Project description:The dentate gyrus of the hippocampus continues generating new neurons throughout life. These nerve cells originate from radial astrocytes within the subgranular zone (SGZ). We find that Sox1, a member of the SoxB1 family of transcription factors, is expressed in a subset of radial astrocytes. Lineage tracing using Sox1 driven reporter mice shows that the Sox1-expressing cells represent an activated neural stem/progenitor population. We used microarrays to evaluate the molecular constitution of neural stem cells with aging. Hippocampi were isolated from Sox1-GFP mice at 5-7 months (n=3) or 1 year 9 months (n=4) using a Leica dissecting scope. Cells were immediately dissociated using papain (Worthington Biochemical Corporation. Lakewood, NJ, USA) and sorted for the GFP high or GFP negative population using a FACSAria Flow Cytometer (BD Biosciences, San Diego, CA, USA). RNA was harvested using the Arcturus PicoPure RNA isolation kit (Life Technologies Corp, Carlsbad, CA, USA) and the quality and quantity of each sample was checked using Agilent BioAnalyzer (Santa Clara, CA, USA) and Nanodrop spectrophotometer (Wilmington, DE, USA). Samples were amplified using the NuGEN FFPE kit (San Carlos, CA, USA) and cDNA was prepared using an oligo(dT)-T7 RNA polymerase primer before biotinylation by in vitro transcription, partial hydrolysis and final hybridization to the Affymetrix (Santa Clara, CA, USA) GeneChip Mouse Gene 1.0 ST Array (Gladstone Genomics Core). Arrays were normalized using Robust Multichip Average (RMA) and the latest probe map to MM9 RefSeq genes (Irizarry et al., 2003; Dai et al., 2005). To detect potential outlier arrays, the distributions of fold changes between all possible pairs within each group were analyzed. One sample from a young mouse was found to have a skewed distribution when compared to other biological replicate samples and was removed from further analysis. The statistical package R was used to perform average linkage hierarchical clustering of the remaining samples. Significance Analysis of Microarrays (SAM) was performed to determine significant differentially expressed genes between the old and young mice with a False Discovery Rate cutoff of 5% (Tusher et al., 2001).

Project description:We sought to find molecular signatures of the SGZ cell types, and to characterize the molecular pathways and transcription factor cascades that define the neurogenic niche. We used laser capture microdissection and DNA microarrays to profile gene expression in the inner (SGZ) and outer portions of the dentate gyrus (DG). Since the vast majority of the cells in the DG are mature granule cells, we compared the expression of the inner and outer portions to reveal molecular markers for the less numerous populations of the SGZ. This data set is part of a larger study assessing conserved molecular signatures of neurogenesis in the hippocampal subgranular zone of rodents and primates. Using a combination of selective SGZ transcriptional profiling with laser microdissection and DNA microarrays as well as in situ hybridization (ISH), we developed an extensive molecular characterization of the mouse SGZ, identifying 367 genes enriched in the SGZ compared to mature granule neurons. These genes displayed a wide range of cellular expression patterns reflecting the cellular milieu of the SGZ, including progenitor and dividing cells, immature granule cells, astrocytes, oligodendrocytes, and GABAergic interneurons. We next used a comparable microarray data set in rhesus monkey that profiled the SGZ across postnatal development to identify genes related to developmentally regulated granule cell neurogenesis. The rhesus monkey SGZ showed highly significant similarity to mouse, whereas network analysis of these data identified SGZ-enriched gene sets with different temporal profiles reflecting differential time-courses for maturation of glia and granule neurons. One neurogenesis-related gene network showed a steady decrease in expression across postnatal rhesus development from birth to adulthood. This temporal pattern is highly correlated with the number of proliferating cells in the dentate gyrus, and the neurogenic transcription factors Sox4 and Sox11 are central hub genes for this gene network. A number of the genes in this network showed a similar postnatal downregulation in mouse, suggesting a general conservation of molecular mechanisms underlying developmental and adult neurogenesis in rodents and primates. Primate data available at: http://www.blueprintnhpatlas.org/ Brains from 10- to 11-week-old C57BL/6 male mice housed in three conditions (no running, 4 days of running, 30 days of running) were sectioned and the dentate gyrus was isolated. For each brain, the inner granule cell layer (containing the subgranular zone) and outer GCL were separated using laser capture microdissection, and RNA from each region was collected and processed as described in the protocols.

