L null males whereas dmrt1.S null males presented no obvious difference in sperm density compared to wildtype males. X. tropicalis dmrt1 and X. laevis dmrt1.L null females were found to completely lack reproductive organs and are infertile whereas dmrt1.S null females appeared unaffected. The contrasting results between dmrt1.L and dmrt1.S in X. laevis provides evidence of both neofunctionalization and subfunctionalization following gene duplication and suggest that gene duplication is a major contributor to evolutionary change. Additional investigation of the transcriptome of these frogs and the role of dmrt1 in the secondary sex characteristic vocalization provides further evidence of the role of dmrt1 in development. Comprehensively, this investigation provides further knowledge of the role of dmrt1 and homeologs of this gene in sexual differentiation and introduces a novel aspect of this gene in female development. Future efforts are focused on generating double knockouts for dmrt1.L and dmrt1.S, further examining the role of dmrt1.S in somatic cell function and developing additional mutant lines in other Xenopus for comparative analysis. / Thesis / Master of Science (MSc) / In many species sexual differentiation is a crucial developmental event. Surprisingly, however, the systems orchestrating sexual differentiation are highly variable among species. The doublesex and mab-3 related transcription factor 1 (dmrt1) gene plays a role in sexual differentiation in many groups, but its specific roles in this process are incompletely characterized and potentially diverse. We used genetic engineering in two species of African clawed frog (Xenopus) to disable function of dmrt1 in order to explore effects on gonadal development and the development of secondary sex characteristics. We found that dmrt1 is required for normal ovary or testis development in both Xenopus species, and that functional divergence occurred following duplication of dmrt1 by whole genome duplication. Taken together, these findings identify previously uncharacterized roles of dmrt1 in Xenopus and provide evidence of dynamic functional evolution of this important gene.

No description available.

<p>Evolutionary innovations, at the molecular level, represent the novel establishment of regulation networks among previously unconnected genes. Understanding the cellular and molecular mechanisms that underlies the development of such innovations is of central importance in evolutionary-developmental research (evo-devo). The sex comb of <em>Drosophila</em> is an excellent model to study the molecular basis of evolutionary innovations. Highline and Lowline are two artificial selected <em>D. melanogaster</em> lines differing in the number of sex comb bristles. It was expected that the “cross-regulation loop” between two transcription factors, <em>Doublesex</em> male isoform (DSX^M) and <em>Sexcombs reduced</em> (SCR), evolves rapidly and promotes the morphological evolution of sex combs. We used immunofluorescent technique (antibody staining) to compare the expression of DSX^M and SCR in the forelegs of three different lines (Highline, Wildtype and Lowline). We hypothesized that artificial selection will increase expression of DSX^Mand SCR in the Highline and reduce expression in the Lowline. The fluorescent pictures of antibody staining experiments indicate that the expression region of DSX^M in the Highline is significantly higher than the expression region in the Lowline, and the expression levels of SCR has minor difference among the three lines. DSX^M expression is altered by the artificial selection, but SCR expression is not. The influence of artificial selection appears to have been constrained by development. Our investigation provides an approach to test the validity of the models of cross-regulation s between SCR and DSX^M during development.</p> / Master of Science (MSc)

Introdução: O gene DMRT1 é um fator muito importante, o qual induz a determinação sexual masculina. Estudos mais recentes têm demonstrado que o Dmrt1 possui um papel significante no desenvolvimento ovariano. Deleções restritas ao gene DMRT1 têm sido raramente identificadas em pacientes com disgenesia gonadal (DG) sem outras características sindrômicas. Objetivo: Pesquisar a presença de haploinsuficiência do gene DMRT1 (deleções e/ou mutações inativadoras) em um grupo grande de pacientes não sindrômicos com distúrbios do desenvolvimento sexual (DDS) por anormalidades gonadais. Polimorfismos do DMRT1, como fatores potenciais pelas anormalidades gonadais, foram também identificados. Pacientes e Métodos: Foram avaliados cerca de 39 pacientes portadores de DDS por anormalidades do desenvolvimento gonadal 46,XY: 24 com disgenesia gonadal parcial e 15 pacientes com disgenesia gonadal completa. As regiões codificadoras do DMRT1 e o domínio DM (exon 1) foram amplificados e sequenciados. A análise de Multiplex ligation probe amplification (MLPA) do DMRT1 foi realizada usando um kit comercial. Resultados: Deleção parcial ou total do DMRT1 não foi identificada pela técnica de MLPA. Oito variantes alélicas do DMRT1 foram identificados. Uma nova variante c.968-15insTTCTCTCT foi identificada em 6,4% e em 14,3% dos alelos dos pacientes 46,XY e indivíduos controles, respectivamente. Conclusão: Este estudo sugere que deleções parciais ou completas no DMRT1 e mutações inativadoras não são frequentemente encontradas em pacientes com anormalidades do desenvolvimento gonadal. Além disso, nenhuma das variantes alélicas identificadas neste grupo de pacientes poderia ser considerada como um marcador potencial polimórfico para disgenesia gonadal / Introduction Dmrt1 gene is a very important factor in inducing male sex determination, and more recently it has been demonstrated that Dmrt1 plays a significant role in ovary development. DMRT1 deletions have rarely been identified in patients with 46,XY gonadal dysgenesis (GD) without syndromic features. Objective- To screen for the presence of DMRT1 haploinsufficiency (deletions and/or inactivating mutations) in a large cohort of non-syndromic patients with disorder of sex development (DSD) due to abnormalities of gonadal development. DMRT1 polymorphisms, as potential susceptibility factors for gonadal abnormalities, were also investigated. Subjects and Methods- We evaluated 39 patients with 46,XY GD: 24 patients with the partial, and 15 with the complete form. The entire coding region (éxons 2-5) of DMRT1 and the DM domain (exon 1) were PCR-amplified and direct sequenced. Multiplex ligation probe amplification (MLPA) analysis of DMRT1 was carried out using a commercial kit. Results- Partial or total deletion of DMRT1 was not identified by MLPA technique. Eight allelic variants of DMRT1 were identified. The novel variant c.968-15insTTCTCTCT was identified in 6.4% and in 14.3% of the alleles of 46,XY patients and control subjects, respectively Conclusion- This study suggest that complete or partial DMRT1 deletions and inactivating mutations are not frequently found in patients with abnormalities of gonadal development. Additionally, none of the allelic variants identified in this cohort of patients could be considered a potential polymorphic susceptibility marker for gonadal dysgenesis

