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

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. Overall design: (a) Tissues around Mouse wound bed was collected (b) Macrophage (in vitro) was collected. We have submitted mouse digit amputation wound bed RNA-seq data (<33% amputation level) to GEO with accession number: GSE130438 before (regenerative digits). In this study, we submitted mouse digit amputation wound bed RNA-seq data (>34% amputation level) and macrophage in vitro (M0, M1 and M2 phenotypes) RNA-seq data to GEO with accession number: GSE279540