Sequential transcriptional programs underpin activation of quiescent hippocampal stem cells.

Postnatal neural stem cells are primarily quiescent, which is a cellular state that exists as a continuum from deep to shallow quiescence. The molecular changes that occur along this continuum are beginning to be understood but the transcription factor network governing these changes has not been defined. We show that these transitions are regulated by sequential transcription factor programs. Single-cell transcriptomic analyses of mice with loss- or gain-of-function of the essential activation factor Ascl1, reveal that Ascl1 promotes the activation of hippocampal neural stem cells by driving these cells out of deep quiescence, despite its low protein expression. Subsequently, during the transition from deep to shallow quiescence, Ascl1 induces the expression of Mycn, which drives progression through shallow states of quiescence towards an active state. Together, these results define the required sequence of transcription factors during hippocampal neural stem cell activation. Each independent scRNA-seq experiment comprised a pooled group of 1-2 conditional knockout mice (of either Ascl1, Huwe1 or Mycn cKO genotype) with 1-2 controls that were prepared simultaneously to minimise processing artefacts. In total, 3 independent experiments were performed on Ascl1 cKO mice and controls, 2 independent experiments on Huwe1 cKO mice and controls and 2 independent experiments on Mycn cKO mice and controls. In the Ascl1 experiments, the cKO mice were homozygous for the floxed allele, heterozygous for Glast-creERT2 and homozygous for the cre-reporter YFP allele, while the the control mice were genetically identical but were wildtype for Ascl1 alleles. In the Huwe1 experiments, the male cKO mice were hemizygous for the floxed allele, heterozygous for Glast-creERT2 and homozygous for the cre-reporter YFP allele, while the male control mice were genetically identical but were wildtype for Huwe1. All mice received tamoxifen via oral gavage (100mg/kg) for 5 days starting from P30 (range from P27-P33) and were euthanised 12-days later. For the Mycn experiments, the cKO and control mice were homozygous for the floxed allele, heterozygous for Glast-creERT2 and heterozygous for Nestin-GFP. The Mycn cKO mice received tamoxifen and Mycn control mice received corn-oil via oral gavage for 5-days at P30 (range from P27-P30) before being euthanised 12-days later. Mice were euthanised by cervical dislocation, and the hippocampal dentate gyri were dissected. The dentate gyrus was disassociated using the Neural Tissue dissociation kit (P). The cells were then sorted on a MoFlo XDP (Bechman Coulter) using a 100um nozzle. Debris were removed, followed by two gates to remove aggregates and dead cells, based on DAPI fluorescence. Cells were then gated for YFP or GFP expression. The single-cell suspension (to a maximum of 10,000 cells) was then loaded into the 10x Chromium. On each experimental day, two libraries were prepared, one for each of the experimental groups to control for batch effects, which are strong in this type of data (i.e., cKO and control). All libraries were prepared with 10x Genomics Chemistry, Single Cell version 3.0.1. We also performed CUT&RUN on primary adult mouse hippocampal NSC cultures (AHNSCs), in two replicates. Cultured AHNSCs were collected and captured with ConA beads and incubated with anti-ASCL1 monoclonal antibody overnight at 4 °C in antibody buffer before completion of the protocol, preparation of the sequencing library and sequencing on the NovaSeq 6000 instrument, PE100. **Please note that the processed data files for GSM8594263 sample have been replaced on April 29, 2025**

产后神经干细胞主要处于静息状态，这是一种以从深度静息到浅度静息为连续谱的细胞状态。沿该连续谱发生的分子变化逐渐为人所知，但调控这些变化的转录因子网络尚未被阐明。我们的研究表明，这些转变受时序性转录因子程序调控。对必需激活因子Ascl1功能缺失或功能获得的小鼠进行单细胞转录组分析显示，尽管Ascl1的蛋白表达水平较低，但其可通过驱使海马神经干细胞脱离深度静息状态，促进其激活。随后，在从深度静息向浅度静息的转变过程中，Ascl1诱导Mycn的表达，而Mycn可驱动细胞从浅静息状态向激活状态进展。综上，这些结果明确了海马神经干细胞激活过程中必需的转录因子时序。

每项独立的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）实验均包含1~2只条件性敲除（conditional knockout, cKO）小鼠（基因型为Ascl1、Huwe1或Mycn cKO）与1~2只同期制备的对照小鼠，以最小化处理过程中的伪迹。总计开展了3项针对Ascl1 cKO小鼠及其对照的独立实验，2项针对Huwe1 cKO小鼠及其对照的独立实验，以及2项针对Mycn cKO小鼠及其对照的独立实验。在Ascl1相关实验中，cKO小鼠为floxed等位基因纯合子、Glast-creERT2杂合子以及cre报告基因YFP等位基因纯合子，而对照小鼠遗传背景一致，但Ascl1等位基因为野生型。在Huwe1相关实验中，雄性cKO小鼠为floxed等位基因半合子、Glast-creERT2杂合子以及YFP等位基因纯合子，而雄性对照小鼠遗传背景一致，但Huwe1等位基因为野生型。所有小鼠均于P30（范围为P27~P33）起通过灌胃给予他莫昔芬（100mg/kg），连续给药5天后，于12天后实施安乐死。在Mycn相关实验中，cKO与对照小鼠均为floxed等位基因纯合子、Glast-creERT2杂合子以及Nestin-GFP杂合子。Mycn cKO小鼠接受他莫昔芬灌胃，而Mycn对照小鼠则于P30（范围为P27~P30）起连续5天通过灌胃给予玉米油，12天后实施安乐死。小鼠经颈椎脱臼法安乐死后，分离海马齿状回。使用神经组织解离试剂盒（P）解离齿状回组织。随后使用MoFlo XDP（贝克曼库尔特，Beckman Coulter）细胞分选仪，配以100μm喷嘴对细胞进行分选。基于DAPI荧光信号去除细胞碎片，随后通过两道门控排除聚集细胞与死细胞。再对细胞进行YFP或GFP表达的门控筛选。将单细胞悬液（最多10000个细胞）上样至10x Genomics Chromium系统。每个实验日均制备两份文库，分别对应实验组与对照组，以控制批次效应——此类数据中的批次效应往往较为显著。所有文库均采用10x Genomics单细胞3.0.1版化学试剂制备。我们还采用CUT&RUN技术对原代成年小鼠海马神经干细胞（adult hippocampal neural stem cells, AHNSCs）培养物开展实验，设置两次生物学重复。收集培养的AHNSCs，用ConA磁珠捕获后，于4℃下在抗体缓冲液中与抗ASCL1单克隆抗体孵育过夜，随后完成后续实验流程、制备测序文库，并在NovaSeq 6000测序仪上进行PE100测序。

请注意：GSM8594263样本的处理后数据文件已于2025年4月29日完成替换。