Pioneer neurons are molecularly distinct, and their axon targeting is regulated by retinoic acid signaling
Ontology highlight
ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE264323 | GEO | 2024/06/01
REPOSITORIES: GEO
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Project description:During nervous system development, pioneer neurons are the first to extend their axons into target tissues, creating a scaffold for follower neurons. Despite years of study, whether pioneer neurons are a molecularly distinct population is unknown. Analysis of zebrafish posterior lateral line (pLL) sensory neurons during axon growth using single-cell RNA sequencing (scRNA-seq) revealed that pioneer and follower neurons are transcriptionally distinct. Expression profiling of differentiating pLL progenitors defined follower as the ground state, whereas “pioneer” is a later developmental state. The scRNA-seq data revealed active retinoic acid (RA) signaling in followers, but not in pioneers. Modulation of RA signaling within single pLL neurons showed that its downregulation in pioneers is necessary for expression of neurotrophic factor receptor ret, which is required for correct targeting of pioneer axons. Our study provided insights into the molecular landscape of pioneer neurons and revealed the regulatory role of RA signaling in their development.
2024-06-01 | GSE266312 | GEO
Project description:While minimizing lung cancer metastasis can improve overall survival, few models exist where subpopulations with stable high and low metastatic potential can be easily and reliably isolated. Our previous work characterized two stable subpopulations, termed Leaders and Followers, from the H1299 Non-Small Cell Lung Cancer model. H1299 Leaders and Followers have different transcriptional profiles, biomarkers, genomic methylation patterns, metabolic states, and morphologies.Of note, H1299 Leaders express high levels of Jagged1 (JAG1) and Myosin 10 (MYO10) and drive collective invasion and metastasis. To determine how JAG1 and MYO10 contribute to Leader cell biology and lung cancer metastasis, we utilized CRISPR-Cas9 to generate edited pools of H1299 cells targeting either JAG1 or MYO10. Fluorescence-activated cell sorting using a Leader-Follower biomarker, IL13R2, was used to generate single cell derived candidate clones. Candidate clones were screened using western blot for Leader and Follower cell biomarkers (JAG1, MYO10, HTATIP2 and IL13R2) followed by Sanger sequencing of candidate clones was also conducted to confirm target gene status. Using this method, 2 JAG1 knockout, 2 MYO10 knockout, 1 MYO10 knockdown and, 2 wildtype MYO10 JAG1 H1299 single-cell derived clones were generated.
2025-08-10 | GSE297208 | GEO
Project description:Cell fates are controlled by ‘pioneers’, sequence-specific transcription factors (TFs) that bind recognition motifs on nucleosomes (‘pioneer binding’). Pioneers occupy a minority of their recognition sequences in the genome, suggesting that the sequence context regulates their binding. Here, we developed PIONEAR-seq, a high-throughput biochemical assay to characterize pioneer binding to nucleosomes. We used PIONEAR-seq to assay 11 human TFs for binding to nucleosomes based on Widom 601 versus genomic sequences. We found that pioneer binding, while mediated primarily by TFs' recognition motifs, senses the broader nucleosome sequence context and that TFs previously found to be dyad or periodic binders on nucleosomes assembled on synthetic sequences exhibit exclusively end binding to nucleosomes based on genomic sequences. We propose a model where the nucleosome exploits the local bendability of the DNA sequence to position pioneer binding, revealing another cis-regulatory layer in eukaryotes.
2025-05-01 | GSE293487 | GEO
Project description:Cell fates are controlled by ‘pioneers’, sequence-specific transcription factors (TFs) that bind recognition motifs on nucleosomes (‘pioneer binding’). Pioneers occupy a minority of their recognition sequences in the genome, suggesting that the sequence context regulates their binding. Here, we developed PIONEAR-seq, a high-throughput biochemical assay to characterize pioneer binding to nucleosomes. We used PIONEAR-seq to assay 11 human TFs for binding to nucleosomes based on Widom 601 versus genomic sequences. We found that pioneer binding, while mediated primarily by TFs' recognition motifs, senses the broader nucleosome sequence context and that TFs previously found to be dyad or periodic binders on nucleosomes assembled on synthetic sequences exhibit exclusively end binding to nucleosomes based on genomic sequences. We propose a model where the nucleosome exploits the local bendability of the DNA sequence to position pioneer binding, revealing another cis-regulatory layer in eukaryotes.
