Project description:RNA sequencing was applied to examine changes in gene expression in reactive astrocytes following traumatic spinal cord injury (SCI) or neuroinflammatory insult by systemic administration of lipopolysaccharide (LPS). Transgenic Ribotag technology was used to isolate astrocyte ribosome-associated mRNAs from the spinal cord of wild type mice and mice with astrocyte-specific conditional gene deletion (cKO) of test-case transcriptional regulators of reactivity, SMARCA4 (Smarca4-astro-cKO) or STAT3 knockout (Stat3-astro-cKO). Examination of differentially expressed gene (DEG) profiles by upstream transcriptional regulator enrichment analysis (TREA) was used identify transcriptional regulators of reactivity in each condition. Together, findings from this study show that transcriptional changes associated with astrocyte reactivity are exquisitely heterogeneous and are customized from vast numbers of potential DEGs via context-specific combinatorial TR interactions. These data have also been made accessible in a open-access, searchable database: http://tr.astrocytereactivity.com
Project description:Single-nuclei Assay for Transposase-Accessible Chromatin with sequencing (snATACseq) was applied to examine chromatin landscape changes and transcriptional regulator (TR) DNA motif accessibility in reactive astrocytes following traumatic spinal cord injury (SCI). Astrocyte nuclei were isolated from the spinal cord of wild type mice and mice with astrocyte-specific conditional gene deletion (cKO) of test-case TRs, SMARCA4 (Smarca4-astro-cKO) or STAT3 knockout (Stat3-astro-cKO). Comparison of differential chromatin accessibility revealed substantial remodeling during astrocyte reactivity, with more chromatin opening than closing. Marked alterations in access to SCI reactivity-associated TR motifs were also detected.
Project description:Analysis of gene expression by astrocytes or non-astrocyte cells in spinal cord injury (SCI) lesions may lead to the identification of molecules that impact on axon regrowth. We conducted genome-wide RNA sequencing of (i) immunoprecipitated astrocyte-specific ribosome-associated RNA (ramRNA) from WT or STAT3-CKO astrocytes, and (ii) the non-precipitated (flow-through) RNA deriving from non-astrocyte cells in the same tissue samples 14 days following SCI. DOI: 10.1038/nature17623 Young adult female mGFAP-Cre-RiboTag or mGFAP-Cre-RiboTag-STAT3-LoxP mice underwent severe crush SCI at thoracic level 10. 14 days following SCI, the central 3mm of the SCI lesion was extracted, homogenized and (i) astrocyte-specific ribosome-associated RNA (ramRNA) precipitated via a hemagglutinin (HA) tag targeted to either WT (n=4) or STAT3-CKO (n=3) astrocytes, and (ii) the non-precipitated (flow-through) RNA deriving from non-astrocyte cells in the same tissue samples. Sex and age-matched mGFAP-Cre-RiboTag mice served as uninjured controls (n=4).
Project description:We conducted snRNAseq of mouse astrocytes after traumatic spinal cord injury (SCI). These data reveal transcriptomic similarities and differences among astrocytes in healthy spinal cord and after spinal cord injury.
Project description:Analysis of gene expression by astrocytes or non-astrocyte cells in spinal cord injury (SCI) lesions may lead to the identification of molecules that impact on axon regrowth. We conducted genome-wide RNA sequencing of (i) immunoprecipitated astrocyte-specific ribosome-associated RNA (ramRNA) from WT or STAT3-CKO astrocytes, and (ii) the non-precipitated (flow-through) RNA deriving from non-astrocyte cells in the same tissue samples 14 days following SCI. DOI: 10.1038/nature17623
Project description:Adult zebrafish have the ability to recover from spinal cord injury and exhibit re-growth of descending axons from the brainstem to the spinal cord. We performed gene expression analysis using microarray to find damage-induced genes after spinal cord injury, which shows that Sox11b mRNA is up-regulated at 11 days after injury. However, the functional relevance of Sox11b for regeneration is not known. Here, we report that the up-regulation of Sox11b mRNA after spinal cord injury is mainly localized in ependymal cells lining the central canal and in newly differentiating neuronal precursors or immature neurons. Using an in vivo morpholino-based gene knockout approach, we demonstrate that Sox11b is essential for locomotor recovery after spinal cord injury. In the injured spinal cord, expression of the neural stem cell associated gene, Nestin, and the proneural gene Ascl1a (Mash1a), which are involved in the self-renewal and cell fate specification of endogenous neural stem cells, respectively, is regulated by Sox11b. Our data indicate that Sox11b promotes neuronal determination of endogenous stem cells and regenerative neurogenesis after spinal cord injury in the adult zebrafish. Enhancing Sox11b expression to promote proliferation and neurogenic determination of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after spinal cord injury in mammals. Spinal cord injury or control sham injury was performed on adult zebrafish. After 4, 12, or 264 hrs, a 5 mm segment of spinal cord was dissected and processed (as a pool from 5 animals) in three replicate groups for each time point and treatment.
