Medical Biology - Theses
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ItemSteady-state and emergency dendritic cell development at a clonal level( 2019)Recent clonal fate and single cell RNA-sequencing studies demonstrate that significant lineage imprinting is already in place within individual haematopoietic stem and progenitor cells (HSPCs). Dendritic cells (DCs) represent one such branch of haematopoiesis and are responsible for pathogen-sensing and activation of the adaptive immune response. At the population level, all three major DC subtypes including type 1 conventional DCs (cDC1s), type 2 conventional DCs (cDC2s) and plasmacytoid DCs (pDCs), can be generated from a restricted common DC progenitor (CDP) population downstream of HSPCs. However, recent clonal evidence has suggested earlier subtype-specific imprinting within the CDP and even early HSPC populations. Therefore, the current hierarchical model of haematopoiesis is insufficient to explain the complexity and dynamics of DC development. The aim of this thesis was to investigate the development of DCs at the single cell level. One caveat of most prior single cell lineage tracing studies was that clonal fate was only measured at a single time point. Therefore, questions remain as to whether the fate bias observed at a snapshot in time is consistent with earlier or later times. Here, using cellular barcoding, I develop an experimental and computational framework to allow robust periodical examination of lineage outputs of thousands of transient clones during DC development in vitro. I reveal that single HSPC clones are largely programmed regarding the types of DCs to make (fate), the number of DCs to produce (size), and when DC generation occurs (timing). Together, I define these unique properties as a clone’s cellular trajectory. Importantly, I demonstrate that a large proportion of early HSPCs are already committed towards either cDC or pDC generation, even when clonal output is measured over time. This finding is consistent with and further complements the most recent evidence of DC subtype imprinting during early haematopoiesis. Exogenous administration of Flt3 ligand (FL) is known to preferentially induce ‘emergency’ DC development, and is shown to provide promising therapeutic benefits in various conditions such as infection and cancer. However, how FL signals regulate cell proliferation and differentiation during early DC development is largely unknown. In this thesis, I investigate the clonal aetiology of this process. Using cellular barcoding, I demonstrate that emergency DC generation is predominantly driven by increased expansion of pre-existing HSPC clones that are already primed with DC potential. Consistently, enhanced cell cycle activity is found to be prominent within most early HSPCs after short exposure to FL. In particular, using a single cell multi-omics profiling approach, I identify key cellular and molecular events within a unique group of early HSPCs that are most responsive to FL stimulation, which include increased cell division, maintenance of hyper-proliferative potential and establishment of a DC lineage program. Collectively, the findings presented in this thesis provide new insights into the control and regulation of DC fate within individual HSPCs during steady-state and emergency haematopoiesis, with important implications regarding the maintenance or manipulation of DC generation in health and disease.
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ItemScreening for breath: identifying Aurkb as a novel regulator of lung development( 2019)The development of the lung is a highly regulated and complex process that is not fully characterised. Although there have been many studies into the development of the lung many of the mechanisms regulating lung organogenesis are still unclear. In recent years, the importance of epigenetic regulators in embryogenesis has been established but epigenetic control of lung morphogenesis is largely underexplored. I have developed a novel in vitro assay to grow embryonic lung stem cells. These cells, called pneumospheres express the early lung progenitor factor Sox9 and recapitulate the E11.5 lung throughout their time in culture. Pneumospheres can be genetically and chemically manipulated to assess the role of signalling pathways and genes-of-interest on lung progenitor cell self-renewal or differentiation. Using pneumospheres I performed a shRNA knockdown screen, targeting 130 genes involved in epigenetic regulation. Nineteen genes were identified in the screen and validated using in vitro and ex vivo culture systems to determine their role lung stem cells and branching morphogenesis. These experiments identified Aurora kinase B (Aurkb) as an interesting candidate gene. Aurkb exerts a dual role as a regulator of cell cycle and epigenetic control through phosphorylation of histones. Disruption of Aurkb either by short-hairpin RNA or by chemical inhibition in vitro abrogates growth of lung epithelial progenitor cells and causes defects in cell cycle, leading to an accumulation of cells in G2/M of the cell cycle. Conditional deletion of Aurkb in the embryonic lung, leads to a complete lack of lung tissue at birth and severe epithelial growth retardation can be seen as early as midgestation. Understanding the regulation of lung development will provide a better understanding of the lung organogenesis and how disruptions in normal biology can cause early lung diseases such as bronchopulmonary dysplasia or can have an impact on lung disorders later in life such as COPD or lung cancer.