Transcriptional Dynamics of Motor Neuron Maturation in vivo and in vitro
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE198767 | GEO | 2022/08/02
REPOSITORIES: GEO
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Project description:Familial amyotrophic lateral sclerosis (ALS) represents about 10% of ALS cases. In about 20% of familial ALS patients, a mutation in superoxide dismutase-1 (SOD1) can be found. The ubiquitous SOD1 protein converts superoxide radical anions to oxygen and hydrogen peroxide. Patients with familial ALS caused by mutations in SOD1 can show comorbidity with frontotemporal dementia and develop cognitive impairment, including apathy, inattention, verbal deficits, and hypersexuality. At the cellular level, pathological signs of ALS may include tau immunoreactive astrocytic and neuronal inclusions, suggesting that cognitive dysfunction in ALS may also reflect abnormal protein metabolism of the microtubule associated protein (MAP), tau. To identify cell-specific expression changes, we performed laser capture microdissection (LCM) to isolate anterior horn motor neurons and surrounding cells. To determine common pathways for the development of ALS due to dysfunction of SOD1 or TAU, we performed whole transcriptome analysis in SOD1 and TAU mouse models. Because ALS is a neurodegenerative disease that specifically affects motor neurons, these cells were the main investigative target. Global transcriptomes of glial cells surrounding the motor neurons were also assessed, because these cells have been implicated as triggers of neurodegeneration. Mouse experiments were performed at the presymptomatic stage, prior to the onset of cell loss, in order to reduce false positive signals due to tissue reactive changes. Lumbar anterior horn anterior horn motor neurons and surrounding cells (glia) were isolated from presymptomatic TAU-P301L and SOD1-G93A transgenic mice using laser capture microdissection (LCM; PixCell® IIe LCM System, Arcturus, Molecular Devices, CA). On average, 500 motor neuron bodies or surrounding glial cells from 20 tissue sections per animal were collected. RNA isolation from LCM collected cells was performed using the Qiagen RNeasy Micro Kit. Only RNA with RNA Integrity Numbers (RIN) above 7.5 were taken for further analysis (Agilent Bioanalyzer 2100). A two-round T7-based amplification and labeling protocol (Agilent Low RNA Input Linear Amplification Kit PLUS) was used to generate high quality labeled cRNA. Agilent Whole Mouse Genome Microarray comparisons of motor neurons and surrounding glia were performed between transgenic (SOD1G93A and TAUP301L) and corresponding nontransgenic (control) littermate animals, producing four independent comparison groups: SOD1G93A motor neurons versus control motor neurons (SOD1mn), SOD1G93A motor neuron surrounding glia versus control glia (SOD1gl), TAUP301L motor neurons versus control motor neurons (TAUmn) and TAUP301L glia versus control glia (TAUgl). Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Raw microarray data were acquired using the Agilent DNA Microarray scanner and processed with the accompanying Agilent Feature Extraction 10.5 Image Analysis software using default settings. Normalized signal intensities were used to identify gene expression changes in SOD1G93A and TAUP301L motor neurons and surrounding glial cells, generating four partially overlapping sets of data. For the identification of differential expression, the genes were required to pass two conservative criteria: a ratio beyond the 99.5% confidence interval observed in homotypic comparisons, which corresponded to an approximately 1.5-fold expression change, and a paired t-test (P<0.01) computed using 100 permutations of the data for each gene. Correction for multiple comparisons was performed using the adjusted Bonferroni test. The analysis was performed in the TM4: Microarray Software Suite. These techniques identified 251 transcripts representing 186 known genes for which expression was altered in at least one of the four comparisons. Four-condition experiment, SOD1G93A motor neurons versus control motor neurons (SODmn), SOD1G93A motor neuron surrounding glia versus control glia (SODgl), TAUP301L motor neurons versus control motor neurons (TAUmn) and TAUP301L glia versus control glia (TAUgl). Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Biological replicates: 4 SOD1G93A motor neurons (SODmn), 4 non SOD littermate motor neurons (nonSODmn), 4 SOD1G93A motor neuron surrounding glia (SODgl), 4 non SOD littermate motor neuron surrounding glia (nonSODgl), 4 TAUP301L motor neurons (TAUmn), 4 non TAU littermate motor neurons (nonTAUmn), 4 TAUP301L motor neuron surrounding glia (TAUgl), 4 non TAU littermate motor neuron surrounding glia (nonTAUgl). Four independent comparison groups were generated: SOD1G93A motor neurons versus control nonSOD motor neurons, SOD1G93A motor neuron surrounding glia versus control nonSOD glia, TAUP301L motor neurons versus control nonTAU motor neurons and TAUP301L glia versus control nonTAU glia. Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Each replicate was repeated twice with dye flip to correct for unequal dye incorporation rates. Therefore, eight microarray hybridizations were performed for each biological comparison, for a total of 32 microarray slides and generating four groups of differentially expressed genes in SOD1G93A motor neurons (SODmn), SOD1G93A motor neuron surrounding glial cells (SODgl), TAUP301L motor neurons (TAUmn), and TAUP301L surrounding glial cells (TAUgl).
