Development of the lung is a highly regulated and complex process that is not fully characterised. Lung development starts at embryonic day (E)9.5 in the mouse where progenitor cells drive morphogenesis of lung lobes and future airways. Progenitor cells express Sox9 but how they are regulated remains to be elucidated. We have developed a novel in vitro assay to expand Sox9-positive cells from E11.5 lung. These cells form pneumospheres and maintain their progenitor activity, enabling the study of factors regulating proliferation and differentiation. RNAseq analysis of pneumospheres overtime revealed an increase in Sox9 expression, and Sox9-downstream targets such as Clusterin and Annexin A4. Alveolar differentiation markers such as surfactant protein c and Rage are downregulated, indicating an enrichment for progenitor cells. Pneumospheres can be maintained in culture for several weeks enabling us to perform genetic or drug screens.
Using this platform, we performed a short-hairpin RNA (shRNA) knockdown screen, targeting 130 genes involved in enzymatic epigenetic regulation. Sequencing at three time-points enabled identification of hairpins that changed overtime. The top genes were validated using in vitro and ex vivo culture systems to determine their role in lung progenitor cells and branching morphogenesis.
These experiments identified Aurora kinase B (Aurkb) as an interesting candidate gene. Aurkb functions as a regulator of cell cycle and epigenetic control through phosphorylation of histones. Disruption of Aurkb by shRNA or by chemical inhibition abrogates growth of lung progenitor cells and causes defects in cell cycle. Future studies aim to decipher the role of Aurkb in lung development using mice carrying a conditional allele of Aurkb.
These studies in the embryo help us understand how developmental genes can become dysregulated later in life leading to disease. Our work highlights the power of interrogating lung development to provide insight into lung diseases and develop novel therapeutic targets.