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Abstract

Stem cells have the remarkable ability to constantly self-renew and create cellular diversity. Adult stem cells in particular are of great interest as they form the pool of cells that provides continuous replacement of vanished cells both in physiological conditions and upon injury. The neuromast of the posterior lateral line in medaka (Oryzias latipes) is an exceptional model to study adult stem cells. As fish grow throughout their entire lives and the neuromasts of the lateral line are continuously exposed to the environment, the neuromasts have constantly active stem cells that react to the need of new cells. So far, the dynamics of neuromast stem cells (NSCs) has not been studied in detail and their regulation has remained elusive. In this thesis, I investigated the proliferation of NSCs by timelapse imaging using keratin 15 (k15) expression as a stem cell marker. This showed that NSCs are highly proliferatively active stem cells in homeostasis. Furthermore, nuclei of the NSCs migrate within the cell in a temporally correlated manner at certain cell cycle steps, resembling interkinetic nuclear migration in which mitosis occurs exclusively on the apical side of the cell. Following the expression of k15 during the cell cycle also revealed that k15 expression is heterogeneous among NSCs, being upregulated in dividing stem cells. Keratins are generally known as markers for stem cells, but their function in stem cell regulation remains unclear. In this work, I therefore investigated the link between the stem cell marker K15 and the behaviour of NSCs by combining fluorescent transgenic lines, 4D imaging and clonal genetic approaches for gain- and loss-of-function of K15. Overexpression of k15 in NSCs resulted in reduced stem cell proliferation visualised by the incorporation of the base analogue bromodeoxyuridine (BrdU). This showed that fewer NSCs underwent S-phase. This result was further confirmed by timelapse imaging, in which fewer stem cells underwent mitosis compared to the wildtype. In contrast, the NSCs of the K15 mutants exhibited increased proliferation detected by BrdU incorporation and timelapse imaging. In addition, neuromast with a mutant K15 showed morphological changes ranging from very small to very large and elongated neuromasts. Clonal analyses in chimeric neuromasts revealed a differential short and long-term behaviour for overexpressing K15 in NSCs. My results suggest that K15 not only serves as a stem cell marker but is also involved in stem cell regulation and stem cell fate. Furthermore, a single-cell RNA sequencing atlas of medaka epithelial cells was generated which forms the basis to further elucidate the regulation of all cell types in the neuromast. Taken together, this work provides new insights into how stem cells are regulated under homeostatic conditions and emphasises the importance of keratins as stem cell markers as regulators of stem cells.