2025-05-01 | GSE293024 | GEO
Project description:Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is only displayed by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we demonstrated that Pitx3-/- embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA-signaling in Pitx3-/- embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and TH are regulated by Pitx3 and RA signaling, influencing the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA-signaling represents only one aspect of the Pitx3 downstream cascade, since Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons. RNA was isolated from dissected ventral midbrains of E14.5 Pitx3-/- and Pitx3+/+ mouse embryos. 3 Experimental samples each consisting of 3 Pitx3-/- ventral midbrains were hybridized to reference RNA derived from 10 Pitx3+/+ ventral midbrains
2011-11-22 | E-GEOD-32940 | biostudies-arrayexpress
Project description:To probe the phenotypic heterogeneity found in cell populations, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3-D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signaling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signaling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape. A single tumor can harbor distinct genetic and epigenetic cellular sub-populations that drive tumor initiation and progression. This intratumor heterogeneity is proposed to be one of the major confounding factors of treatment causing relapse and poor clinical outcome1. Genomic instability and epigenetic modifications generate intratumor heterogeneity creating distinct genetic and epigenetic sub-populations or clones. A branched tumor evolutionary architecture can emerge containing the plasticity to progress under harsh environmental conditions and thwart therapeutic attempts to eradicate the tumor. It can be argued that until we discover how intratumor heterogeneity can be circumvented, precision oncology initiatives may fall short of expectations. Single cell sequencing methodologies have improved the genomic, transcriptomic, and epigenomic resolution of clonal tumor populations; however, the phenotypic implications of these alterations remain unclear. This is partly due to experimental challenges and is compounded by phenotypic plasticity that allows cancer cells to adapt to local changes in the microenvironment, without changes to the genome itself (e.g., epithelial to mesenchymal transition). Despite repeated observations that a small number of rare cancer cells or clones, hidden within a larger tumor population can drive tumor growth and spread, studies linking single cell or clonal phenotypes with genomic data have been limited. To probe the biology of a rare and phenotypically heterogeneous cell populations, single cells or subclones need to be isolated based upon user-defined criteria, instead of a random isolation approach; therefore, we developed a technique to image live cells within a biologically relevant 3-D environment, select a cell or cellular group based upon user-defined criteria, extract the cell(s), and subject the cell(s) to genomic and molecular analyses. In this way, we can purify, amplify, and systematically dissect the biologies of rare cells. This new technique, termed spatiotemporal genomic and cellular analysis (SaGA), was used to dissect the phenotypic heterogeneity of collective cancer cell invasion in a 3-D lung cancer model. These data incorporate the first SaGA-derived leader and follower cell lines to reveal that leader cells utilize atypical vasculogenesis signaling machinery by secreting VEGF to attract follower cells in invasive cell chains. In contrast, follower cells support leader cell growth by increasing their mitotic efficiency. This relationship argues for a cellular symbiosis within the collective invasion pack. Furthermore, these data provide proof of concept that SaGA is a powerful technology for dissecting phenotypic heterogeneity within cancer cell populations.
2017-02-10 | GSE93865 | GEO
Project description:Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is only displayed by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we demonstrated that Pitx3-/- embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA-signaling in Pitx3-/- embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and TH are regulated by Pitx3 and RA signaling, influencing the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA-signaling represents only one aspect of the Pitx3 downstream cascade, since Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.
2011-11-22 | GSE32940 | GEO
Project description:Adult zebrafish regenerate about half of cerebrospinal axons by six weeks after a complete spinal cord transection. We isolated the two populations of neurons that successfully regenerated their axons versus those that did not and isolated the total RNA. Cerebrospinal neurons from unlesioned animals were also collected to serve as controls. Whole transcriptome microarray was performed to determine axonal regeneration-associated genes. For each condition (regenerated, non-regenerated and control neurons) we had 3 biological replicates that consisted of neurons pooled from 3 to 9 zebrafish. A complete spinal cord transection with Fluororuby retrograde tracing was performed at the 8th vertebra level in adult zebrafish. Fluororuby labeled all the neurons in the brain that project their axons to the 8th vertebra. Three weeks later Fluoroemerald was injected 4mm distally from the spinal cord transection site, thereby labeling all neurons that regenerated their axons to this level. The zebrafish were sacrificed 1 week after Fluoroemerald tracing, brain enzymatically dissociated and cells sorted using fluorescence-activated cell sorter. After sorting the total RNA was extracted, amplified and labeled with biotin and then hybridized to Affymetrix 3' IVT gene expression microarray. Unlesioned fish were traced with Fluororuby at the 8th vertebra level and labeled neurons isolated 1 week after to serve as control for gene expression microarray.
2015-04-17 | E-GEOD-56842 | 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. ChIP-seq studies: We characterize the binding of Cdx2 in progenitor motor neurons using a V5 tagged doxycycline inducible Cdx2 ESC line (iCdx2). Progenitor motor neurons were generated after 4 days of in vitro differentiation of mouse embryonic stem cells using retinoic acid (RA) and hedgehog (Hh) signaling exposure at day 2. On day 3, the cells are exposed to Dox with and without accompanying FGF signaling. The genome-wide binding of the induced Cdx2 transcription factor is profiled using ChIP-seq with an anti-V5 antibody. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included. We also examine the effect of induced Cdx2 expression on polycomb-associated chromatin structure in the resulting cellular populations by profiling the H3K27me3 chromatin mark using ChIP-seq. H3K27me3 experiments were performed after 5 days of in vitro differentiation using cells exposed to either: 1) RA & Hh to derive progenitor motor neurons, followed by Dox & FGF; 2) Dox & FGF alone; or 3) RA and Hh alone. There are 6 Illumina sequencing datasets included in this submission: two biological replicates of iCdx2 ChIP-seq in the presence of FGF; one sample of iCdx2 ChIP-seq in the absence of FGF; one H3K27me3 ChIP-seq in the presence of RA, Hh, Dox, and FGF; one H3K27me3 ChIP-seq in the presence of Dox and FGF; and one H3K27me3 ChIP-seq in the presence of RA and Hh.
2013-07-04 | E-GEOD-39433 | biostudies-arrayexpress
Project description:We have conducted quantitative proteomic analyses of the axons of cultured rat cortical neurons. Axons are isolated by using glass chips that enable the axons and their cell bodies of neurons to grow in separated regions on the chip surface. Proteins extracted from the isolated axons, as well as those extracted from whole cortical neurons are subjected to two-dimensional liquid chromatography (2D-LC)-mass spectrometry (MS)-MS analyses. The abundances of proteins in the axon are found to be strongly correlated with their average abundances in whole neurons. Based upon these data, a quantitative description of the protein distribution among various subcellular structures in the axon has been generated. The proteins extracted from the axons and whole neurons are also subjected to stable isotope dimethyl labeling reaction and then to 2D-LC-MS/MS analysis. Proteins enriched in the axon compartment of rat cortical neurons are thus identified.
2018-04-17 | PXD005527 | Pride