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Plastic Surgery Research Council

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A Multi-Species Approach To Limb Regeneration, From Fins To Feet
David A. Brown, MD, PhD, Grant Darner, BS, Timothy Curtis, Jr., BS, Jianhong Ou, PhD, Thomas Dvergsten, BS, Sydney Jeffs, BS, Kenneth Poss, PhD.
Duke University, Durham, NC, USA.

PURPOSE: The study of limb and appendage regeneration, which is prevalent in various forms in the animal kingdom, may inform novel strategies for wound healing and regenerative medicine. The first critical step in appendage regeneration is re-epithelialization to form a specialized epidermis, known as the regeneration epidermis, which functions as a signaling center to induce reformation of the structure from underlying mesenchyme in the amputation stump. Despite its universal necessity in appendage regeneration among different species, widely conserved factors defining the regeneration epidermis have not been identified.
METHODS: Zebrafish caudal fin and mouse digit were used as models for appendage regeneration. The regeneration epidermis of zebrafish caudal fin was profiled with single-cell RNA sequencing. Immunofluorescence and in situ hybridization were used to characterize gene expression. Zebrafish null mutants were generated with the CRISPR/Cas-9 system. Epidermal-specific, inducible deletion mouse mutants were produced by crossing mice expressing CreER driven by the Krt14 promoter to others harboring floxed alleles of the target genes. Discarded leg segments from staged human amputations were obtained to assay for Sp expression.
RESULTS: Single-cell RNA sequencing in regenerating zebrafish fin revealed an enriched cell cluster marked by the Sp transcription factor family. Immunofluorescence and in situ hybridization confirmed the expression of Sp family members in the basal epidermis of regenerating caudal fin as well as regenerating mouse digits. CRISPR/Cas-9 was used to generate null mutants in zebrafish for several Sp genes, which demonstrate a variety of skeletal defects. Mice deficient for Sp genes exhibit distal limb defects, while induced deletion of in the basal epidermis of adult mice before digit amputation display impaired bone outgrowth, suggesting a critical role in this process. Intriguingly, we also detected Sp transcripts in the wound epidermis of human leg amputations. Upcoming work will identify transcriptional targets of Sp genes in regenerating mouse digits and human leg amputation wounds with CUT&RUN sequencing.
CONCLUSION: Sp transcription factors may represent conserved genes that participate in appendage regeneration across various species. Future work will define the role of these factors in regeneration-linked gene expression, as well as explore the idea that their expression may be remnants of an incomplete limb regeneration program in humans.


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