The role of translation in keratinocyte cell fate determination.
Mammalian epidermis is both maintained and repaired after injury by a population of proliferating progenitor cells localised in the basal layer of the tissue. While progenitor cells normally remain in the basal layer throughout their lifetime, differentiating cells move up the tissue and are eventually shed off, helping to maintain tissue homeostasis. Cultured human keratinocytes generate a range of colony sizes, but can be separated into two groups based on the ratio of outcomes after cell division. On average, ‘balanced’ colonies give rise to equal numbers of proliferative and differentiating cells, whereas ‘expanding’ colonies grow in an exponential manner, with the majority of divisions resulting in the generation of two proliferative daughter cells. Previous work has demonstrated that cells in the middle third of a growing ‘expanding’ colony spontaneously switch their behaviour towards the ‘balanced’ state. Cells are also able to switch back from ‘balanced’ to ‘expanding’ after a scratch injury. These results suggest the existence of a tuneable mechanism determining the fate of proliferative keratinocytes, but the molecular identity of this switch remains unknown. In this thesis I will discuss the potential role of protein translation levels in defining the division outcomes of cultured human keratinocytes. Using a combination of cell imaging and RNA sequencing technologies, I show that the two modes of proliferation produce distinct transcriptional and translational profiles. I am able to distinguish between the ‘balanced’ and ‘expanding’ colonies at a very early stage during colony growth using specific markers for translation and a global transcription marker suggesting a colony’s fate is set early on. Finally, use of RNA interference allowed me to knock-down a specific Eukaryotic translation initiation factor, leading to an apparent switch in the colony’s proliferative potential. This suggests that translation level may actually determine the cell fate of human keratinocytes and is not only a marker of keratinocyte differentiation. Overall, these findings bring us closer to understanding the molecular basis of progenitor fate determination, and may give us insight into how epidermal homeostasis is maintained and how it is modulated in tissue repair and cancer.