Chx10 consolidates V2a interneuron identity through two distinct gene repression modes [ChIP-Seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Jae Lee
PROVIDER: E-GEOD-83872 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:During development, two cell-types born from closely related progenitor pools often express the identical transcriptional regulators despite their completely distinct characteristics. This phenomenon highlights the necessity of the mechanism that operates to segregate the identities of the two cell-types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). Here we demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate V2a and MN pathways. Together, our study uncovers a widely applicable gene regulatory principle for segregating related cell fates. RNA samples from Chx10-ESC-derived MNs were prepared for sequencing according to the Illumina protocol, and sequenced on the Illumina HiSeq 2000. We will then compare the transcriptome changes between -Dox (no Chx10) and +Dox (Chx10) in order to identify genes rregulated by Chx10.
2016-07-28 | E-GEOD-83873 | biostudies-arrayexpress
Project description:During development, two cell-types born from closely related progenitor pools often express the identical transcriptional regulators despite their completely distinct characteristics. This phenomenon highlights the necessity of the mechanism that operates to segregate the identities of the two cell-types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). Here we demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate V2a and MN pathways. Together, our study uncovers a widely applicable gene regulatory principle for segregating related cell fates.
2016-07-28 | GSE83873 | GEO
Project description:During development, two cell-types born from closely related progenitor pools often express the identical transcriptional regulators despite their completely distinct characteristics. This phenomenon highlights the necessity of the mechanism that operates to segregate the identities of the two cell-types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). Here we demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate V2a and MN pathways. Together, our study uncovers a widely applicable gene regulatory principle for segregating related cell fates.
2016-07-28 | GSE83872 | GEO
Project description:Combinatorial transcription codes generate the myriad of cell types during development, and thus likely provide crucial insights into directed differentiation of stem cells to a specific cell type. The LIM-complex composed of Isl1 and Lhx3 directs the specification of spinal motor neurons (MNs) in embryos. Here, we report that Isl1-Lhx3, a LIMcomplex-mimicking fusion, induces a signature of MN transcriptome and concomitantly suppresses interneuron differentiation programs, thereby serving as a potent and specific inducer of MNs in stem cells. We show that an equimolar ratio of Isl1 and Lhx3 and the LIM-domain of Lhx3 are crucial for generating MNs without upregulating interneuron genes. These led us to design Isl1-Lhx3, which maintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM-domain of Lhx3. Isl1-Lhx3 drives MN differentiation with high specificity and efficiency in the spinal cord and embryonic stem cells, bypassing the need for sonic hedgehog. RNA-seq analysis revealed that Isl1-Lhx3 induces the expression of a battery of MN genes that control various functional aspects of MNs, while suppressing key interneuron genes. Our studies uncover a highly efficient method for directed MN generation and MN gene networks. Our results also demonstrate a general strategy of utilizing embryonic transcription complexes for producing specific cell types from stem cells. Examine the RNA expression profiles of inducible motor neurons-embryonic stem cells (iMN-ESCs) with or without Dox treatment, with biological duplicates.
2012-02-03 | E-GEOD-35510 | biostudies-arrayexpress
Project description:Combinatorial transcription codes generate the myriad of cell types during development, and thus likely provide crucial insights into directed differentiation of stem cells to a specific cell type. The LIM-complex composed of Isl1 and Lhx3 directs the specification of spinal motor neurons (MNs) in embryos. Here, we report that Isl1-Lhx3, a LIMcomplex-mimicking fusion, induces a signature of MN transcriptome and concomitantly suppresses interneuron differentiation programs, thereby serving as a potent and specific inducer of MNs in stem cells. We show that an equimolar ratio of Isl1 and Lhx3 and the LIM-domain of Lhx3 are crucial for generating MNs without upregulating interneuron genes. These led us to design Isl1-Lhx3, which maintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM-domain of Lhx3. Isl1-Lhx3 drives MN differentiation with high specificity and efficiency in the spinal cord and embryonic stem cells, bypassing the need for sonic hedgehog. RNA-seq analysis revealed that Isl1-Lhx3 induces the expression of a battery of MN genes that control various functional aspects of MNs, while suppressing key interneuron genes. Our studies uncover a highly efficient method for directed MN generation and MN gene networks. Our results also demonstrate a general strategy of utilizing embryonic transcription complexes for producing specific cell types from stem cells.
