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Continuous development of the cerebral cortex from the prenatal to postnatal period depends on neurons and glial cells, both of which are generated from neural progenitor cells (NPCs). Owing to technical limitations regarding the transfer of genes into mouse brain, the mechanisms behind the long-term development of the cerebral cortex have not been well studied. Plasmid transfection into NPCs in embryonic mouse brains by in utero electroporation (IUE) is a widely used technique aimed at expressing transgenes in NPCs and their recent progeny neurons. Because the plasmids in NPCs are attenuated with each cell division, the transgene is not expressed in their descendants, including glial cells. The present study shows that an Epstein-Barr virus-based plasmid (EB-oriP plasmid) is helpful for studying long-term cerebral cortex development. The use of the EB-oriP plasmid for IUE allowed transgene expression even in the descendant progeny cells of adult mouse brains. Combining the EB-oriP plasmid with the shRNA expression cassette allowed examination of the genes of interest in the continuous development of the cerebral cortex. Furthermore, preferential transgene expression was achieved in combination with cell type-specific promoter-driven transgene expression. Meanwhile, introducing the EB-oriP plasmid twice into the same individual embryos during separate embryonic development stages suggested heterogeneity of NPCs. In summary, IUE using the EB-oriP plasmid is a novel option to study the long-term development of the cerebral cortex in mice.

The variety of microtubule arrays observed across different cell types should require a diverse group of proteins that control microtubule organization. Nevertheless, mainly because of the intrinsic propensity of microtubules to easily form bundles upon stabilization, only a small number of microtubule crosslinking proteins have been identified, especially in postmitotic cells. Among them is microtubule crosslinking factor 1 (MTCL1) that not only interconnects microtubules via its N-terminal microtubule-binding domain (N-MTBD), but also stabilizes microtubules via its C-terminal microtubule-binding domain (C-MTBD). Here, we comprehensively analyzed the assembly structure of MTCL1 to elucidate the molecular basis of this dual activity in microtubule regulation. Our results indicate that MTCL1 forms a parallel dimer not only through multiple homo-interactions of the central coiled-coil motifs, but also the most C-terminal non-coiled-coil region immediately downstream of the C-MTBD. Among these homo-interaction regions, the first coiled-coil motif adjacent to N-MTBD is sufficient for the MTCL1 function to crosslink microtubules without affecting the dynamic property, and disruption of this motif drastically transformed MTCL1-induced microtubule assembly from tight to network-like bundles. Notably, suppression of the homo-interaction of this motif inhibited the endogenous MTCL1 function to stabilize Golgi-associated microtubules that are essential for Golgi-ribbon formation. Because the microtubule-stabilizing activity of MTCL1 is completely attributed to C-MTBD, the present study suggests possible interplay between N-MTBD and C-MTBD, in which normal crosslinking and accumulation of microtubules by N-MTBD is essential for microtubule stabilization by C-MTBD.