Identifying the driving factors for regenerative fibroblasts in the mouse digit tip

Abstract

Composite tissue regeneration is very limited in mammals; however, humans and mice can fully regenerate the distal tips of the digits following amputation. This process involves the formation of a blastema, a cellular structure that is the source of the regenerated tissue and is integral to successful regeneration. Proximal amputations beyond the nail do not form a blastema and result in fibrotic wound healing. This differential behavior makes the mouse digit tip an ideal model system to investigate the cellular and molecular factors driving each response and why complex regeneration is so limited in mammals. Chapter 1 is an overview of mouse digit fibrosis vs regeneration, discussing the roles of each cell type established in each distinct wound healing process in the digit. In particular, fibroblasts are a major contributor to the blastema and play an integral part in fibrosis; thus, they may be a cell population that drives the decision between fibrosis and regeneration. My thesis focused on fibroblast subtypes and their role in fibrosis versus regeneration, with specific attention on the blastema enriched gene Mest. To understand Mest’s role in digit tip regeneration (Chapter 2), I further verified that it is not expressed in fibrotic tissue, indicating blastemal-specific expression, and found that Mest knock-out mice exhibited delayed bone regeneration due to an impaired neutrophil recruitment and clearance response. Additionally, I established that the locus could utilize an alternative promote to evade methylation-based transcriptional repression. While this study further established Mest as a pro-regenerative factor, I aimed to identify additional candidate genes and functionally test them (Chapter 3). To achieve these goals, I performed single-cell sequencing of fibrosing digits and computationally compared them to blastemal tissue. From this, we were able to identify fibroblast subtypes and genes specific to fibrosis or regeneration. We then developed an AAV gene delivery technique to functionally assess our candidate genes in vivo, which successfully drove differing wound healing outcomes. Ultimately, my thesis work advances our understanding of fibroblast heterogeneity in digit wound healing and provides a robust framework for investigating and modulating molecular factors that govern fibrosis and regeneration.