2012-02-03 | GSE35510 | 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:To discover RBPs with increased insolubility in a human ALS model, we applied a well established dual-SMAD inhibition-based protocol (Fang et al., 2019; Markmiller et al., 2021; Markmiller et al., 2018; Martinez et al., 2016) to generate iPSC-MN from six control iPSC lines, from four iPSC lines originating from two sALS patients, and from two iPSC lines originating from fALS patients with pathogenic variants in the TARDBP gene (Table S1; Figure S1A). No difference in differentiation capacity was observed (Figure S1B-G), resulting in average 40% ISL1+ MN (Figures S1G), comparable to numbers observed in large scale MN differentation studies (Baxi et al., 2022). The susceptibility of ALS MN to sodium arsenite-induced stress was not changed (Figure S1H and I). Next, we asked which proteins exhibit an increased insolubility in our ALS iPSC-MN. We fractioned iPSC- MN by lysis in radio-immunoprecipitation assay (RIPA) buffer, followed by ultracentrifugation and solubilization of RIPA insoluble proteins in urea buffer. The ultracentrifugation-cleared RIPA insoluble protein fraction is widely used to study protein insolubility in the context of neurodegeneration (Nuber et al., 2013; Walker et al., 2015). Label-free mass spectrometry of the insoluble protein fraction was utilized to identify proteins that are insoluble in sALS and fALS, relative to control iPSC-MNs (Figure 1A). Gene ontology (GO) analysis of the 100 proteins (top 2.9% of all detected proteins) with the highest label free quantification (LFQ) intensities in controls (Figure S1J) revealed that ‘unfolded protein binding’ (corrected P = 7.95 x 10-16) and ‘structural constituent of cytoskeleton’ (corrected P = 1.47 x 10-10) were among the 10 most significantly enriched GO terms, indicating enrichment of insoluble proteins (Figure S1K). Principle component analysis of the insoluble fractions did not distinguish ALS from control samples, suggesting that the overall insoluble proteome is not changed (Figure S1L). At threshold P ≤ 0.05 (Welch’s t-test) and fold change ≥ 1.5, we identified 88 proteins enriched in the insoluble fraction in ALS samples relative to control (Figure 1B). When the sample labels were randomly shuffled, we observed an average of 7.5 proteins (~12-fold lower) as differentially enriched at the same statistical thresholds, indicative of an ALS-specific protein insolubility pattern (Figure 1C). The 88 candidate proteins included cytoskeletal components and motor proteins, functional categories associated with prominent ALS in vitro phenotypes (Akiyama et al., 2019; Egawa et al., 2012; Fazal et al., 2021; Guo et al., 2017; Kreiter et al., 2018) (Figure 1D). Notably, 5 RBPs, NOVA1, ELAVL4, FXR2, RBFOX2, and RBFOX3 were also enriched (Figure 1D). The NOVA1 paralog NOVA2 was significantly enriched (P = 0.03) but did not meet our enrichment threshold (fold change = 1.36). Interestingly, insoluble TDP-43 protein was not significantly different in ALS and control (P = 0.98; fold change = 0.97). Western blot analysis confirmed the increase in insolubility of NOVA1, NOVA2, ELAVL4, RBFOX2 and RBFOX3 (Figure 1E and 1F). The soluble protein levels of NOVA1 and NOVA2 were also increased (Figure 1F). In conclusion, we identified 5 RBPs with elevated insoluble protein levels of ALS-iPSC-MNs.
