Summary Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage-tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage-tracing in vivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multi-lineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal, but ultimately leads to terminal astrocytic differentiation and depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into in vivo properties of adult neural stem cells.

Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

DNA methylation has been traditionally viewed as a highly stable epigenetic mark in post-mitotic cells, however, postnatal brains appear to exhibit stimulus-induced methylation changes, at least in a few identified CpG dinucleotides. How extensively the neuronal DNA methylome is regulated by neuronal activity is unknown. Using a next-generation sequencing-based method for genome-wide analysis at a single-nucleotide resolution, we quantitatively compared the CpG methylation landscape of adult mouse dentate granule neurons in vivo before and after synchronous neuronal activation. About 1.4% of 219,991 CpGs measured show rapid active demethylation or de novo methylation. Some modifications remain stable for at least 24 hours. These activity-modified CpGs exhibit a broad genomic distribution with significant enrichment in low-CpG density regions, and are associated with brain-specific genes related to neuronal plasticity. Our study implicates modification of the neuronal DNA methylome as a previously under-appreciated mechanism for activity-dependent epigenetic regulation in the adult nervous system.