Thesis advisor: John Wing / Thesis advisor: Stephen Wicks / Molecular biology and genetics, single nucleotide polymorphism genetic mapping, phenotypic assays including behavioral assessment, and fluorescent microscopy of GFP-tagged proteins were used to study ciliary defects in the nematode Caenorhabditis elegans. Mammalian cilia are multifunctional. Some of the physiological roles in which they are involved include sensing developmental signaling molecules and ligands as well as creating flows of mucus and cerebrospinal fluid that function as flow meters and mechanosensors. Due to the multifunctional nature of cilia, it is not surprising that many human diseases can be caused by ciliary defects. Bardet-Biedl Syndrome is a rare genetic ciliopathy characterized by retinal degeneration, polydactyly, obesity, cystic kidneys, mental retardation, and many other ailments. We have identified osm- 12/bbs-7 to be a C. elegans homologue of human BBS7, a gene known to cause Bardet-Biedl Syndrome when mutated. With the help of Michel Leroux’s group, I showed the BBS-7 protein to be localized to the base of cilia and to undergo intraflagellar transport along the ciliary axoneme. Our findings suggest that BBS- 7 plays a role in the assembly and/or functioning of the IFT complex. I also performed a mutagenesis and phenotypic screen for animals defective in the uptake of DiI into a subset of their ciliated neurons in order to identify new components involved in ciliogenesis and IFT. I describe an extended bulked segregant analysis (BSA) mapping methodology, which can save time and resources by filtering out alleles of previously known genes without performing time-consuming interval mapping. In addition, I identified one of the 11 dyefilling defective alleles from the screen to be a novel allele of dyf-3, which encodes a protein required for sensory cilia formation. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The maintenance of the airway/respiratory epithelium during adult homeostasis and repair and its construction during embryonic development require tightly regulated cell fate decisions. This regulation takes the form of complex transcription factor and signalling cascades, much of which are unknown, particularly in human lung development. Multiciliogenesis describes the process of specification/differentiation of airway epithelial progenitors/stem cells into mature multiciliated cells (MCCs). Here, I have identified 2 novel transcription factors, Fank1 and Jazf1 which form part of the transcription factor cascade regulating multiciliogenesis in adult and embryonic mouse tracheas. Mouse tracheal epithelium is representative of epithelium lining the entire human airway and it is possible that we will also be able to extrapolate these findings to the human airway. It is not until we fully understand the regulation of multiciliogenesis that it will be possible to look at ways of pushing basal cells towards a MCC fate for purposes of cell replacement therapy, for example in patients with mucociliary disease. As well as exploring cell fate decisions in the mouse upper airway epithelium using embryonic tracheal explants and mouse tracheal epithelial cell (MTEC) cultures, I have also explored the regulation of cell fate decisions in distal human lung epithelium at the pseudoglandular stage of development. At this stage SOX9+ distal tip cells are self-renewing and multipotent and give rise to SOX2+ stalk descendents, which differentiate into airway epithelium. The regulation of SOX9+ lung tip cell multipotency and migration of SOX2+ stalk descendents during human lung development is poorly understood. I have compared human tip (SOX9+) versus stalk (SOX2+) transcriptomes using gene ontology (GO), which has highlighted some key signalling pathways enriched in tip cells which could be important in maintaining distal tip cell multipotency. These pathways have been utilised in optimising conditions for propagating self-renewing tip-derived organoids. These organoids have the potential to be differentiated into bronchiolar and alveolar fates and as such are an invaluable research tool for studying human lung epithelial development, whilst minimising the use of human embryos and its associated ethical implications. I have also performed human tip versus mouse tip transcriptome GO analysis which highlights that although there are many similarities, there are also differences between human and mouse lung epithelium development, emphasising the need for research on human tissue.