2010-07-30 | E-GEOD-22482 | biostudies-arrayexpress
Project description:We found hereditary congenital facial paralysis type 1 (HCFP1) is caused by variants that duplicate or change cis regulatory elements (cREs) controlling the neuronal expression of the GATA2 transcription factor. We generated an HCFP1 mouse model in which tandem copies of the human version of a GATA2 regulatory element we term cRE1 were inserted proximal to the mouse cRE1 (cRE1dup/+; hg19: chr3:128,175,708-128,176,563). We report the use of single cell RNA sequencing (scRNAseq) for transcriptomic analysis of developing rhombomere 4(r4)-derived wild type (WT) and cRE1dup/+ facial branchial motor neurons (FBMNs) and inner ear efferent neurons (IEEs), as well as surrounding tissue, from embryos aged 9.5-12.5 post coitus (E9.5-12.5).
2023-04-04 | GSE171337 | GEO
Project description:Microarray analysis has been applied to the study of ALS in order to investigate gene expression in whole spinal cord homogenates of SOD1 G93A mice and human ALS cases, although the massive presence of glial cells and inflammatory factors has made it difficult to define which gene expression changes were motor neuron specific. Recently, laser capture microdissection (LCM), combined with microarray analysis, has allowed the identification of motor neuron specific changes in gene expression in human ALS cases. The aim of the present study is to combine LCM and microarray analysis to study how motor neurons in the spinal cord of transgenic SOD1 G93A mice and transgenic SOD1 WT respond to stimuli determined by the presence of the human mutant protein throughout the evolution of the stages in motor neuron injury Experiment Overall Design: Motor neurons have been isolated from the spinal cord of G93A mice and non transgenic littermates at different time points and the transcription expression profile of the isolated motor neurons has been analysed
2008-06-11 | E-GEOD-10953 | biostudies-arrayexpress
Project description:Gene expression profiling has been performed on motor cortex and spinal cord homogenates and of sporadic ALS cases and controls, to identify genes and pathways differentially expressed in ALS. More recent studies have combined the use of laser capture microdissection (LCM) with gene expression profiling to isolate the motor neurons from the surrounding cells, such as microglia and astrocytes, in order to determine those genes differentially expressed in the vulnerable cell population â i.e. motor neuron. The aim of this study was to determine the gene expression profiles from a small subset of cases which all carry mutations in the CHMP2B gene. These mutations have been found to be associated with the lower motor neuron dominant variant of ALS. Expression profiles from isolated motor neurons in CHMP2B-related ALS cases were compared to those from control motor neurons, in order to establish the pathways implicated in CHMP2B-related motor neuronal cell death.
