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Single-cell RNA sequencing of primary cultures of neonatal digit amputation wound cells 4 days post-amputation

ABSTRACT: Single-cell RNA sequencing of primary cultures of neonatal digit amputation wound cells 4 days post-amputation

PROVIDER: PRJNA768252 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16173005_1.fastq.gz	Fastqsanger.gz
	SRR16173005_2.fastq.gz	Fastqsanger.gz
	SRR16173006_1.fastq.gz	Fastqsanger.gz
	SRR16173006_2.fastq.gz	Fastqsanger.gz
	SRR16173007_1.fastq.gz	Fastqsanger.gz

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Single-cell RNA sequencing of primary cultures of neonatal digit amputation wound cells 4 days post-amputation

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Transcriptomic Landscape Around Wound Bed Defines Regenerative Versus Non-regenerative Outcomes in Mouse Digit Amputation

Project description:In the mouse distal terminal phalanx (P3), it remains mystery why amputation at less than 33% of the digit results in regeneration, while amputation exceeding 67% leads to non-regeneration. Unraveling the molecular mechanisms underlying this disparity could provide crucial insights for regenerative medicine. In this study, we aim to investigate the tissues within the wound bed to understand the tissue microenvironment associated with regenerative versus non-regenerative outcomes. We employed a P3-specific amputation model in mice, integrated with time-series RNA-seq and a macrophage assay challenged with pro- and anti-inflammatory cytokines, to explore these mechanisms. Our findings revealed that non-regenerative digits exhibit a greater intense early transcriptional response in the wound bed compared to regenerative ones. Furthermore, early macrophage phenotypes differ distinctly between regenerative and non-regenerative outcomes. Regenerative digits also display unique co-expression modules related to Bone Morphogenetic Protein 2 (Bmp2). The differentially expressed genes (DEGs) between regenerative and non-regenerative digits are enriched in targets of several transcription factors, such as HOXA11 and HOXD11 from the HOX gene family, showing a time-dependent pattern of enrichment. These transcription factors, known for their roles in bone regeneration, skeletal patterning, osteoblast activity, fracture healing, angiogenesis, and key signaling pathways, may act as master regulators of the regenerative gene signatures. Additionally, we developed a deep learning AI model capable of predicting post-amputation time and level from RNA-seq data, indicating that the regenerative probability may be "encoded" in the transcriptomic response to amputation.

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Transcriptomic Landscape Around Wound Bed Defines Regenerative Versus Non-regenerative Outcomes in Mouse Digit Amputation

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Single cell RNA sequencing of adult regenerating mouse digit tips

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 one additional single cell RNA sequencing sample (28 days post-amputation) to add to our already published 38,000 cells from mouse digit tip blastemas and unamputated control digit tips used to generate an atlas of the cell types participating in digit tip regeneration.

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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: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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