Les flagelles sont des organites essentiels chez la plupart des eucaryotes, y compris chez l’Homme. Ils possèdent une structure cylindrique composée de neuf doublets de microtubules appelée axonème qui est conservée au cours de l’évolution. Ils sont construits par un mécanisme appelé Transport IntraFlagellaire (IFT). Malgré des variations de composition et de longueur entre différents types de cils, la longueur des cils d’un type cellulaire donné est étroitement contrôlée. Toute anomalie de la longueur du flagelle ou de la machinerie IFT peut entraîner de graves dysfonctionnements cellulaires, y compris chez l'homme, où elles sont associées à des maladies génétiques appelées ciliopathies. Au cours de ma thèse, nous avons dans un premier temps étudié le rôle et le fonctionnement de l'IFT chez Trypanosoma brucei, un parasite protozoaire flagellé qui est un excellent modèle pour étudier les cils. En utilisant le FIB-SEM, nous avons démontré que les trains IFT n’étaient présents presque exclusivement que sur deux des neuf doublets microtubules de l'axonème. Puis, l'utilisation de la microscopie à haute résolution nous a permis de démontrer dans des cellules vivantes que ces deux voies sont utilisées pour l’IFT dans les deux sens sur chacun de ces doublets. Nous avons ensuite étudié les mécanismes contrôlant la longueur du flagelle et proposé un nouveau modèle appelé «grow and lock» où le flagelle s'allonge avec un taux de croissance constant jusqu'à ce qu'un signal bloque son élongation ou son raccourcissement. Pour finir nous avons étudié l’implication ce modèle durant le cycle parasitaire, lorsque de la construction de flagelles de très longueurs différentes. / Cilia and flagella are essential organelles in most eukaryotes including humans. They share a canonical cylindrical structure composed of nine doublets of microtubules called the axoneme that is conserved during evolution. They are built by an active mechanism termed Intraflagellar Transport or IFT. Despite some variations in composition and length between different types of cilia, the length for a given cell type is tightly controlled. Any defect in flagellum length or IFT machinery can lead to serious cellular dysfunctions, including in humans where it is associated to genetic diseases called ciliopathies. During my thesis, we have first investigated the role and functioning of IFT in Trypanosoma brucei a flagellated protozoan parasite that is a powerful model to investigate cilia. Using Focus Ion Beam-Scanning Electron Microscopy (FIB-SEM), we have demonstrated that IFT trains are present almost exclusively on only two out of nine microtubules doublets of the axoneme. Then, the use of high-resolution microscopy allowed us to observe in live cells that two tracks are actually used for bidirectional IFT trafficking. We have investigated mechanisms controlling flagellum length and propose a new model named “grow and lock” where the flagellum elongates at a constant growth-rate until a signal blocks further elongation or shortening. Finally this and other models have been investigated during the parasite cycle, when trypanosomes construct flagella with very different lengths.

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La polarité cellulaire est un déterminant essentiel dans le maintien de l’architecture tissulaire et la fonction de l’organe. Ainsi, la division cellulaire, la ciliogenèse, la prolifération, et la migration sont des évènements étroitement associés au processus de la polarisation cellulaire. L’altération de la polarité cellulaire contribue à la perte de l’intégrité des épithéliums et favorise le développement des cancers. La signalisation des lipides, telle que des phosphatidylinositols (PtdIns) joue un rôle vital dans la polarité apico-basale. Dans cette étude, nous avons développé des recherches pour mieux comprendre les mécanismes impliqués dans les effets de la phosphatase SHIP2 sur la polarité cellulaire. Nous avons pu démontrer que SHIP2 est impliquée dans la formation du site d’initiation de la formation de la lumière (AMIS) en régulant d’une part la contractilité acto-myosine induite par RhoA kinase et d’autre part YAP, un composant de la voie de signalisation Hippo. De plus, nous avons montré que l'inhibition de SHIP2 contribue à un défaut dans la formation de fuseau mitotique et dans le clivage de ce fuseau mitotique. La surexpression de SHIP2 induit une lumière large et des cils allongés attribuables à la diminution de l’expression de YAP, Aurora A et HEF1. Par contre, la diminution de l’expression de SHIP2 inhibe la formation des cils en provoquant la surexpression de YAP, Aurora A et HEF1 et ainsi l’apparition d’un phénotype multilumens. L’ensemble de nos travaux définissent un nouveau rôle de SHIP2 dans le maintien de l’intégrité et de l’homéostasie des cellules épithéliales. Nous avons aussi pu démontrer que l’expression de SHIP2 peut discriminer les différents cancers du foie (HCC, ICC et mixte) et que SHIP2 et Merlin/NF2, une protéine de la voie de signalisation Hippo, ont une forte expression dans le cholangiocarcinome (ICC) qui s’oppose à celle de YAP et de RhoA kinase. / Cell polarity is critical caracteristic for the maintenance of tissue architecture. Cell division, ciliogenesis, cell proliferation and migration are events tightly associated to cell polarization processes. Alteration in cell polarity contributes to loss of epithelium integrity and enhances cancer development. Lipids signaling, such as phosphatidylinositol (PtdIns), play a vital role in apico-basal polarity. In this study, we developed researches to better understand mechanisms implicated the role of the phosphatase SHIP2 in cell polarity. We demonstrated that SHIP2 is implicated in formation of the apical membrane initiation site (AMIS) by regulating YAP, a component of Hippo pathway, and RhoA-dependant acto-myosin contractility. Furthermore, we demonstrated that inhibition of SHIP2 contributes to defect in the formation and cleavage of the mitotic spindle. Overexpression of SHIP2 induced a large lumen with long cilia due to a decrease in YAP, Aurora A and HEF1 luminal localization. On the contrary, down regulation of SHIP2 impaired cilia outgrowth by increasing Aurora A, HEF1 and YAP luminal localization with appearance of a multilumens phenotype. Thus, our results reinforced the role of SHIP2 in maintain of integrity and homeostasis of epithelial cells. In this study, we also demonstrated that expression of SHIP2 distinguished the different types of liver cancer (HCC, ICC and mixte), and that SHIP2 and Merlin/NF2 are overexpressed in ICC which is the opposite of YAP and RhoA expression.