2010-03-07 | E-GEOD-19332 | biostudies-arrayexpress
Project description:Human pluripotent stem cells are a promising source of diverse cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons, is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that induce up to 50% motor neurons within 3 weeks from human pluripotent stem cells with defined subtype identities that are relevant to neurodegenerative diseases. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3-). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays. We analyze 3 samples including 2 positive samples and 1 negative sample. Descriptions are as follow: a) Positive Sample 1: SHH-derived, day 21 GFP-high FACS purified motor neurons.b) Positive Sample 2: S+P-derived, day 21 GFP-high FACS purified motor neurons. c) Negative: S+P condition, day 21 no GFP FACS purified motor neurons
2013-02-06 | E-GEOD-41795 | biostudies-arrayexpress
Project description:Egr3 is a zinc-finger transcription factor involved in growth and development. Egr3-deficient mice have severe sensory ataxia due to failed development of muscle spindle stretch receptors. Sensory and motor neurons that normally innervate spindles are absent in Egr3-deficient mice, presumably as a secondary consequence to the loss of trophic signals produced by spindles during development that are required for innervation and neuron survival. The molecular mechanisms involving motor neuron fate specification, target derived growth factor dependencies, and specification of target innervation have been difficult to study since select markers for functionally specific motor neurons are very poorly characterized. A more thorough understanding of the molecular mediators of motor neuron biology will be important to evaluate the efficacy of new strategies devised to thwart neuron death that occurs in a variety of human motor neuronopathies and neuropathies. To identify genes specifically expressed by spinal cord fusimotor neurons: Many motor neuron specific genes have been described over the years. However, none have been described that distinguish fusimotor neurons from skeletomotor neurons despite the fact that they have distinct muscle targets (muscle spindle stretch receptors) and comprise 25-30% of the spinal motor neuron populations. Since these motor neurons have remarkably different target innervation and function, we hypothesize that they express genes that establish their specific phenotypes during development. We hypothesize that fusimotor neurons can be distinguished in the spinal cord by characterizing fusimotor neuron specific gene expression. Once fusimotor neuron specific genes are identified, they will be used as markers to identify fusimotor neurons in complex neuroglial cell populations in vivo and in vitro. We hypothesize that by characterizing fusimotor neuron specific genes, unique marker molecules will be identified for in vivo and in vitro study of this functionally distinct and important motor neuron subtype. Moreover, we hypothesize that many of the genes that are specifically expressed by fusimotor neurons will be involved in mechanisms related to their fate specification, target innervation and growth factor dependent biology. We will use the Affymetrix microarray platform to identify genes that are specifically expressed by fusimotor neurons in mouse spinal cord. The differential expression analysis will be performed on microdissected segments of spinal cord (L3-L5) from wild type and Egr3-deficient mice. Postnatal Egr3-deficient mice lack muscle spindles and fusimotor neurons in their spinal cords. By comparing gene expression from microdissected segments of spinal cord (L3-L5) between wild type and Egr3-deficient mice, we hypothesize that fusimotor neuron selective genes can be identified. We will microdissect L3-L5 segments of spinal cord using precise anatomical landmarks to ensure that comparable spinal cord regions are anlayzed from each animal. For each microarray experiment, total RNA will be extracted from L3-L5 cords (approximately 2 mm length of spinal cord). The integrity of each RNA sample will be verified by gel electrophoresis. The intact RNA samples from mice of similar genotype will be pooled from three (3) 27-day old animals. The intact cord dissection is easier in young animals (eg: 27-day old) and the phenotype is known to exist at this developmental stage. The RNA from each animal of a similar genotype will be pooled into a single sample to minimize false positive gene calls that may represent genes related to the specific state of vigilance of a particular animal at the time of sacrifice (eg: activity dependent genes). Thus, each of the two RNA samples to be analyzed for a particular microarray experiment will represent RNA from three (3) spinal cords of each genotype. RNA amplification for probe synthesis should not be necessary since we will provide 7 ug of intact pooled total RNA for each sample. For statistical analysis, the experiment will be performed twice. Since the RNA samples are precious, they will be provided to the Array Consortium in two shipments with each of the experiments performed independently.
2008-06-11 | E-GEOD-1830 | biostudies-arrayexpress
Project description:We aim to understand the role that Cdx2 plays in specifying the rostro-caudal identity of differentiating motor neurons. We find that expressing Cdx2 in combination with FGF signaling is sufficient to produce motor neurons with a more caudal identity. ChIP-seq analysis of Cdx2 finds that it binds extensively throughout the Hox regions in progenitor motor neurons. Analysis of polycomb-associated chromatin over Hox regions in the subsequently generated motor neurons finds that Cdx2 binding corresponds to chromatin domains encompassing de-repressed caudal Hox genes. These results suggest a direct role for Cdx2 in specifying caudal motor neuron identity. Expression studies: Affymetrix arrays are used to profile gene expression in ES cells, RA/Hh-derived Day 5 motor neurons, and RA/Hh-derived motor neurons that have also been exposed to Dox (to activate iCdx2) and FGF.
2013-07-04 | E-GEOD-39422 | biostudies-arrayexpress
Project description:We report cell type specific Nova HITS-CLIP using BAC-transgenic lines expressing GFP-Nova under the motor neuron specific choline acetyltransferase (Chat) promoter. By comparing transcriptome wide Nova binding map in motor neurons and that in the whole spinal cord, we identified differential Nova binding sites in motor neurons, which correlate with motor neuron specific RNA processing.
2018-07-04 | GSE71294 | GEO
Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).
2013-09-19 | E-GEOD-50993 | biostudies-arrayexpress
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.
2019-12-13 | GSE141949 | GEO