Project description:This assay assessed genome-wide chromatin occupancy by transcription factor PBX1, alongside activity-associated histone mark H3K27ac in a primary adult neurogenic population (adult neurospheres, aNS, isolated from the ventricular-subventricular zone, V-SVZ) capable of differentiating into all neural lineages.
Project description:We used microarrays to detail the global programme of gene expression underlying neuronal differentiation of SVZ-neurospheres when manipulating Pax6 expression in the presence or absence of Pbx1
Project description:Comparative gene expression in adult SVZ-neurospheres differentiated under standard conditions and upon manipulations of Pax6 (OE) and/or Pbx1 (KD/OE)
Project description:The relevance of DNA-dependent poly-ADP ribose production for neuronal differentiation of adult stem- and progenitor cells from the SVZ was studied. To identify genes whose up- or downregulation during neuronal differentiation requires the activity of poly-ADP-Ribosylase (PARP) 1 or 2, SVZ-derived adult neurosphere cultures were differentiated in the presence or absence of Olaparib.
Project description:Developmental transcription factors act in networks, but how these networks achieve cell- and tissue specificity is still poorly understood. We here explored pre-B-cell leukemia homeobox 1 (PBX1) in adult neurogenesis combining genomic, transcriptomic, and proteomic approaches. ChIP-Seq analysis uncovered PBX1 binding to a wide range of different genes. Integration of PBX1 ChIP-seq with ATAC-seq data predicted interaction partners, which were subsequently validated by mass-spectrometry. Spatial transcriptomics revealed distinct temporal expression dynamics of Pbx1 and interacting factors. Among these were class I bHLH proteins TCF3, TCF4 and TCF12. RNA-seq upon Pbx1, Tcf3 and Tcf4 knockdown identified proliferation and differentiation associated genes as shared targets. Neuronal differentiation was reduced upon depletion of either factor, suggesting functional cooperation between PBX1 and TCF3/4. Notably, while physiological PBX1-TCF interactions have not yet been described, chromosomal translocation resulting in genomic TCF3::PBX1 fusion characterizes a subtype of acute lymphoblastic leukemia. Introducing Pbx1 into Nalm6 cells, a pre B-cell line expressing TCF3 but lacking PBX1, upregulated leukemogenic genes including BLK and NOTCH3, arguing that functional PBX1-TCF cooperation likely extends to hematopoietic contexts. Our study hence uncovers a PBX1-TCF module orchestrating the balance between progenitor cell proliferation and differentiation in adult neurogenesis with implications for leukemia etiology.
Project description:The mammalian adult brain contains two neural stem and precursor (NPC) niches: subventricular zone [SVZ] lining the lateral ventricles and subgranular zone [SGZ] in the hippocampus. From these SVZ NPCs represent the largest NPC pool. Notably, while SGZ NPCs typically only produce neurons and astrocytes, SVZ NPCs produce neurons, astrocytes and oligodendrocytes throughout life. Of particular importance is the generation and replacement of oligodendrocytes, the only myelinating cells of the central nervous system (CNS). SVZ NPCs contribute to myelination by regenerating oligodendrocyte precursor cell (OPC) pool and by differentiating into oligodendrocytes in the developing and demyelinated brain. The neurosphere assay has been widely adopted by the scientific community to facilitate the study of NPCs in vitro. Here, we present a streamlined protocol for culturing postnatal and adult SVZ NPCs and OPCs from primary neurosphere cells. We characterize the purity and differentiation potential as well as provide RNA-sequencing profiles of postnatal SVZ NPCs, postnatal SVZ OPCs and adult SVZ NPCs. We show that primary neurospheres cells generated from postnatal and adult SVZ differentiate into neurons, astrocytes and oligodendrocytes concurrently and at comparable levels. SVZ OPCs are generated by subjecting primary neurosphere cells to OPC growth factors fibroblast growth factor (FGF) and platelet-derived growth factor-AA (PDGF-AA). We further show SVZ OPCs can differentiate into oligodendrocytes in the absence and presence of thyroid hormone T3. Transcriptomic analysis confirmed the identities of each cell population and revealed novel immune and signalling pathways expressed in an age and cell type specific manner.
Project description:We report two sets of potential circadian clock target genes by high-throughput transcriptome profiling (RNA-sequencing): one set from the corpus callosum of the adult mouse brain and the other set from adult neural stem cell cultured from the SVZ (neurospheres) of the mouse brain. First, the corpus callosum was dissected from three adult wild-type and two Bmal1 knockout (Bmal1-/-) mice, mRNA profiles were generated by deep sequencing using BGISEQ-500 RNA-Seq platform. The sequence reads that passed quality control were analyzed: we mapped more than 21 million total clean reads, more than 96% total Mapping Ratio, more than 86% Uniquely Mapping Ratio per sample to mouse genome. qRT-PCR validation with primers covering at least 3 known housekeeping genes and at least 5 well-known circadian clock genes were performed with SYBR Green assay. 103 differentially expressed genes (DEGs) were identified in the corpus callosum of Bmal1-/- mice (58 up-regulated and 45 down-regulated genes; |log2FC| > 1 & adjusted P-value < 0.05). Hypergeometric test revealed that potential functions of circadian clock target genes in the corpus callosum are secreted proteins as signaling molecules. For the second set of data, neurospheres were cultured from the SVZ of three adult wild-type and three adult Bmal1-/- mice, mRNA profiles were generated by deep sequencing using BGISEQ-500 RNA-Seq platform. The sequence reads that passed quality control were analyzed: we mapped more than 28 million total clean reads, more than 97% total Mapping Ratio, more than 68% Uniquely Mapping Ratio per sample to mouse genome. qRT-PCR validation with at least 3 known housekeeping genes and at least 5 well-known circadian clock genes using SYBR Green assay. 30 DEGs were identified in neurospheres cultured from Bmal1-/- mice (17 up-regulated and 13 down-regulated genes; |log2FC| > 1 & adjusted P-value < 0.05). Gene Set Enrichment Analysis (GSEA) revealed that potential circadian clock associated functions in the SVZ (neurospheres) are controlling diverse signal transductions.
Project description:Down syndrome is the most common form of genetic mental retardation. How Trisomy 21 causes mental retardation remains unclear and its effects on adult neurogenesis have not been addressed. To gain insight into the mechanisms causing mental retardation we used microarrays to investigate gene expression differences between Ts1Cje (a mouse model of Down syndrome) and C57BL/6 littermate control neurospheres. The neurospheres were generated from neural stem cells and progenitors isolated from the lateral walls of the lateral ventricles from adult mice.
Project description:Down syndrome is the most common form of genetic mental retardation. How Trisomy 21 causes mental retardation remains unclear and its effects on adult neurogenesis have not been addressed. To gain insight into the mechanisms causing mental retardation we used microarrays to investigate gene expression differences between Ts1Cje (a mouse model of Down syndrome) and C57BL/6 littermate control neurospheres. The neurospheres were generated from neural stem cells and progenitors isolated from the lateral walls of the lateral ventricles from adult mice. RNA was extracted for hybridization to arrays from 3 pairs of Ts1Cje and disomic C57BL/6 littermate control 7-day old adult neurosphere cultures.