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Walcher, Tessa (2013): Molecular dissection of Pax6 DNA-binding domains and their roles in mouse cerebral cortex development. Dissertation, LMU München: Fakultät für Biologie



Generation of the brain depends on proper regulation of progenitor proliferation and differentiation during development. Many such factors known to affect proliferation and differentiation are transcription factors. In particular, the transcription factor Pax6 has received much attention because of its potency to control various aspects of brain development. During development of the telencephalon Pax6 regulates patterning, cell proliferation and neurogenesis, but how Pax6 mediates and coordinates these diverse functions at the molecular level is not well understood. It has previously been demonstrated that the homeodomain of Pax6 plays a role in establishing the pallial-subpallial boundary. However it is not involved in other processes during telencephalic development as shown by the analysis of Pax64NEU mice, which are characterized by a point-mutation in the DNA-binding helix of the homeodomain. In order to gain more insights into the molecular network underlying the mild homeodomain function in the developing telencephalon, transcriptome analysis with Pax64NEU mice was performed. Almost no transcriptional changes were detected, suggesting that transcriptional regulation by the homeodomain of Pax6 has no major impact on forebrain development. Additionally, these results implied that the majority of effects exerted through Pax6 during telencephalic development are mediated by the bipartite paired-domain (PD). Therefore the main focus of this thesis was to examine the specific roles of the Pax6 paired-domain and its individual DNA-binding subdomains (PAI and RED) during forebrain development. The role of these DNA-binding domains was examined using mice with point-mutations in the PAI (Pax6Leca4, N50K) and RED (Pax6Leca2 R128C) subdomains and showed that the mutations in these subdomains exert opposing roles regulating proliferation in the developing cortex. While the mutated PAI domain resulted in reduced proliferation of both apical and basal progenitors, the mutated RED domain provoked increased proliferation. However, the PAI domain largely mediates the neurogenic function of Pax6. Additionally, genome-wide transcriptome analysis was able to unravel the key signatures mediated by the distinct domains. In summary, Pax6 exerts its key roles during forebrain development by use of distinct subdomains to regulate proliferation and differentiation. Thus Pax6 is able to coordinate and fine tune patterning, neurogenesis and proliferation in a simultaneous manner in different radial glial subpopulations. The transcriptional regulation through Pax6 may not only be restricted to protein coding genes, but may also include control of microRNA (miRNA) expression. Such small RNA molecules have recently been implicated in proliferation and differentiation during development, however expression and the role of single microRNAs is still poorly understood. Towards this end, miRNA expression profiling was performed using an embryonic stem cell differentiation system at different stages of neuronal differentiation in order to identify new miRNAs involved in radial glia specification and differentiation. This analysis revealed a number of microRNAs induced during differentiation from neural progenitors to neurons. Most strikingly only four miRNA candidates were found with exclusively high expression in progenitor cells. These data suggest that also Pax6 may play a role in transcriptional regulation beyond mRNAs.