[Introduction] Expansion and folding in the neocortex are associated with unique cognitive abilities that distinguish human from other mammalian species. At present, a set of human- or primate-specific genes have been proven that promote cortical expansion and folding, such as TMEM14B. Since cortical folding emerges progressively during evolution, multiple genes, not only specific genes, but also conserved genes, should be involved in this process. In parallel, we have previously reported that promoter-associated non-coding RNAs (pancRNAs) transcribed from bidirectional promoter act on cis-acting elements in the transcriptional regulation of neighboring genes. Among such pancRNAs, we are focusing on pancCD63 because it is expressed in the human, but not in the mouse neural stem cells (NSCs). CD63, known as an exosomal marker, is also expressed much higher in human NSCs. [Materials and Methods] Human NSCs, AF22 cell line, were infected by lentivirus in knockdown experiments. Immunostaining detecting active caspase3 and cell cycle labeling assay using EdU were performed to analyze cell apoptosis and proliferation, respectively. As a gain-of-function experiment, in-utero electroporation was performed on E13.5 mouse embryos, and plasmid DNA was microinjected through the uterus into the lateral ventricle. [Results] Knockdown of pancCD63 reduced expression level of CD63, suggesting that pancCD63 can be as a regulatory molecule to affect CD63 expression. Knockdown of either CD63 or pancCD63 resulted in a dramatical decrease in EdU+ cells and an increase in active caspase3+ cells, which suggests that pancCD63-CD63 pair promote human NSC proliferation. Overexpression of CD63 in mouse brain increased number of basal progenitors marked by Pax6. It is to be noted that, at E18.5, CD63 overexpression generated a large number of upper layer neuron and showed a folding-like structure. In summary, pancCD63-CD63 pair plays a key role in cortical development and folding.
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