Cilia are polarized extensions of the cells microtubule-based cytoskeleton dedicated to sensory, signaling and motility-related functions. In mammals, there are two main types of cilia, immotile and motile, where motile cilia generate/modulate fluid flow at the embryonic node, in respiratory airways, cerebral ventricles and the oviduct in addition to sperm propulsion via the flagellum. Defects in cilia motility cause a rare genetic disorder called Primary Ciliary Dyskinesia (PCD). In this thesis, I present functional and molecular characterisation of two PCD causing genes HEATR2 and ZMYND10. Core cilia genes are transcriptionally activated by members of the winged-helix transcription factors of the RFX family. The forkhead transcription factor FOXJ1, additionally activates motility genes such as the ones encoding components of axonemal dynein motors which transfer the chemical energy released from ATP hydrolysis to kinetic motion necessary for ciliary motility. I present data in this thesis which show that Heatr2 and Zmynd10 are both targets of the RFX3-FOXJ1 transcriptional module which co-operatively switches on genes required to make motile cilia Mutations in both HEATR2 and ZMYND10 cause the same subtype of PCD (loss of inner and outer arm dyneins in cilia). I characterise a human PCD causing mutation in HEATR2 in this thesis. Additionally, using genetic null mouse models generated using the CRISPR technology, I describe the phenotypic effects of complete loss of Zmynd10 in mice. Zmynd10 mutant mice display characteristic PCD-like features. Adding to my functional studies, I present proteomic data to propose mechanisms by which HEATR2 and ZMYND10 proteins control cilia motility. Mass spectrometry and protein interaction studies support distinct roles for HEATR2 and ZMYND10 in intracellular transport and pre-assembly of axonemal dynein motors. The multisystem approaches described in this thesis to characterise the roles of HEATR2 and ZMYND10 highlight the molecular complexity underlying the assembly and delivery of axonemal dyneins to motile cilia and provide novel functional and molecular insights into the pathophysiology of PCD.

During embryogenesis, airway epithelial cells possess primary cilia, and HH signaling guides lung development. As epithelial cells mature, they produce hundreds of motile cilia and continue to produce the sonic hedgehog (SHH) ligand, which is found apically in the thin layer of liquid covering airways. However, whether ciliated airway cells express apical HH signaling components and what their function might be have remained unknown. Here we show that motile cilia are enriched for HH signaling proteins, including patched 1 and smoothened. These cilia are also enriched for proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Surprisingly, SHH in differentiated airway epithelia did not elicit the canonical SHH signaling pathway that regulates transcription during development. But instead, activating HH signaling decreases intracellular levels of cAMP, which reduces ciliary beat frequency and airway surface liquid pH, similar to changes that have been observed in the airway of people with chronic obstructive pulmonary disease (COPD). Furthermore, we observed that significant increase of SHH ligand expression in differentiated airway epithelia with COPD, suggesting a potential role of SHH signaling in the pathogenesis of airway disease. Collectively, our study indicates that airway cilia detect apical SHH to mediate airway physiology through noncanonical HH signaling. SHH may dampen defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. This may suggest a potential role of SHH signaling in the pathogenesis of airway disease, such as COPD.

