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.

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Les voies aériennes humaines sont bordées d'un épithélium pseudostratifié composé principalement de cellules basales et de cellules pyramidales parmi lesquelles figurent les cellules sécrétrices de mucus et les cellules multiciliées. Toutes ces cellules contribuent à la clairance mucociliaire des voies respiratoires. Cet épithélium se régénère lentement dans des conditions homéostatiques, mais il est capable de se régénérer rapidement après agression grâce à des processus de prolifération, de migration, de polarisation et de différenciation. Chez les patients atteints de maladies respiratoires chroniques telles que la broncho-pneumopathie chronique obstructive, l'asthme ou la mucoviscidose, la réparation tissulaire est souvent défectueuse, caractérisée par une perte de cellules multiciliées et une hyperplasie des cellules sécrétrices, ayant pour conséquence une clairance mucociliaire affectée. La séquence des événements cellulaires conduisant à un tissu fonctionnel ou remodelé est encore mal décrite. Notre principal objectif a été d’identifier les types cellulaires successifs mis en jeu lors de la régénération tissulaire et les événements moléculaires responsables d'une régénération saine ou pathologique. Nous avons analysé la composition cellulaire de l’épithélium des voies respiratoires à plusieurs stades de différenciation en utilisant un modèle de culture 3D in vitro qui reproduit la composition cellulaire in vivo. En appliquant une méthode de transcriptomique sur cellule unique couplée à des méthodes bioinformatiques, nous avons établi les hiérarchies cellulaires permettant de reconstruire les différentes trajectoires cellulaires mises en jeu lors de la régénération de l’épithélium des voies respiratoires humaines. Après avoir confirmé les lignages cellulaires qui ont été précédemment décrits, nous avons découvert une nouvelle trajectoire reliant les cellules sécrétrices de mucus aux cellules multiciliées. Nous avons également caractérisé de nouvelles populations cellulaires et de nouveaux acteurs moléculaires impliqués dans le processus de régénération de l'épithélium des voies respiratoires humaines. Enfin, grâce à ces approches, nous avons mis en évidence des réponses spécifiques de chaque type cellulaire survenant dans des situations pathologiques d’hyperplasie sécrétoire. Ainsi, nos données, en apportant d'importantes contributions à la compréhension de la dynamique de différenciation de l’épithélium des voies respiratoires humaines, ouvrent de nouvelles voies vers l’identification de cibles thérapeutiques. / Human airways are lined by a pseudostratified epithelium mainly composed of basal and columnar cells, among these cells we can find multiciliated, secretory cells and goblet cells. All these cells work together in the mucociliary clearance of the airways. This epithelium regenerates slowly under homeostatic conditions but is able to recover quickly after aggressions through proliferation, migration, polarization and differentiation processes. However, in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, asthma or cystic fibrosis, epithelial repair is defective, tissue remodeling occurs, leading to loss of multiciliated cells and goblet cell hyperplasia, impairing correct mucociliary clearance. The sequence of cellular events leading to a functional or remodelled tissue are still poorly described. Hence, we aim at identifying the successive cell types appearing during tissue regeneration and the molecular events that are responsible for healthy or pathological regeneration. We have analysed airway epithelial cell composition at several stages of differentiation using an in vitro 3D culture model which reproduces in vivo epithelial cell composition. Applying single cell transcriptomics and computational methods, we have identified cell lineage hierarchies and thus constructed a comprehensive cell trajectory roadmap in human airways. We have confirmed the cell lineages that have been previously described and have discovered a novel trajectory linking goblet cells to multiciliated cells. We have also discovered novel cell populations and molecular interactors involved in the process of healthy human airway epithelium regeneration. Using these approaches, we have finally shed light on cell-type specific responses involved in pathological goblet cell hyperplasia. Our data, by bringing significant contributions to the understanding of differentiation’s dynamics in the context of healthy and pathological human airway epithelium, may lead to the identification of novel therapeutic targets.