Defining Epithelial Cell Fate Dynamics of the Oesophagus during Postnatal Development and Homeostasis

Epithelial cells can respond rapidly to changing tissue demands to ensure organism survival. However, the mechanisms that finely control how cells adapt their behaviour remain largely unknown. To uncover the rules that govern epithelial cell fate, it is critical to understand their dynamic nature by exploring their response to situations away from homeostasis. Postnatal development provides an ideal physiological model of rapid but restricted tissue growth, in which cells switch from a state of expansion to the steady state of homeostasis. In this project, I investigated the cellular and molecular mechanisms that govern this transition in the mouse oesophageal epithelium. Using wholemounting techniques to explore the whole tissue at 3D single cell resolution, I investigated the postnatal-to-homeostatic transition through studies of morphological features and cell fate dynamics. Comprehensive immunofluorescence studies revealed a defined period in time when homeostatic features become established. Typical adult marker gene expression and tissue morphology, such as keratinization and cornification, were evident from P28, indicating the transition point towards homeostasis. By looking at the localisation of transcription factors associated with cell fate, I identified a temporal gradient of expression that spans postnatal development. After birth, during a period of rapid tissue expansion, a fraction of the basal population was seen to express the regenerative marker SOX9. This population progressively reduced as the tissue reached the transition point, coinciding with the emergence of a new basal population expressing KLF4, which is known to mark keratinocyte differentiation. Next, I questioned the relevance of the KLF4+ basal population by analysing proliferative capacity and the associated keratin profile. The results indicated KLF4 as a marker of commitment towards differentiation in the oesophageal basal layer, balancing progenitor cell behaviour and defining the establishment of tissue homeostasis. To investigate the molecular signature of the basal cell population throughout postnatal development, I executed an in-depth single cell RNA-sequencing analysis, revealing that the onset of homeostasis occurs simultaneously with changes in the expression of genes associated with mechanics. I was able to identify that the postnatal oesophagus is defined by a differential growth, leading to the build-up of longitudinal mechanical strain as the tissue matures. To further study the relevance of tissue strain for epithelial cell behaviour, I developed and applied an in vitro system of whole-organ stretching. This approach unveiled a mechanism whereby mechanical stress at the organ level triggers the establishment of the KLF4-expressing committed basal population in the oesophageal epithelium in a YAP dependent fashion. Together, the results of this project indicate a simple mechanism for the control of epithelial cell fate, whereby mechanical changes at the whole organ level orchestrate the establishment of tissue homeostasis at the cellular level.CRUK Cambridge Cancer Centre PhD fellowshi