Résumé confidentiel / Confidential abstract

Résumé confidentiel / Confidential abstract

Le cil primaire est une antenne sensorielle présente à la surface de la plupart des cellules qui contrôle des voies de signalisation clés au cours du développement et de l’homéostasie tissulaire. Des défauts de formation ou de fonctionnement des cils sont responsables de maladies génétiques complexes appelées ciliopathies. La néphronophtise (NPH) est une ciliopathie caractérisée par une néphropathie tubulo-interstitielle chronique évoluant généralement vers l’insuffisance rénale terminale (IRT) avant l’âge adulte. La NPH peut être isolée ou associée à des signes extra-rénaux tels que la rétinite pigmentaire et des défauts du squelette permettant de définir des syndromes comme celui de Saldino-Mainzer (MZSDS). La NPH est une maladie à transmission autosomique récessive très hétérogène sur le plan génétique et les protéines codées par les gènes identifiés ont quasiment toutes été impliquées dans des fonctions ciliaires. Le séquençage d’exome de patients, ciblant plus de 1300 gènes ciliaires (ciliome), a permis de mettre en évidence des mutations dans deux nouveaux gènes candidats pour la NPH : CEP83 et TEKT1. Mon travail de thèse a consisté à caractériser l’effet des mutations et à valider leur implication dans les phénotypes des patients. CEP83 a été retrouvé muté chez plusieurs patients non-apparentés présentant une NPH avec IRT précoce (< 5 ans). CEP83 est un composant des appendices distaux du centriole père qui joue un rôle clé dans les étapes précoces de la formation du cil. J’ai montré que les mutations identifiées entraînaient une désorganisation des appendices distaux qui pourrait expliquer les défauts de ciliogénèse observés dans les fibroblastes et les biopsies rénales de patients. Ces résultats ont permis de démontrer l’implication d’une nouvelle protéine centriolaire dans la physiopathologie des formes sévères de NPH. TEKT1 présente des mutations hétérozygotes composites chez un patient ayant un tableau clinique complexe associant un MZSDS et une dyskinésie ciliaire primitive (PCD) due à des défauts de cils motiles. Une analyse génétique détaillée a mis en évidence des mutations sévères dans un second gène, WDR19, déjà caractérisé dans les formes de NPH associées à des défauts osseux. TEKT1 code la protéine Tektine-1, un membre encore non caractérisé de la famille des tektines impliquées dans les cils motiles. L’analyse de cellules nasales multiciliées a montré que Tektine-1 était localisée le long de l’axoneme des cils motiles contrôles et absent des cils des cellules du patient qui présentaient aussi des anomalies sévères de battement. En parallèle, des défauts de ciliogénèse, typiques de mutations de WDR19, ont été observés dans les fibroblastes du patient. Ces résultats suggèrent que ce phénotype complexe est dû aux effets complémentaires des mutations des deux gènes TEKT1 et WDR19, responsables des défauts dans les cils motiles et primaires, respectivement. / The primary cilium is a sensory antenna present on the surface of most of the cells. It controls key signaling pathways during development and tissue homeostasis. Defects in cilia growth or activity are responsible for complex genetic diseases called ciliopathies. Nephronophthisis (NPH) is a ciliopathy characterized by chronic tubulointerstitial nephritis which usually progresses to end-stage renal disease (ESRD) before adulthood. NPH may be isolated or associated with extra-renal defects such as retinitis pigmentosa and skeleton involvement. The combination of these symptoms defines syndromes such as Saldino-Mainzer (MZSDS). NPH is an autosomal recessive disorder highly genetically heterogeneous and almost all of proteins encoded by the identified genes have been involved in ciliary function. The exome sequencing in patients, targeting up to 1300 ciliary genes (ciliome), highlighted new mutations in 2 NPH candidate genes: CEP83 and TEKT1. My work was to characterize the effects of the mutations and validate their involvement in patient phenotypes. CEP83 was found mutated in several unrelated patients with early-onset of NPH (IRT<5 years). CEP83 is a component of distal appendages on the mother centriole which play a crucial role in the early steps of cilia formation. I have shown that the identified mutations perturbed the distal appendages formation which might explain the defects in ciliogenesis observed in fibroblasts and kidney biopsies from patients. These results have demonstrated the involvement of a new centriolar protein in the pathophysiology of NPH severe forms. TEKT1 presents compound heterozygous mutations in a patient with a complex phenotype combining a MZSDS and primary ciliary dyskinesia (PCD) due to defects in motile cilia. The genetic analysis showed mutations in a second gene, WDR19, already characterized in NPH associated with bone defects. TEKT1 encodes the Tektin-1 protein, an uncharacterized member of the tektin family involved in motile cilia. The nasal multiciliated cells analysis showed that Tektin-1 was localized along the axoneme of control motile cilia and absent from the cilia in patient cells, which also had severe beating impairment. In parallel, defects in ciliogenesis, typical of WDR19 mutations, were observed in the fibroblasts from the patient. These results suggest that this dual ciliary phenotype is rather due to the additional effect of mutations in both TEKT1 and WDR19, responsible for the defects in motile and primary cilia, respectively.

