Single-cell RNA sequencing of primary cultures of neonatal digit amputation wound cells 4 days post-amputation
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ABSTRACT:
PROVIDER: PRJNA768252 | ENA |
REPOSITORIES: ENA
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Project description:Here we present single-cell RNA sequencing (scRNA-seq) data of untreated digit amputation wound cells that can be induced by BMP9 to undergo chondrogenesis to determine the responsive cell type
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Project description:Purpose: Investigate the transcriptomic landscape throughout the time course of murine digit regeneration after level-dependent amputation. Methods: The terminal phalanx bone of hind limb digits was subjected to either a distal amputation (25% bone length loss) or proximal amputation (65% bone length loss). Total RNA was isolated from bone and fibrous tissues distal to the distal interphalangeal joint at 12, 14, and 21 days post-amputation (DPA). mRNA library was sequenced using Illumina HiSeq 3000. Trimmed reads were aligned to the mouse genome mm10 and batch-corrected. Results: A limb-specific developmental signaling pathway is transiently upregulated at 14 DPA after distal amputation, corresponding with regeneration of digit tip tissues. Absence of a limb-specific pathway after proximal amputation corresponded with minimal regeneration and fibrotic scarring. Conclusions: Digit regeneration is a level-dependent and spatiotemporally controlled process, with distal and proximal amputations showing significant differences in gene expression and tissue regrowth over time.
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Project description:Innate regeneration following digit tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses in mice, the digit tip blastema has been defined as a population of heterogeneous, lineage restructed progenitor cells. These previous studies, however, do not comprehensively evaluate blastema heterogeneity or address lineage restruction of closely related cell types. Here we report single cell RNA sequencing of over 38,000 cells from mouse digit tip blastemas and unamputated control digit tips and generate an atlas of the cell types participating in digit tip regeneration.
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Project description:The distal mouse digit tip undergoes complex tissue regeneration following amputation, a process facilitated by the formation of a cellular structure called the blastema. Through single-cell RNA sequencing of the blastema, we identified the gene Mest (mesoderm specific transcript) highly expressed in a subset of blastemal fibroblasts. To determine if Mest expression is necessary for mouse digit tip regeneration, we analyzed Mest wildtype (WT) versus homozygous knockout (KO) post amputation digits. Through single-cell sequencing and FACS analysis of WT and KO regenerating tissues, we determined that this phenomenon was due to a prolonged immune response in the KO digits.
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Project description:Expression of genes encoding multiple keratin- and keratin-associated proteins varies between C57BL/6 and MRL/MpJ (superhealer) mice following digit amputation. We characterized changes in gene expression by microarray analysis of regenerating digits in these mice at various timepoints following surgical amputation. These changes were validated by quantitative rtPCR analyses.
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Project description:Transcriptional profiling of zebrafish rergenerating fins comparing control fins and regenerating fins (3 days following fin amputation)
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Project description:Adult zebrafish can completely regenerate their caudal fin following amputation. This complex process is initiated by the formation of an epithelial would cap over the amputation site by 12 hours post amputation (hpa). Once the cap is formed, mesenchymal cells proliferate and migrate from sites distal to the wound plane and accumulate under the epithelial cap forming the blastemal structure within 48 hpa. Blastemal cells proliferate and differentiate, replacing the amputated tissues, which are populated with angiogenic vessels and innervating nerves during the regenerative outgrowth phase which is completed around 14 days post amputation (dpa). Regenerative outgrowth does not occur in TCDD-exposed zebrafish. To identify the molecular pathways that are perturbed by TCDD exposure, male zebrafish were i.p. injected with 50 ng/g TCDD or vehicle and caudal fins were amputated. Regenerating fin tissue was collected at 1, 3 and 5 dpa for mRNA abundance analysis. Microarray analysis and quantitative real time PCR revealed that wound healing and regeneration alone altered the expression of nearly 900 genes by at least two fold between 1 and 5 dpa. TCDD altered the abundance of 370 genes at least two fold. Among these, several known aryl hydrocarbon responsive genes were identified in addition to several genes involved in extracellular matrix composition and metabolism. The profile of misexpressed genes is suggestive of impaired cellular differentiation and extracellular matrix composition potentially regulated by Sox9b. Keywords: Time course and TCDD exposure response
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