Next Generation Sequencing of zebrafish V2a interneurons in spinal segments rostral to the lesion after spinal cord injury
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ABSTRACT:
PROVIDER: PRJNA879067 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21518655_1.fastq.gz | Fastqsanger.gz | |||
| SRR21518655_2.fastq.gz | Fastqsanger.gz | |||
| SRR21518656_1.fastq.gz | Fastqsanger.gz | |||
| SRR21518656_2.fastq.gz | Fastqsanger.gz | |||
| SRR21518657_1.fastq.gz | Fastqsanger.gz |
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Project description:The goals of this study is to compare transcriptome profiles (RNA-seq) of zebrafish V2a interneurons with regrown axon and those without regrown axon in the spinal segments rostral to the lesion after spinal cord injury. For purification of V2a interneurons with regrown axon, the fluorescent tracer Rhodamine Dextran (RD) was retrogradely applied to Tg(Chx10:GFP) fish at three or eight weeks post injury. Spinal cord segments rostral to the lesion site was collected from 20 fish at 3- or 8 wpi, and corresponding spinal cord segments from 20 uninjured fish were collected as control material. GFP+/RD+ and GFP+/RD- cells were FAC-sorted and subjected to RNA-sequencing. Total RNA was isolated using SMART-SeqTM v4 UltraTM Low Input RNA Kit for Sequencing (Clontech). Sequencing libraries (N=5-6) were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina following the manufacturer’s instructions (NEB). We mapped about 40-80 million sequence reads per sample to the zebrafish genome and identified 42,370 transcripts in the zebrafish V2a interneurons in the spinal cord. Our study represents the detailed analysis of transcriptomes of zebrafish V2a interneurons with regrown axon and those without regrown axon in the spinal segments rostral to the lesion after spinal cord injury.
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Project description:The spinal cord contains neural networks that enable regionally-distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both fore and hindlimb movements and here we identified two major types that emerge during development: Type-I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons, and Type-II that downregulate Chx10 and project to supraspinal structures. Type-I and -II V2a interneurons are arrayed in counter-gradients and this network activates different patterns of motor output at cervical and lumbar levels. Single cell RNA-seq revealed Type-I and -II V2a neurons are each comprised of multiple subtypes. Our findings uncover a molecular and anatomical organization of V2a interneurons reminiscent of the orderly way motor neurons are divided into columns and pools.
2018-02-22 | GSE108788 | GEO
Project description:The spinal cord contains neural networks that enable regionally-distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both fore and hindlimb movements and here we identified two major types that emerge during development: Type-I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons, and Type-II that downregulate Chx10 and project to supraspinal structures. Type-I and -II V2a interneurons are arrayed in counter-gradients and this network activates different patterns of motor output at cervical and lumbar levels. Single cell RNA-seq revealed Type-I and -II V2a neurons are each comprised of multiple subtypes. Our findings uncover a molecular and anatomical organization of V2a interneurons reminiscent of the orderly way motor neurons are divided into columns and pools.
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Project description:The V2a neurons contribute significantly to recovery of locomotor and respiratory function after injury in animal models. We used single nucleus RNA sequencing of enriched cervical V2a neurons to analyze the diversity of these neurons and their response to spinal cord injury.
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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. ChIP DNA samples from Chx10-ESC-derived MNs were prepared for sequencing according to the Illumina protocol, and sequenced on the Illumina HiSeq 2000. The peak calling was conducted with MACS software (Zhang et al., 2008). MEME-ChIP Suite (Bailey et al., 2009; Machanick and Bailey, 2011) and TOMTOM algorithm (Gupta et al., 2007) was used for motif analysis.
2016-07-28 | E-GEOD-83872 | 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.
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Project description:In development, early regionalization steps segregate populations of cells and direct them to specific cell fates. In some instances, this process separates populations of progenitors which eventually become analogous cell types. V2a neurons are a population of excitatory interneurons which are found throughout the hindbrain and spinal cord, two regions of the CNS that arise from different progenitors in development. Plasticity in the V2a population after spinal cord injury has increased interest in this population as a therapeutic target. We therefore set out to examine how progenitor lineage influences V2a gene regulation and ultimately function. To do so, we differentiated V2a neurons from human stem cells via two distinct progenitor populations. Single nucleus multiomic analysis showed that different V2a lineages were enriched for different transcription factor motifs and differentially expressed hundreds of genes relevant to neuron function, such as axon growth and calcium handling. Attempting to ‘skip’ developmental patterning by induced transcription factor expression yielded a population unlike either developmentally relevant population, highlighting the importance of following developmental steps in establishing cell identities in vitro. Lastly, we employed the CellOracle tool and lentiviral knockdown to perturb lineage-specific gene regulatory networks and identify CREB5 and TCF7L2 as regulators unique to a spinal-like lineage. Overall, this work highlights how small differences in differentiation protocols can influence mature cell fate and uncovers new regulators of neural diversity along the anterior-posterior axis of the central nervous system.
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