Le processus de formation des cils mobiles multiples (multiciliogénèse) est composé de nombreuses étapes. Récemment, nous avons démontré que les microARNs de la famille miR-449 contrôlent plusieurs de ces étapes. Au cours de mon travail, je me suis concentré sur le rôle joué par miR-449 dans deux aspects du développement de l'épithélium multicilié. Dans les cellules multiciliées, un réseau dense d'actine sous-jacent l'aspect apicale de la membrane cellulaire (coiffe d'actine) est nécessaire pour l'ancrage des multiples corps basaux, et donc pour une ciliogenèse approprié. Dans le cadre de mon travail, j'ai participé à l' identification de la petite GTPase R-Ras comme une des véritables cibles de miR-449. J'ai démontré que la réorganisation de la coiffe d'actine et l'ensemble du processus de multiciliogénèse étaient compromis lorsque l'ARN messager de R-Ras se trouve protégé de la liaison avec miR-449. J'ai aussi contribué à identifier une nouvelle cible de miR-449, le gène Steel, qui code pour le ligand du récepteur transmembranaire à tyrosine-kinase KIT. La repression de Steel par miR449 est impliquée dans le processus par lequel les cellules multiciliées atteignent leur position finale dans l'épiderme embryonnaire de Xenopus. STEEL, qui agit probablement comme une molécule de guidage pour les cellules multiciliées qui expriment KIT, doit être réprimé par miR-449 dans ces mêmes cellules en cours de migration pour assurer leur deplacement directionnel approprié. En conclusion, mon travail a contribué à élucider le rôle complexe joué par le miARN miR-449 dans le processus de multiciliogénèse chez les vertébrés. / The process of multiple motile cilia formation (multiciliogenesis) is composed of many different steps. Recently, we demonstrated that microRNAs of the miR-449 family control several of these steps. During my work, I focused on the role played by miR-449 in two aspects of the development of the mucociliary epithelium. In multiciliated cells, a dense actin network underlying the apical aspect of the cell membrane (actin cap) is required for the anchoring of the multiple basal bodies, and therefore for proper ciliogenesis. Small GTPases play important role in the formation of the actin cap. In the course of my work, I took part in the identification of transcripts coding the small GTPase R-Ras as bona fide targets of miR-449. I demonstrated that apical and subapical actin network reorganization and multiciliogenesis were impaired when R-Ras mRNA was protected from miR-449 binding. Moreover, the actin cap formation and multiciliogenesis were rescued when the translation of protected R-Ras transcripts was prevented. I also contributed to the finding that a new miR-449 target, the KIT receptor tyrosin kinase ligand STEEL, is involved in the process through which the multiciliated cells reach their final position within the developing frog epidermis. STEEL, which likely acts as a guidance molecule for the KIT-expressing multiciliated cells, needs to be repressed by miR-449 within the migrating cells to ensure their proper directional migration. Altogether, my work contributed to elucidate the complex role played by the miR-449 miRNA in the process of vertebrate multiciliogenesis.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18427号 / 生博第307号 / 新制||生||41(附属図書館) / 31285 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 根岸 学, 教授 豊島 文子, 教授 見学 美根子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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<p> The ErbB2/Neu orphan receptor tyrosine kinase is amplified in 20-30% of breast and ovarian cancers and predicates poor patient prognosis. Five conserved tyrosine residues, autophosphorylated by Neu catalytic activity, "dock" adaptors and second messengers that activate discrete signalling pathways, most prominently the Ras/MAPK pathway, to regulate cell survival and proliferation. </p> <p> Genetic analysis using Drosophila provides an efficient means for identifying evolutionarily conserved signalling components. Neu and Drosophila EGFR overexpression directs Drosophila tissue development synonymously. Consistent with biochemical evidence, genetic analysis ofNeu signalling through individual pTyr revealed activating signals for 4(Yl144, YB; Y1201, YC; Y1226/7, YD; Y1253, YE) of the 5 sites. Strong Ras-dependent signalling was mediated through adaptors Grb-2 (YB) and SHC (YD). In contrast to biochemical evidence, a strong Ras component was not genetically detected for YC or YE. </p> <p> We have conducted two enhancer-suppressor screens to identify novel Ras and non-Ras requirements for YC signalling. For the first screen, a quantitative approach was designed to identify modification of an YC-specific wing notch phenotype. Thirty-two members of the Ras/MAPK signalling cassette were assessed. Sensitivity to Ras, Raf, MAPK and the Ras-related GTPase R-Ras was identified downstream ofYC. The second screen, a large-scale mutagenesis, took advantage of an YC-induced rough eye phenotype. From over 19 000 screened flies, 11 enhancers and 6 suppressors were isolated. One strong suppressor has been genetically mapped to the dual transcription factor and phosphatase eyes absent. Other promising Ras-dependent and Ras-independent modifiers await farther mapping. Results from both screens point to YC as a unique pTyr that uses both Ras-dependent and Ras-independent outputs. </p> / Thesis / Doctor of Philosophy (PhD)