Cell polarity is an essential property of adult neurons, which rely on asymmetric distribution of receptors and transmitters for proper signal propagation and cell function. In the retina, loss of photoreceptor (PR) polarity can lead to retinal dystrophies such as Leber Congenital Amaurosis, but the molecular mechanisms involved in regulating PR polarity remain unclear. A highly conserved protein complex involved in the establishment of cell polarity from C. elegans to mammals is the Par complex. Localized at the subapical region of polarized cells, it is composed of the “partitioning defective” PDZ domain-containing proteins Par3/Par6 and the atypical protein kinase C (aPKC). Although extensively studied in epithelial cells, the role of the Par complex in mammalian neurons remains poorly understood. Our unpublished results indicate that conditional inactivation (cKO) of Par3 in the developing retina interferes with the polarized growth of the photosensitive cilium at the apical tip of PR cells, eventually leading to PR degeneration. To uncover how Par3 might regulate ciliogenesis in PR cells, we immunoprecipitated Par3 from mouse retinal extracts and carried out mass spectrometry analysis. We found a cluster of calcium/calmodulin-dependent protein kinase II (CaMKII) proteins as potential Par3-interacting partners in the retina. CaMKII is one of the most abundant proteins found in the central nervous system, where it constitutes 1-2% of total proteins. While extensive studies have demonstrated the importance of CaMKII in long-term potentiation (LTP), long term depression (LTD) and dendrite arborisation, its role in cell polarity remains unknown. Using tagged versions of Par3 and CaMKIID, we validated their interaction in vivo and in vitro by co-immunoprecipitation. Interestingly, we found that CaMKIID localizes to the ciliary region of PRs, suggesting that Par3 might recruit CaMKIID at the apical membrane of PR cells, where it could be involved in ciliogenesis. To explore this hypothesis, we investigated whether dominant-negative or constitutively active forms of CaMKIID could impact cilia formation in PRs. Interestingly, overexpression of both mutant forms of CaMKIID during PR development resulted in shortening of the photosensitive cilia (outer segments), similar to what we observed in Par3 cKO retinas. This study suggests that a CaMKIID/Par3 protein complex regulates the establishment of PR cell polarity, raising the possibility that this complex may be generally involved in controlling neuronal polarity throughout the nervous system. / Le traitement et la propagation de l’information nerveuse repose sur une distribution asymétrique de récepteurs et d’émetteurs à la surface de chaque neurone. Ce cloisonnement en domaines sous-cellulaires distincts est également appelé polarité cellulaire. Dans la rétine, la perte de polarité des photorécepteurs peut entraîner des dystrophies rétiniennes telle que l'amaurose congénitale de Leber, mais les mécanismes moléculaires impliqués restent flous. Un complexe protéique impliqué dans l'établissement de la polarité cellulaire, hautement conservé de C. elegans aux mammifères, est le complexe PAR. Localisé au niveau de la région sous-apicale des cellules polarisées, le coeur de ce complexe est constitué des protéines de la famille partitioning defective Par3 / Par6 et de la protéine kinase C atypique aPKC. Bien que largement étudié dans les cellules épithéliales, le rôle du complexe Par dans les neurones de mammifères reste mal compris. Nos résultats indiquent que l'inactivation conditionnelle (cKO) de Par3 dans la rétine de souris en développement interfère avec la croissance polarisée du cil photosensible à la pointe apicale des cellules photoréceptrices (PR), conduisant finalement à une dégénérescence des PRs. Pour découvrir comment Par3 pourrait réguler la ciliogenèse des PRs, nous avons immunoprécipité Par3 à partir d'extraits rétiniens de souris et effectué une analyse par spectrométrie de masse. Nous avons trouvé un ensemble de protéines appartenant à la famille des calcium-calmoduline-dépendantes de la protéine kinase II (CaMKII) comme partenaires potentiels de Par3 dans la rétine. Les CaMKII figurent parmi les protéines les plus abondantes du système nerveux central où elles constituent 1 à 2% des protéines totales. Alors que des études approfondies ont démontré l'importance de CaMKII dans la potentialisation et la dépression à long terme (LTP et LTD), et l'arborisation des dendrites, son rôle dans la polarité cellulaire reste inconnu. En utilisant des versions étiquetées de Par3 et CaMKIID, nous avons validé leur interaction in vivo et in vitro par co-immunoprécipitation. Nous avons mis en évidence une localisation de CaMKIID dans la région ciliaire des PR, suggérant que Par3 pourrait recruter CaMKIID à la membrane apicale des cellules PR, où il pourrait être impliqué dans la ciliogenèse. Pour explorer cette hypothèse, nous avons étudié si les formes dominantes négatives ou constitutivement actives de CaMKIID pouvaient avoir un impact sur la formation des cils des PRs. vii La surexpression des deux formes mutantes au cours du développement des PRs a entrainé un raccourcissement des segments externes, semblable à ce que nous avons observé dans les rétines Par3 cKO. Cette étude montre qu'un complexe de protéines CaMKIID / Par3 pourrait réguler l’établissement et le maintien de polarité des PRs, suggérant l’implication ce complexe dans le contrôle de la polarité neuronale de l’ensemble du système nerveux central.