Project description:In the embryonic spinal cord, motor neurons diversify into different subsets characterized by distinct molecular identity, localization and connectivity. However, the factors that control MN diversification remain poorly known. To identify genes downstream of Onecut transcription factors in motor neurons that may contribute to motor neuron diversification, we performed a RNA-sequencing comparison of control and of OC-deficient motor neuron transcriptome at embryonic day 10.5.
Project description:Background: Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo. Results: Fluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which ~1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons. Conclusion: We have described a microarray-based method, MAPCeL, for profiling gene expression in specific C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system. Experiment Overall Design: Our goal is to profile gene expression throughout the nervous system of the model organism Caenorhabditis elegans. As a first goal, we profiled a single class of embryonic motor neurons. To isolate transcripts from thesec neurons we developed the MAPCeL (Microarray Profiling C. elegans Cells) technique in which unc-4::GFP+ cells are captured by FACS for RNA isolation. We verified these data by bioinformatic means and by in vivo validation by creating GFP reporters for a random set of genes in our enriched gene list.
Project description:The aim of this study is to profile gene expression dynamics during the in vitro differentiation of embryonic stem cells into ventral motor neurons. Expression levels were profiled using Affymetrix microarrays at six timepoints during in vitro differentiation: ES cells (Day 0), embryoid bodies (Day 2), retinoid induction of neurogenesis (Day 2 +8hours of exposure to retinoic acid), neural precursors (Day 3), progenitor motor neurons (Day 4), postmitotic motor neurons (Day 7). The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog agonist. Here, gene expression patterns are profiled at various defined stages during the differentiation process using Affymetrix expression arrays.
Project description:Gene expression changes in spinal motor neurons of the SOD1G93A-transgenic model for ALS after treatment with G-CSF. To gain insight into the mode of action of G-CSF, we performed gene expression profiling on isolated lumbar motor neurons from SOD1G93A mice, the most frequently studied animal model for ALS, with and without G-CSF treatment. A first group of SOD1G93A and WT mice was included in the study at week 11 of age when SOD1G93A mice present no signs of motor dysfunction but subtle signs of denervation detectable by electromyography. The second cohort of mice was treated with G-CSF or vehicle from week 11 to week 15. At the time of study completion, SOD1G93A mice presented clear motor impairment and motor neuron degeneration is documented. This design should provide information on genes altered in motor neurons of SOD1G93A mice from the clinically non-symptomatic to an early symptomatic stage, and give insight into genes influenced by G-CSF treatment. We sampled 300 motoneurons per mouse spinal cord by laser microdissection.
Project description:Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy’s Disease, is a slowly progressive adult-onset neuromuscular disease which results from a polyglutamine (polyQ) encoding CAG repeat expansion within the androgen receptor gene (AR). Despite the ubiquitous expression of the androgen receptor, it is unclear why motor neurons selectively degenerate and there are no effective treatments or disease modifying therapies for this debilitating disease. In order to identify potential therapeutic targets, we set out to establish the genes and molecular pathways involved in early motor neuron dysfunction in SBMA. We therefore undertook global transcriptomic profiling of cultured primary embryonic motor neurons from the spinal cord of AR100 mice, which model SBMA.. Four biological replicate samples were used for genome wide analysis using Affymetrix 430 v2.0 mouse arrays. Data was normalised using therobust multichip average (RMA) algorithm.
Project description:Progenitor motor neurons can be generated with high-efficiency by differentiating ES cells in vitro in the presence of retinoic acid and hedgehog signalling. Here, we characterize the chromatin landscape associated with progenitor motor neurons (pMNs) in order to assess how histone modification domains shift during the differentiation process. In this study, we characterize the genomic occupancy of H3K27me3, H3K4me3, H3K79me2 and Pol2 using ChIP-seq in progenitor motor neurons that have been differentiated in vitro from ES cells. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included.
Project description:Background: Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo. Results: Fluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which ~1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons. Conclusion: We have described a microarray-based method, MAPCeL, for profiling gene expression in specific C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system. Keywords: expression profile