The role of Dlk1 in the gene regulatory network underlying motor neuron diversification

Wasser, Yasmine; Marquardt, Till (Thesis advisor); Zimmer-Bensch, Geraldine Marion (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

Delta-like homologue 1 (Dlk1) has been established as molecular determinant of motor neuron (MN) functional diversification. Previous studies in mouse and chicken embryo revealed Dlk1 dependent gene expression during MN development, which promotes fast alpha MN subtype properties. However, previous experiments to identify Dlk1 interaction partners and components of the Dlk1 pathway were not sufficient to fully resolve the pathway. This thesis addresses the mechanism through which Dlk1 promotes the fast alpha MN properties. Based on previous observations, the major focus of the thesis was hypoxia dependent Dlk1 regulation and its contribution to MN diversification during embryogenesis. Prior, E12.5 embryos were analysed with regards to Dlk1 protein expression and the evidence of lower oxygen conditions, as different oxygen levels could have an influence during neurogenesis. Dlk1 is influenced by lower oxygen levels in cancer-dependent gene regulations. Therefore, Dlk1 might also be influenced by low oxygen levels during neurogenesis. However, since no meaningful differences in hypoxia could be observed in the spinal cord of the embryo, the focus was shifted to MN development in vitro. Therefore, mouse embryonic stem cells were differentiated into MNs and analysed. With this approach, a decrease in the amount of full-length Dlk1 protein could be observed, triggered by hypoxic stimulation. Next, Dlk1 regulation was also investigated in situ in murine brain slices. Here, it was possible to verify the effects obtained in embryonic stem cell-derived MNs. It has been shown, that full-length Dlk1 protein levels decrease after a hypoxic stimulus, regardless of the age and gender of the mice. To further investigate the decrease of Dlk1 protein after hypoxia, a genetically modified embryonic stem cell line was used to investigate Dlk1 processing. It was found that Dlk1 undergoes cleavage after hypoxic stimulation. Furthermore, it could be shown that the cleavage is likely dependent on the TACE/Adam17 protease. Evidence was found for the nuclear translocation of the cleaved Dlk1 intracellular fragment upon hypoxia-mediated cleavage. Taken together, Dlk1 is cleaved by Adam17 in an extracellular and intracellular fragment, triggered by an external hypoxic stimulation. The Dlk1 intracellular fragment in part translocated to the nucleus, where it could function as a transcriptional co factor and mediate the gene expression signature implemented by Dlk1. Further, the thesis analyses if Mmp9 gene regulation is might be associated with the hypoxic-stimulated Dlk1-dependent process. Dlk1 processing in cancer-dependent cell regulation could indicate an influence on the extracellular matrix reorganisation. Hereafter, Dlk1 might be involved in the Mmp9 secretion mediated by hypoxic induction.