RNA-seq analysis of FACS-sorted control and Tsc2-deleted nociceptors
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA447995 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6915340.fastq.gz | Fastqsanger.gz | |||
| SRR6915350.fastq.gz | Fastqsanger.gz | |||
| SRR6915331.fastq.gz | Fastqsanger.gz | |||
| SRR6915341.fastq.gz | Fastqsanger.gz | |||
| SRR6915351.fastq.gz | Fastqsanger.gz |
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Project description:The goals of this study are to compare the transcriptomes of control heterozygous and Tuberous sclerosis 2 (Tsc2) homozygous nociceptors that have been enriched by fluorescence-associated cell sorting (FACS) with the aim of identifying differences in genes associated with sensory behavior. Nav1.8-Cre transgenic mice were used to delete Tsc2 as well as express Green fluorescent protein from the Rosa26 locus. Tsc2 deletion constitutively activates mTORC1 signaling cell autonomously. Dissociated dorsal root ganglia from adult mice were FACS-sorted for GFP and analyzed by RNA-seq. We find that a number of sensory behavior associated genes are affected by Tsc2 deletion including Calca, Ntrk1, Mrgpra3, and Nppb. Additionally, profiling of ion channel expression showed a reduction in almost half of expressed sodium, potassium and calcium channel as well as G-protein coupled receptors. Other categories of genes such as transcription factors and ligand-gated ion channels contained an even numbers of up- and downregulated genes with many that did not change as a result of Tsc2 deletion. In addition, expression of most marker genes enriched in Isolectin B4 (IB4) negative neurons were strongly reduced in Tsc2 mutant nociceptors, consistent with reduced sensitivity to heat, which is a modality related to these neurons. We conclude Tsc2 deletion and consequent mTORC1 activation alters gene expression of nociceptors resulting in altered sensory thresholds.
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Project description:The goals of this study are to compare the transcriptomes of control heterozygous and Tuberous sclerosis 2 (Tsc2) homozygous nociceptors 3 days after sciatic nerve injury that have been enriched by fluorescence-associated cell sorting (FACS) with the aim of identifying differences in genes associated with axon regeneration. Nav1.8-Cre transgenic mice were used to delete Tsc2 as well as express Green fluorescent protein from the Rosa26 locus. Tsc2 deletion constitutively activates mTORC1 signaling cell autonomously. Dissociated dorsal root ganglia from adult mice were FACS-sorted for GFP and analyzed by RNA-seq. We find that the expression of a number of regeneration-associated transcription factors including cJun, Atf3, Sox11 and other are upregulated in Tsc2 mutant neurons in the absence of an injury. Additionally, the target genes of several other normally expressed regeneration-associated transcription factors are differentially expressed in Tsc2 mutant neurons, such as Creb1 and Klf’s. We conclude Tsc2 deletion and consequent mTORC1 activation establishes a pro-regenerative gene expression profile in nociceptors in the absence of an injury.
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Project description:RNA-seq analysis of FACS-sorted control and Tsc2 cKO nociceptors with and without sciatic nerve crush
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Project description:We explored the effects of nociceptors on DC under various conditions by means of in vitro coculture of Flt3L-induced bone marrow-derived DCs and primary mouse nociceptors.
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Project description:Silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. We had previously shown that mouse silent nociceptors express the nicotinic acetylcholine receptor alpha-3 subunit (CHRNA3) and can thus readily be identified in Tg(Chrna3-EGFP)BZ135Gsat reporter mice (Prato et al. Cell Reports 2017). Most importantly, we had shown that CHRNA3+ slient nociceptors acquire mechanosensitivity upon treatment with the inflammatory mediator nerve growth factor and demonstrated that this process requires de-novo gene transcription. Here, we performed paired-end RNA-sequencing to identify transcripts that might be involved in the acquisition of mechanosenstivity in mouse silent nociceptors.
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