Single cell transcriptomics reveal regulators of progenitor cell fate and postmitotic maturation during brain development
In this thesis the NanoString nCounter technology was applied to validate an improved PCR-based strategy for quantitative and qualitative global single cell transcriptome analysis. RNA isolates from whole tissue were quantitatively compared with cDNA amplificates prepared from single cell equivalent dilutions of the same RNA. A significant correlation was obtained between the values measured for RNA and cDNA amplificates for transcript copy numbers from 10 to several thousand. This method was then used to investigate two distinct neurodevelopmental issues. The first neuroscientific question dealt with the generation of cortical projection neurons, whose fate is specified at the progenitor level and depends on the mode of division. With the help of the high resolution of the single cell amplification method, it was possible to provide evidence for an extra-cortical fine-tuning of cortical progenitor output via the Eph-receptor/ephrin-ligand system. The method was further applied to investigate cortical interneuron development, especially to their post-mitotic phase of tangential migration. To identify factors orchestrating the migration, we analyzed single cell transcriptomes of cells derived from distinct proliferative niches of interneurons in the basal telencephalon. Interestingly, we observed DNA methyltransferase 1 (Dnmt1) expression in a fraction of post-mitotic POA-derived cortical interneurons. Deletion of Dnmt1 in this interneurons caused defective migration, resulting in drastically reduced numbers of cortical interneurons in adults. Next generation sequencing analysis of FAC-sorted Dnmt1 wildtype and knockout mice revealed DNMT1-dependent repression of genes involved in late maturational processes like neurite outgrowth. In this context, further experiments provide evidence that DNMT1 preserves the migratory shape of postmitotic GABAergic interneurons in part through negative regulation of Pak6, which stimulates neuritogenesis at post-migratory stages.
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