Project description:We sought to find molecular signatures of the SGZ cell types, and to characterize the molecular pathways and transcription factor cascades that define the neurogenic niche. We used laser capture microdissection and DNA microarrays to profile gene expression in the inner (SGZ) and outer portions of the dentate gyrus (DG). Since the vast majority of the cells in the DG are mature granule cells, we compared the expression of the inner and outer portions to reveal molecular markers for the less numerous populations of the SGZ. This data set is part of a larger study assessing conserved molecular signatures of neurogenesis in the hippocampal subgranular zone of rodents and primates. Using a combination of selective SGZ transcriptional profiling with laser microdissection and DNA microarrays as well as in situ hybridization (ISH), we developed an extensive molecular characterization of the mouse SGZ, identifying 367 genes enriched in the SGZ compared to mature granule neurons. These genes displayed a wide range of cellular expression patterns reflecting the cellular milieu of the SGZ, including progenitor and dividing cells, immature granule cells, astrocytes, oligodendrocytes, and GABAergic interneurons. We next used a comparable microarray data set in rhesus monkey that profiled the SGZ across postnatal development to identify genes related to developmentally regulated granule cell neurogenesis. The rhesus monkey SGZ showed highly significant similarity to mouse, whereas network analysis of these data identified SGZ-enriched gene sets with different temporal profiles reflecting differential time-courses for maturation of glia and granule neurons. One neurogenesis-related gene network showed a steady decrease in expression across postnatal rhesus development from birth to adulthood. This temporal pattern is highly correlated with the number of proliferating cells in the dentate gyrus, and the neurogenic transcription factors Sox4 and Sox11 are central hub genes for this gene network. A number of the genes in this network showed a similar postnatal downregulation in mouse, suggesting a general conservation of molecular mechanisms underlying developmental and adult neurogenesis in rodents and primates. Primate data available at: http://www.blueprintnhpatlas.org/

Project description:It remains controversial whether the routes from differentiated cells to iPSCs are related to the reverse order of normal developmental processes or independent of them. Here, we generated iPSCs from mouse astrocytes by three (Oct3/4, Klf4 and Sox2 (OKS)), two (OK), or four (OKS plus c-Myc) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently upregulated during reprogramming and Sox1-positive cells become iPSCs. The upregulation of Sox1 is essential for OK-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during MEF reprogramming, neither Sox1 upregulation nor an increase in neurogenic potential occurs. Thus, astrocytes are reprogrammed through an NSC-like state, suggesting that reprogramming partially follows the retrograde pathway of normal developmental processes. To investigate the gene expression profile of intermediate-state cells during astrocyte reprogramming, we performed genome-wide gene expression analysis in five samples; starting astrocytes, intermediate-state cells expressing Sox1-GFP, NSCs, iPSCs established from astrocytes, and iPSCs established from MEFs (iPS-MEF-Ng-20D-17) that had previously been reported (Okita, K. et al. Nature 448: 313-317 (2007)). Two (NSCs, iPSCs from astrocytes and MEFs) or three (astrocytes, intermediate-state cells) biological replicates were prepared for microarray samples. Total RNA was extracted with an RNeasy kit (Qiagen). cDNA synthesis and transcriptional amplification were performed using 50-100 ng of total RNA with the GeneChip WT PLUS Reagent Kit (Affymetrix). Fragmented and biotin-labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) according to the manufacturerâ??s protocol. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.

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Expression data from the adult hippocampus of Sox1-GFP mice

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