Intravital imaging identifies long-term self-renewing stem cells in the adult mouse hippocampus

Neural stem cells (NSCs) generate new neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used chronic in vivo 2-photon microscopy and followed single NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI Family Zinc Finger 1 (Gli1) or Achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they becoming exhausted. Using protein expression profiling and single-cell RNA sequencing (scRNA-seq), we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here provide the cellular framework for how functional diversity of NSCs enables the generation of new neurons in the adult hippocampus. Comparison between Gli1- and Ascl1-targeted tdTomato+ cells. 5 days or 12 weeks after tamoxifen injection, tdTomato+ cells were isolated from the dentate gyrus of 3 mice per group per timepoint. Single cells were FACS sorted into 384 -well plates. Seven 384-well plates were processed according to the SmartSeq2 protocol using a microliter pipetting robot and sequenced on an Illumina HiSeq2500 or HiSeq4000.

神经干细胞（Neural stem cells, NSCs）可在哺乳动物整个生命周期内于海马体中产生新生神经元。然而，成体大脑内单个神经干细胞的长期自我更新潜能仍未明确。本研究采用慢性活体双光子显微镜成像技术，对通过干细胞表达基因GLI家族锌指蛋白1（GLI Family Zinc Finger 1, Gli1）或无翅-翅状同源物1（Achaete-scute homolog 1, Ascl1）的调控元件驱动的条件性重组进行遗传标记的单个神经干细胞进行追踪。通过对神经干细胞及其子代细胞开展活体成像，我们鉴定出一类在成体海马体中具有长期自我更新能力的Gli1靶向神经干细胞群体。与之形成对比的是，Ascl1靶向神经干细胞一旦被激活，仅会进行有限的增殖活动，随后便会耗竭。通过蛋白质表达谱分析与单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），我们发现Gli1与Ascl1靶向细胞拥有高度相似却又存在显著差异的转录谱，这证明了具有多样行为特性的异质性神经干细胞群体的存在。综上，本研究为神经干细胞的功能多样性如何促成成体海马体的神经元新生提供了细胞框架。本研究对Gli1与Ascl1靶向的tdTomato阳性细胞进行了比较：在他莫昔芬注射后第5天或第12周，分别从每组每时间点3只小鼠的齿状回中分离tdTomato阳性细胞；通过荧光激活细胞分选（FACS）将单细胞接种至384孔板中；共使用7块384孔板，依照SmartSeq2实验流程，借助微升移液机器人进行处理，随后在Illumina HiSeq2500或HiSeq4000测序平台上完成测序。