Eukaryotic organisms rely on the coordinated beating of motile cilia for a multitude of fundamental reasons. In smaller organisms, such as Paramecium and the single cell alga Chlamydomonas reinhardtii, it is a matter of propulsion, to swim towards a higher concentration of nutrients or away from damaging environments. Larger organisms use instead the coordinated motion of cilia to push fluid along an epithelium: examples common to mammals are the circulation of cerebrospinal fluid in the brain, the transport of ovules in the fallopian tubes, and breaking the left/right symmetry in the embryo. Another notable example, and one that is central to this thesis, is mucociliary clearance in human airways: A carpet of motile cilia helps keeping the cell surface free from pathogens and foreign particles by constantly evacuating from lungs, bronchi, and trachea a barrier of mucus. The question of how motile cilia interact with one another to beat in a coordinated fashion is an open and pressing one, with immediate implications for the medical community. In order for the fluid propulsion to be effective, the motion of cilia needs to be phase-locked across significant distances, in the form of travelling waves (``metachronal waves''). It is still not known how this long-range coordination emerges from local rules, as there is no central node regulating the coordination among cilia. In the first part of this thesis I will focus on studying the coordination in carpets of cilia with a top-down approach, by proposing, implementing, and applying a new method of analysing microscope videos of ciliated epithelia. Chapter 1 provides the reader with an introduction on motile cilia and flagella, treating their structure and motion and reporting the different open questions currently tackled by the scientific community, with particular interest in the coordination mechanisms of cilia and the mucociliary clearance apparatus. Chapter 2 introduces Differential Dynamic Microscopy (DDM), a powerful and versatile image analysis tool that bridges the gap between spectroscopy and microscopy by allowing to perform scattering experiments on a microscope. The most interesting aspects of DDM for this work are that it can be applied to microscope videos where it is not possible to resolve individual objects in the field of view, and it requires no user input. These two characteristics make DDM a perfect candidate for analysing several hundred microscope videos of weakly scattering filaments such as cilia. In Chapter 3 I will present how it is possible to employ DDM to extract a wealth of often-overlooked information from videos of ciliated epithelia: DDM can successfully probe the ciliary beat frequency (CBF) in a sample, measure the direction of beating of the cilia, and detect metachronal waves and read their direction and wavelength. In vitro ciliated epithelia however often do not show perfect coordination or alignment among cilia. For the analysis of these samples, where the metachronal coordination might not be evident, we developed a new approach, called multiscale DDM (multiDDM), to measure a coordination length scale, a characteristic length of the system over which the coordination between cilia is lost. The new technique of multiDDM is employed in Chapter 4 to study how the coordination among cilia changes as a response to changes in the rheology of the mucous layer. In particular, we show that cilia beating under a thick, gel-like mucus layer show a larger coordination length scale, as if the mucus acted as an elastic raft effectively coupling cilia over long distances. This is corroborated by the coordination length scale being larger in samples from patients affected by Cystic Fibrosis than in healthy samples, and much shorter when the mucus layer is washed and cilia therefore beat in a near-Newtonian fluid. We then show how it is possible to employ multiDDM to measure the effectiveness of drugs in recovering, in CF samples, a coordination length scale typical of a healthy phenotype. In the second part I will focus instead on the single cilium scale, showing how we can attempt to link the beating pattern of cilia to numerical simulations studying synchronisation in a model system. In particular in Chapter 5 I will describe our approach to quantitatively describe the beating pattern of single cilia obtained from human airway cells of either healthy individuals or patients affected by Primary Ciliary Dyskinesia. Our description of the beating pattern, and the selection of a few meaningful, summary parameters, are then shown to be accurate enough to discriminate between different mutations within Primary Ciliary Dyskinesia. In Chapter 6 instead I report the results obtained by coarse-graining the ciliary beat pattern into a model system consisting of two ``rotors''. The rotors are simulated colloidal particles driven along closed trajectories while leaving their phase free. In my study, the trajectories followed by the rotors are analytical fits of experimental trajectories of the centre of drag of real cilia. The rotors, that are coupled only via hydrodynamics interactions, are seen to phase-lock, and the shape of the trajectory they are driven along is seen to influence the steady state of the system.

Chez les vertébrés, le battement coordonné des cils motiles présents par centaines à la surface apicale des cellules multiciliées (MCC) est requis pour propulser directionnellement les fluides biologiques à l’intérieur de certains organes (voies respiratoires, ventricules cérébraux, trompes utérines ou certaines structures embryonnaires). De nombreuses pathologies humaines sont associées à des défauts ciliaires ou à une perte des MCC (dyskinésies ciliaires, mucoviscidose, asthme,...). Dans ce contexte, mon travail de thèse a consisté à élucider les mécanismes complexes contrôlant la différenciation des MCC et donc la formation des cils motiles (multiciliogenèse). Par des approches de génomiques fonctionnelles à partir de deux modèles d’épithéliums multiciliés évolutivement éloignés (épithélium respiratoire humain et épiderme d’embryon de Xénope) nous avons identifié la famille des microARN (petits ARN non-codants régulateurs de l’expression génique) miR-449 comme majoritairement exprimée dans les MCC. Nous avons montré que miR-449 contrôle la multiciliogenèse i) en bloquant le cycle cellulaire, ii) en réprimant directement la voie de signalisation Notch et iii) en inhibant l’expression de la petite GTPase R-Ras. Enfin, nos travaux montrent que l’ensemble de ces mécanismes est conservé chez les vertébrés. En conclusion, miR-449 est un nouveau régulateur clé de la multiciliogenèse conservé au cours de l’évolution. Nos résultats pourraient ouvrir la voie à de nouvelles stratégies thérapeutiques utilisant des petits ARN régulateurs dans le traitement de certaines pathologies associées à des défauts ciliaires. / In vertebrates, the coordinated beating of hundreds of motile cilia present at the apical surface of multiciliated cells (MCC) is required for propel directionally flow of biological fluids inside some organs (airways, cerebral ventricles, fallopian tubes or some embryonic structures). Many human diseases are associated with ciliary defects or loss of MCC (ciliary dyskinesia, cystic fibrosis, asthma ...). In this context, my thesis has sought to elucidate the complex mechanisms that control the differentiation of MCC and thus the formation of motile cilia (multiciliogenesis). By functional genomic approaches from two evolutionarily distant models of multiciliated epithelia (human respiratory epithelium and epidermis of Xenopus embryo) we identified the miR-449 family of microRNAs (small non-coding RNAs regulating gene expression) as mainly expressed in MCC. Then, we showed that miR-449 controlled multiciliogenesis by i) blocking the cell cycle ii) directly suppressing the Notch pathway and iii) by inhibiting the expression of the small GTPase R-Ras. Finally, we have demonstrated that all these mechanisms were conserved in vertebrates. In conclusion, miR-449 is a new key and conserved regulator of multiciliogenesis. Our findings could pave the way for new therapeutic strategies using small regulatory RNAs in the treatment of several diseases associated with ciliary defects.