Temporal gene expression changes in neural stem cells during brain development and maturation

Most neurons of the brain are generated from neural stem cells (NSCs) during embryonic period. In the embryonic brain, NSCs exist in the neuroephithelium. At the midgestation, NSCs start to produce neurons. And at the later time point of embryonic period, NSCs then produce glial cells. Finally, most NSCs are transformed to astrocytes and disappeared. However, in some brain regions, a significant number of NSCs are preserved and neurogenesis continues throughout adulthood in the mammalian brain. In the adult mouse brain, NSCs exist mainly in two regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). It has been reported that NSCs continuously generate new neurons over time and new born neurons are integrated into the functional networks of the olfactory bulb and the hippocampal DG, respectively. Several reports indicated the property of NSCs changes during brain development and maturation, however their detailed mechanisms are still largely unknown. In this project, we focused on the temporal gene expression changes in NSCs during brain development and maturation. To specifically label NSCs and their cell cycle phases in the mouse brain by fluorescence proteins, we used transgenic (Tg) mouse lines; pHes1-d2EGFP (Ohtsuka et al., Molecular and Cellular Neuroscience, 2006), Nestin-mCherry-hGem (Furutachi et al., Nature Neuroscience, 2015), Nestin-mKO2-hCdt1 (unpublished), Nestin-mCherry-nls (unpublished), and GFAP-GFP (Suzuki et al., Neuroscience Research, 2003). In pHes1-d2EGFP Tg mice, destabilized EGFP(d2EGFP) is expressed by the mouse Hes1 promoter. In Nestin-mCherry-hGem and Nestin-mKO2-hCdt1 Tg mice, mCherry-hGem and mKO2-hCdt1 are expressed by the rat Nestin promoter/enhancer, respectively. mCherry-hGem is specifically expressed in the S/G2/M-phase of cell cycle. mKO2-hCdt1 is specifically expressed in the G1/G0-phase. In GFAP-GFP Tg mice, GFP is expressed by the mouse GFAP promoter. In Nestin-mCherry-nls Tg mice, nuclear localization signal-attached mCherry (mCherry-nls) is expressed by the rat Nestin promoter/enhancer. We utilized pHes1-d2EGFP; Nestin-mKO2-hCdt1 double Tg mice to purify NSCs in the G1-phase and pHes1-d2EGFP; Nestin-mCherry-hGem double Tg mice to purify NSCs in the S/G2/M-phase with fluorescence-activated cell sorting (FACS) from the embryonic brain (embryonic day 14.5, E14.5). By the blue laser (488nm) and yellow-green laser (561nm), we collected GFP-positive and mCherry/mKO2-double positive cells. We separately isolated NSCs from the dorsal or ventral brain regions. In the adult brain, a part of GFAP-positive cells functions as NSCs. To selectively purify NSCs both expressing GFAP and Nestin, we applied GFAP-GFP; Nestin-mCherry-nls double Tg mouse. Similar to the embryonic samples, we isolated GFP- and mCherry-double positive cells from the adult SVZ or DG (2-3 months old mice) with FACS. We used one brain for each embryonic brain sample (about 2-5 x 10^5 FACS-collected cells) or thirty brain for adult brain sample (about 1 x 10^5 FACS-collected cells). We purified the total RNA by utilizing the RNeasy Micro Kit (Qiagen) under the protocols of the manufacturer and analyzed the quality of the total RNA by Bioanalyzer. Two nano gram RNA samples were used for generating each sequencing library. We used the SMARTer Ultra Low Input RNA for Illumina Sequencing v3 kit (TAKARA) to generate the libraries, and analyzed the quality by Agilent 2200 TapeStation. All samples were sequenced with paired-end sequencing methods using the Illumina HiSeq 2000 Sequencer. Mapping of 100 bp paired-end reads to the mouse reference genome (UCSC, GRCm38/mm10) was performed by TopHat2 under the protocol of Genedata Expressionist (Genedata).