Single-nucleus transcriptomic sequencing and cross integration comparison of multiple species revealed new cell composition and specific gene marker in hippocampal aging of tree shrew. Single-nucleus transcriptomic sequencing and cross integration comparison of multiple species revealed new cell composition and specific gene marker in hippocampal aging of tree shrew

The brain of the tree shrew (Tupaia belangeri chinensis, TS) has drawn considerable attention due to its high similarities to that of humans. The hippocampal formation is one of the main brain regions affected in aging and neurological diseases. However, the cellular compositions of the TS hippocampus involving in aging development remain elusive. Here we established the first single-nucleus transcriptomic profiles of TS hippocampus and identified 16 cell subpopulations. The cross-species comparison of multi-species hippocampus revealed the cell landscape and associated specific gene expression patterns at the single-cell resolution. We validated ROBO2 and FGF14 as a TS/primate-specific NB[1] marker and confirmed TS-specific neural stem cells (NSCs) with SOX5/SOX6 high expression. Then, we identified TS cell types and molecular pathways closely associated with human neurological disorders, bridging the gap between gene mutations and pathogenesis. Importantly, we established the first single-nucleus transcriptomic atlas of TS hippocampal aging, in which the dynamics of the neurogenic lineage and the diversity of oligodendrocyte and microglia was revealed and delineated. Specifically, the regulatory continuum from adult NSCs to immature and mature granule cells was addressed in the neurogenic lineage. Meanwhile, in-depth dissection of gene-expression dynamics revealed impaired neurogenesis along the neurogenesis trajectory; additionally elevated pro-inflammatory responses in the aged microglia and endothelial cells may contribute to a hostile microenvironment for neurogenesis. Together, to our knowledge, this is the first time to report a single-cell atlas of TS in hippocampus aging which therefore provides extensive resources in both cell compositions and specific gene map for future research regarding neural science, evolutionary developmental biology, and regenerative medicine, combined with a comprehensive analysis across species. Overall design: Single-nucleus RNA-sequencing of hippocampus from infancy, adult, and aging tree shrew (Tupaia belangeri chinensis).

树鼩（Tupaia belangeri chinensis, TS）的大脑因与人类大脑具有高度相似性，受到了广泛关注。海马结构是衰老及神经系统疾病主要受累的脑区之一，但目前涉及树鼩海马衰老进程的细胞组成机制仍不甚明确。本研究首次构建了树鼩海马的单细胞核转录组图谱（single-nucleus transcriptomic profiles），鉴定出16种细胞亚群。通过多物种海马的跨物种比较，我们在单细胞分辨率下揭示了其细胞图谱及相关特异性基因表达模式。我们验证了ROBO2与FGF14可作为树鼩/灵长类特异性神经母细胞（neuroblast, NB）[1]标志物，并确认了高表达SOX5/SOX6的树鼩特异性神经干细胞（neural stem cells, NSCs）。随后，我们鉴定出与人类神经系统疾病密切相关的树鼩细胞类型及分子通路，填补了基因突变与发病机制之间的研究空白。尤为重要的是，我们首次构建了树鼩海马衰老的单细胞核转录组图谱，揭示并刻画了神经发生谱系的动态变化，以及少突胶质细胞（oligodendrocyte）和小胶质细胞（microglia）的多样性。具体而言，我们解析了成年神经干细胞向未成熟及成熟颗粒细胞的调控连续体。同时，对基因表达动态的深入分析揭示了神经发生轨迹中受损的神经发生过程；此外，衰老小胶质细胞与内皮细胞的促炎反应增强，可能会构建不利于神经发生的微环境。综上，据我们所知，本研究首次报道了树鼩海马衰老的单细胞图谱，为神经科学、进化发育生物学及再生医学领域的后续研究提供了涵盖细胞组成与特异性基因图谱的丰富资源，并完成了跨物种的综合分析。
整体实验设计：对幼年、成年及衰老树鼩（Tupaia belangeri chinensis）的海马组织开展单细胞核RNA测序（single-nucleus RNA-sequencing）。