Defining the mechanism of Clara cell dedifferentiation into basal stem cells

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DESCRIPTION (provided by applicant): Recently, increasing attention has been paid to defining the progenitor and stem cell populations of the lung and dissecting the molecular mechanisms that govern the behavior of these cells during airway regeneration and repair. Recent studies in multiple different organ systems have demonstrated that well-defined differentiated cells as well as progenitor and stem cell populations are much more plastic than previously recognized. However, the plasticity of airway epithelial cells after injury has not been well documented. Diphtheria toxin-induced cell death in combination with genetic lineage tracing has been used to stringently demonstrate that distinct types of epithelial stem cells in the skin and intestine can interconvert. The same pairing of strategies has also demonstrated that differentiated luminal intestinal epithelial cells can "dedifferentiate" into intestinal stem cells We present preliminary data that suggests that a luminal Clara cell can convert into an airway basal stem cell following injury. We ablated airway basal stem cells by expressing diphtheria (DTA) toxin exclusively in basal cells, which causes those basal cells to undergo apoptosis. In response to this depletion of basal stem cells, luminal Clara cell progenitors begin replicating and then these Clara cells begin to express the basal cell-specific markers p63 and cytokeratin 5. Furthermore, Clara cells that are sorted to purity and cultured ex vivo lose their characteristi Clara cell markers, begin expressing the Yap1 transcriptional co-activator, and then express markers specific to basal stem cells. These dedifferentiated basal-like cells then start replicatin and give rise to colonies. We hypothesize that luminal Clara cell progenitors can convert into basal stem cells when the basal stem cell pool is depleted. To test this hypothesis, we will lineage trace the contribution of Clara cells to the basal stem cell pool after DTA-mediated airway basal stem cell ablation. We will then test whether the dedifferentiated Clara cells possess functional stem cell capacity. We also hypothesize that the Yap1 transcriptional co-activator is necessary for Clara cell dedifferentiation. We will use mouse genetics to test whether Yap1 expression is sufficient and/or necessary for Clara cell plasticity.