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. Overall design: 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**