Project description:To identify pathways regulating NSC quiescence, with a possible link to quiescence depth, we used double transgenic Tg(her4:drfp);Tg(mcm5:gfp) fish and paradigms predicted to highlight deep vs shallower quiescence. In adult fish (3 month-post-fertilization -mpf-), we performed bulk RNA sequencing on FACS sorted RFPhigh,GFPneg qNSCs (expressing strongly her4, signing NSCs transcriptionally remote from activation), and activated NSCs.
Project description:To elucidate the transcriptional and epigenetic alterations underlying the neurogenic defects of FA-NSCs, we conducted gene expression microarray analysis and global DNA methylation profiling. The gene expression pattern of gene-corrected NSCs (C-FA-NSCs) resembled that of control-NSCs but clustered distantly from FA-NSCs (Fig. 6F and Table S1). Hierarchical clustering based on DNA methylation levels in the promoter region (+/-1.5kb from TSS) of genes whose expression levels were rescued in C-FA-NSCs, placed C-FA-NSCs closer to control-NSCs and away from FA-NSCs (Fig. 6G), although this pattern was not seen at the whole genome level (Fig. S4C). This suggests that FANCA gene correction leads to specific methylation changes in a subset of promoters. Examination of the methylomes of NSCs derived from Fanconi Anemia iPSCs before and after gene correction by targeted bisulfite sequencing with padlock probes
Project description: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.
Project description:Multipotent neural stem cells (NSCs) are found in several isolated niches of the adult mammalian brain where they have unique potential to assist in tissue repair. Modern transcriptomics offer high-throughput methods for identifying disease or injury associated gene expression signatures in endogenous adult NSCs, but they require adaptation to accommodate the rarity of NSCs. Bulk RNA sequencing of NSCs requires pooling several mice, which impedes application to labor-intensive injury models. Alternatively, single cell RNA sequencing can profile hundreds to thousands of cells from a single mouse and is increasingly used to study NSCs. However, consequences of the low RNA input from a single NSC on downstream identification of differentially expressed genes (DEGs) remains largely unexplored. Here, to clarify the role that low RNA input plays in DEG identification, we directly compared DEGs in an oxidative stress model of cultured NSCs by bulk and single cell sequencing. While both methods yielded DEGs that were replicable, single cell sequencing DEGs derived from genes with higher relative transcript counts compared to all detected genes and exhibited smaller fold changes than DEGs identified by bulk RNAseq. The loss of high fold-change DEGs in the single cell platform presents an important limitation for identifying disease-relevant genes. To facilitate identification of such genes, we determined an RNA-input threshold that enables transcriptional profiling of NSCs comparable to traditional bulk sequencing and used it to establish a workflow for in vivo profiling of endogenous NSCs. We then applied this workflow to identify DEGs after lateral fluid percussion injury, a labor-intensive animal model of traumatic brain injury. Our work suggests that single cell RNA sequencing may underestimate the diversity of pathologic DEGs but population level transcriptomic analysis can be adapted to capture more of these DEGs with similar efficacy and diversity as traditional bulk sequencing. Together, our data and workflow will be useful for investigators interested in understanding and manipulating adult hippocampal NSC responses to various stimuli.
Project description:Multipotent neural stem cells (NSCs) are found in several isolated niches of the adult mammalian brain where they have unique potential to assist in tissue repair. Modern transcriptomics offer high-throughput methods for identifying disease or injury associated gene expression signatures in endogenous adult NSCs, but they require adaptation to accommodate the rarity of NSCs. Bulk RNA sequencing of NSCs requires pooling several mice, which impedes application to labor-intensive injury models. Alternatively, single cell RNA sequencing can profile hundreds to thousands of cells from a single mouse and is increasingly used to study NSCs. However, consequences of the low RNA input from a single NSC on downstream identification of differentially expressed genes (DEGs) remains largely unexplored. Here, to clarify the role that low RNA input plays in DEG identification, we directly compared DEGs in an oxidative stress model of cultured NSCs by bulk and single cell sequencing. While both methods yielded DEGs that were replicable, single cell sequencing DEGs derived from genes with higher relative transcript counts compared to all detected genes and exhibited smaller fold changes than DEGs identified by bulk RNAseq. The loss of high fold-change DEGs in the single cell platform presents an important limitation for identifying disease-relevant genes. To facilitate identification of such genes, we determined an RNA-input threshold that enables transcriptional profiling of NSCs comparable to traditional bulk sequencing and used it to establish a workflow for in vivo profiling of endogenous NSCs. We then applied this workflow to identify DEGs after lateral fluid percussion injury, a labor-intensive animal model of traumatic brain injury. Our work suggests that single cell RNA sequencing may underestimate the diversity of pathologic DEGs but population level transcriptomic analysis can be adapted to capture more of these DEGs with similar efficacy and diversity as traditional bulk sequencing. Together, our data and workflow will be useful for investigators interested in understanding and manipulating adult hippocampal NSC responses to various stimuli.