2022-06-28 | PXD032140 | Pride
Project description:The goal of this study is to find skate pectoral fin motor neuron markers. Total RNA was extracted from manually sorted pectoral fin MNs, which were retrogradely labeled and tail spinal cord cells. By comparing RNA expression profiles of pectoral fin MNs and tail spinal cord neurons, we could identity general MN, MN columnar, and subset MN pool markers for fin MNs.
2018-02-08 | GSE105156 | GEO
Project description:Single cell dataset of human motor neurons laser-captured from postmortem ALS and control tissues
(1) ALS Pilot dataset
(2) TDP43 stratified dataset
Summary: Unbiased proteomics has been employed to interrogate central nervous system (CNS) tissues (brain, spinal cord) and fluid matrices (CSF, plasma) from amyotrophic lateral sclerosis (ALS) patients; yet, a limitation of conventional bulk tissue studies is that motor neuron (MN) proteome signals may be confounded by admixed non-MN proteins. Recent advances in trace sample proteomics have enabled quantitative protein abundance datasets from single human MNs (Cong et al., 2020b). In this study, we leveraged laser capture microdissection (LCM) and nanoPOTS (Zhu et al., 2018c) single-cell mass spectrometry (MS)-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control donor spinal cord tissues, leading to the identification of 2515 proteins across MNs samples (>900 per single MN) and quantitative comparison of 1870 proteins between disease groups. Furthermore, we studied the impact of enriching/stratifying MN proteome samples based on the presence and extent of immunoreactive, cytoplasmic TDP-43 inclusions, allowing identification of 3368 proteins across MNs samples and profiling of 2238 proteins across TDP-43 strata. We found extensive overlap in differential protein abundance profiles between MNs with or without obvious TDP-43 cytoplasmic inclusions that together point to early and sustained dysregulation of oxidative phosphorylation, mRNA splicing and translation, and retromer-mediated vesicular transport in ALS. Our data are the first unbiased quantification of single MN protein abundance changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein abundance changes in human neurologic diseases.
2023-06-07 | MSV000092119 | MassIVE
Project description:Motor neuron (MN), together with interneuron, are the two major neuronal types in spinal cord. MNs control muscle movement and are essential for breathing, walking and fine motor skills. Malfunction of MNs is frequently associated with neuronal diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. To establish normal function, MNs need to be differentiated properly from neural progenitor cells. Thus, it is of great importance to study the mechanism for MN specification. This study focuses on Mnx1 - a conserved homeobox transcription factor gets expressed during MN specification. Mnx1 is also the most widely used MN marker. Previous studies reported that Mnx1 mutation in mouse causes early lethality - probably by affecting the controlling over respiratory system. However, the exact role of Mnx1 for MN identity remains poorly understood. Taking advantage of a recently developed in vitro model for efficient MN induction from mouse ES cells, we combined experimental and OMICs techniques to systematically investigate the molecular function of Mnx1. We identified thousands of Mnx1 binding sites - many are co-bound by core MN factor Lhx3 and Isl1 and lack active histone marks. Disruption of Mnx1 causes increased expression of 85 genes - while only 14 genes get down-regulated. Up-regulated genes are enriched with neuronal functions, usually get expressed during MN differentiation, and tend to have higher expression in motor neurons and brain compared with other tissues – indicating Mnx1 may fine tune neuronal genes to desired level. However, only a dozen of regions with increased H3K27ac marks are bound by Mnx1, and only two up-regulated genes (Pbx3 and Pou6f2) are identified as putative direct targets of Mnx1 - both are core neuronal factors with the potential to regulate other differential neuronal genes. These results suggest that Mnx1 may contribute to MN identity by fine-tuning the expression of many neuronal genes through direct regulation of a few core neuronal transcription factors. In summary, this study represents the first systematic investigation about the molecular function of the core MN factor Mnx1. It clarifies the mechanism of Mnx1 for MN identity, and also improves the understanding about the regulation mode of homeobox transcription factors.
2021-12-31 | GSE143161 | GEO