Project description:Comparison of genomic data from neural progenitor cells derived from mouse embryonic stem cells under different experimetnal conditions in vitro and invivo. We conducted genome-wide RNA sequencing of immunoprecipitated specific ribosome-associated mRNA using RiboTag methods from: (i) mouse embryonic stem cell (ESC), (ii) derived neural progenitor cells, (iii) differentiated neural progenitor cells (in vitro), (iv) grafted neural progenitor cells (recovered from different in vivo tissue enivornments - healthy spinal cord, spinal cord injury lesions) and (v) host astrocytes using GFAp-Cre RiboTag mice.
Project description:We have previously shown that Il1a-knockout (KO) mice exhibit rapid (at day 1) and persistent improvements in locomotion associated with reduced lesion volume compared with Il1b-KO mice and C57BL/6 controls after traumatic spinal cord injury (SCI). To investigate the mechanism by which Il1a mediates its detrimental effect, we analyzed the transcriptome of the injured spinal cord of Il1a-KO, Il1b-KO and C57BL/6 mice at 24 hours after SCI using GeneChip microarrays. Il1a-KO, Il1b-KO and C57BL/6 mice were subjected to a 50-kdyn SCI and a 6-mm spinal cord segment centered over the site of contusion extracted for RNA isolation and microarray analysis.
Project description:Traumatic spinal cord injury (SCI) often leads to loss of locomotor function. Neuroplasticity of spinal circuitry underlies some functional recovery and therefore represents a therapeutic target to improve locomotor function following SCI. However, the cellular and molecular mechanisms mediating neuroplasticity below the lesion level are not fully understood. The present study performed a gene expression profiling in the rat lumbar spinal cord at 1 and 3 weeks after contusive SCI at T9 compared to control rat that received sham injury (laminectomy). The below-level gene expression profiles were compared with those of animals that were subjected to treadmill locomotor training. Rat lumbar spinal cords were taken for the microarray analysis at 1 and 3 weeks after contusive spinal cord injury at the T9 level. Another group of rats received treadmill locomotor training for 3 weeks, and theirs spinal cords were harvested for the microarray. The changes in gene expression after spinal cord injury were analyzed at the two time points. The influence of treadmill locomotor training was evaluated by comparing gene expression profiles between animals with or without treadmill training.
Project description:Adult zebrafish have the ability to recover from spinal cord injury and exhibit re-growth of descending axons from the brainstem to the spinal cord. We performed gene expression analysis using microarray to find damage-induced genes after spinal cord injury, which shows that Sox11b mRNA is up-regulated at 11 days after injury. However, the functional relevance of Sox11b for regeneration is not known. Here, we report that the up-regulation of Sox11b mRNA after spinal cord injury is mainly localized in ependymal cells lining the central canal and in newly differentiating neuronal precursors or immature neurons. Using an in vivo morpholino-based gene knockout approach, we demonstrate that Sox11b is essential for locomotor recovery after spinal cord injury. In the injured spinal cord, expression of the neural stem cell associated gene, Nestin, and the proneural gene Ascl1a (Mash1a), which are involved in the self-renewal and cell fate specification of endogenous neural stem cells, respectively, is regulated by Sox11b. Our data indicate that Sox11b promotes neuronal determination of endogenous stem cells and regenerative neurogenesis after spinal cord injury in the adult zebrafish. Enhancing Sox11b expression to promote proliferation and neurogenic determination of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after spinal cord injury in mammals.