Introdução: O gene DMRT1 é um fator muito importante, o qual induz a determinação sexual masculina. Estudos mais recentes têm demonstrado que o Dmrt1 possui um papel significante no desenvolvimento ovariano. Deleções restritas ao gene DMRT1 têm sido raramente identificadas em pacientes com disgenesia gonadal (DG) sem outras características sindrômicas. Objetivo: Pesquisar a presença de haploinsuficiência do gene DMRT1 (deleções e/ou mutações inativadoras) em um grupo grande de pacientes não sindrômicos com distúrbios do desenvolvimento sexual (DDS) por anormalidades gonadais. Polimorfismos do DMRT1, como fatores potenciais pelas anormalidades gonadais, foram também identificados. Pacientes e Métodos: Foram avaliados cerca de 39 pacientes portadores de DDS por anormalidades do desenvolvimento gonadal 46,XY: 24 com disgenesia gonadal parcial e 15 pacientes com disgenesia gonadal completa. As regiões codificadoras do DMRT1 e o domínio DM (exon 1) foram amplificados e sequenciados. A análise de Multiplex ligation probe amplification (MLPA) do DMRT1 foi realizada usando um kit comercial. Resultados: Deleção parcial ou total do DMRT1 não foi identificada pela técnica de MLPA. Oito variantes alélicas do DMRT1 foram identificados. Uma nova variante c.968-15insTTCTCTCT foi identificada em 6,4% e em 14,3% dos alelos dos pacientes 46,XY e indivíduos controles, respectivamente. Conclusão: Este estudo sugere que deleções parciais ou completas no DMRT1 e mutações inativadoras não são frequentemente encontradas em pacientes com anormalidades do desenvolvimento gonadal. Além disso, nenhuma das variantes alélicas identificadas neste grupo de pacientes poderia ser considerada como um marcador potencial polimórfico para disgenesia gonadal / Introduction Dmrt1 gene is a very important factor in inducing male sex determination, and more recently it has been demonstrated that Dmrt1 plays a significant role in ovary development. DMRT1 deletions have rarely been identified in patients with 46,XY gonadal dysgenesis (GD) without syndromic features. Objective- To screen for the presence of DMRT1 haploinsufficiency (deletions and/or inactivating mutations) in a large cohort of non-syndromic patients with disorder of sex development (DSD) due to abnormalities of gonadal development. DMRT1 polymorphisms, as potential susceptibility factors for gonadal abnormalities, were also investigated. Subjects and Methods- We evaluated 39 patients with 46,XY GD: 24 patients with the partial, and 15 with the complete form. The entire coding region (éxons 2-5) of DMRT1 and the DM domain (exon 1) were PCR-amplified and direct sequenced. Multiplex ligation probe amplification (MLPA) analysis of DMRT1 was carried out using a commercial kit. Results- Partial or total deletion of DMRT1 was not identified by MLPA technique. Eight allelic variants of DMRT1 were identified. The novel variant c.968-15insTTCTCTCT was identified in 6.4% and in 14.3% of the alleles of 46,XY patients and control subjects, respectively Conclusion- This study suggest that complete or partial DMRT1 deletions and inactivating mutations are not frequently found in patients with abnormalities of gonadal development. Additionally, none of the allelic variants identified in this cohort of patients could be considered a potential polymorphic susceptibility marker for gonadal dysgenesis