Project description:Multipotent neural stem cells (NSCs) are found in several isolated niches of the adult mammalian brain where they have unique potential to assist in tissue repair. Modern transcriptomics offer high-throughput methods for identifying disease or injury associated gene expression signatures in endogenous adult NSCs, but they require adaptation to accommodate the rarity of NSCs. Bulk RNA sequencing of NSCs requires pooling several mice, which impedes application to labor-intensive injury models. Alternatively, single cell RNA sequencing can profile hundreds to thousands of cells from a single mouse and is increasingly used to study NSCs. However, consequences of the low RNA input from a single NSC on downstream identification of differentially expressed genes (DEGs) remains largely unexplored. Here, to clarify the role that low RNA input plays in DEG identification, we directly compared DEGs in an oxidative stress model of cultured NSCs by bulk and single cell sequencing. While both methods yielded DEGs that were replicable, single cell sequencing DEGs derived from genes with higher relative transcript counts compared to all detected genes and exhibited smaller fold changes than DEGs identified by bulk RNAseq. The loss of high fold-change DEGs in the single cell platform presents an important limitation for identifying disease-relevant genes. To facilitate identification of such genes, we determined an RNA-input threshold that enables transcriptional profiling of NSCs comparable to traditional bulk sequencing and used it to establish a workflow for in vivo profiling of endogenous NSCs. We then applied this workflow to identify DEGs after lateral fluid percussion injury, a labor-intensive animal model of traumatic brain injury. Our work suggests that single cell RNA sequencing may underestimate the diversity of pathologic DEGs but population level transcriptomic analysis can be adapted to capture more of these DEGs with similar efficacy and diversity as traditional bulk sequencing. Together, our data and workflow will be useful for investigators interested in understanding and manipulating adult hippocampal NSC responses to various stimuli.
Project description:One of the key regulatory mechanisms of brain size and neuronal diversity is through control of NB identity via cell fate maintenance. Dedifferentiation is the reversion of differentiated cells to a stem cell like fate, whereby, the gene expression program of mature cells is altered and genes associated with multipotency are expressed. Beyond the fact that misexpression of these factors and pathways caused the formation of ectopic NBs, whether these dedifferentiated NBs faithfully produce the correct number and types of neurons or glial cells, or undergo timely terminal differentiation, has not been assessed. These characteristics are key determinants of overall CNS size and function, thus are important parameters when considering whether dedifferentiation leads to tumourigenesis or can be appropriately utilized for regenerative purposes. Appropriate terminal differentiation as well as neuronal diversity are key characteristics of NSCs, and are specified through temporal patterning of the NSCs driven by the successive expression of temporal transcription factors (tTFs). In this study, we found that ectopic NSCs induced via bHLH transcription factor Deadpan (Dpn) expression in the optic lobes of the developing Drosophila CNS fail to undergo appropriate temporal progression, where they express mid-tTF, Sloppy-paired 1 (Slp-1) at the expense of late-tTF Tailless (Tll); consequently generating an excess of Twin of eyeless (Toy) positive neurons and fewer Reversed polarity (Repo) positive glial cells. Dpn overexpression also resulted in stalled progression through the cell cycle, and a failure to undergo timely terminal differentiation. Mechanistically, DamID studies demonstrated that Dpn directly binds to both Dichaete (D), a Sox-box transcription factor known to repress Slp-1, as well as a number of cell cycle genes. Promoting cell cycle progression or overexpression of D were able to re-trigger the progression of the temporal series in dedifferentiated NBs, restoring both neuronal diversity and timely NB terminal differentiation.
Project description:The mature brain contains an incredible number and diversity of cells that are produced and maintained by heterogeneous pools of neural stem cells. Two distinct types of neural stem cells (NSCs) exist in the developing and adult mouse brain: GFAP (Glial Fibrillary Acid Protein)-negative primitive (p)NSCs and downstream GFAP-positive definitive (d)NSCs. To better understand the embryonic functions of NSCs, we performed clonal lineage tracing within neurospheres grown from either pNSCs or dNSCs to enrich for their most immediate downstream neural progenitor cells (NPCs). These clonal progenitor lineage tracing data allowed us to construct a hierarchy of progenitor subtypes downstream of pNSCs and dNSCs that were then validated using single cell transcriptomics. Further, we identify Nexn as required for neuronal specification from neuron/astrocyte progenitor cells downstream of rare pNSCs. Combined, these data provide single cell resolution of NPC lineages downstream of rare pNSCs that likely would be missed from population level analyses in vivo.
Project description:Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain. Single cell RNAseq of cells isolated from their in vivo niche in the subventricular zone, Striatum and Cortex during homeostasis as well as following ischemic injury. In total 272 single cells. (<WT>: homeostasis samples; <Ischemic_injured> and <Ischemic_injured_and_Interferon_gamma_knockout>: samples following ischemic injuried).