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Abstract

miRNAs cooperate and fine tune gene expression on the post-transcriptional level and can therefore be seen as an additional regulatory layer during mouse embryonic stem cell (mESC) self-renewal and differentiation. However, the biological activity of conserved miRNAs during those processes is poorly understood, as most studies cannot uncouple miRNA activity from miRNA expression levels. Therefore, my PhD project focused on studying single cell miRNA dynamics during mESC differentiation towards the three major germ layers and measured miRNA affinities to a miRNA reporter in vivo. I established stable mESC lines expressing fluorescent reporters for the 162 miRNAs conserved in vertebrates and could show that the temporal miRNA activity profile is tightly regulated throughout mESC differentiation. Interestingly, miRNAs exhibit activity changes at early, mid and late stages of stem cell differentiation. Moreover, miRNAs seem to regulate differentiation in a cooperative manner on a global level rather than being germ layer specific. However, this does not exclude single miRNA clusters from being potentially germ layer specific as shown for the highly conserved miR-302 cluster. Strikingly, based on principle component analysis, miRNA activity diverged between germ layer fates already 48 hours after onset of differentiation. In addition, this PhD project experimentally determined miRNA affinities for 119 conserved miRNAs by integrating measurements of miRNA activity and expression levels. I could show that miRNA affinities span several orders of magnitude and are negatively correlated to miRNA expression levels, which suggests that weakly expressed miRNAs can be as potent as highly expressed ones. Knowing the affinity and expression levels in a given cell type, enabled me to rank miRNAs according to their effective potency. This will potentially help to determine which genes are targets of a given miRNA. In summary, this thesis project provides a comprehensive picture of changes in miRNA activity upon mESC differentiation in addition to experimentally determined miRNA affinities. Future gain and loss-of-function experiments of interesting miRNAs in fluorescently labeled fate marker cell lines will potentially reveal miRNAs indispensable for stem cell differentiation.