Interrogating mechanisms of limb regeneration using a viral toolbox for molecular gain- and loss-of-function

Incomplete healing and limb loss impact millions of Americans each year. However, animals such as the Mexican axolotl salamander (Ambystoma mexicanum) have an exceptional ability to regenerate complex organs and tissues, including full limbs. Understanding the molecular mechanisms that make regeneration possible in the axolotl has the potential to advance therapies for improved human healing and tissue regeneration. An integral component of regeneration is the potential for contributing cells to revert back to a developmental-like state in a process called dedifferentiation. Despite its crucial role in limb regeneration, we know little about the molecular underpinnings of regenerative dedifferentiation. A key limitation to testing potential regulators of cellular dedifferentiation is a lack of tools that allow gain- or loss-of-function in complex, post-developmental limb tissue. The work outlined in this application leverages expertise in creating and applying molecular tools in the axolotl to overcome these challenges by i) validating new viral gene delivery methods in the axolotl limb and ii) combining viral delivery with novel transgenic strategies for gain-of-function, CRISPR/Cas-mediated loss-of-function, and cell type-specific nuclei isolation. Using these tools, we will test the role of a candidate transcriptional regulator in driving dedifferentiation and successful limb regrowth. The outcome of this work will not only establish new tools to elucidate mechanisms of regenerative dedifferentiation but will also provide a framework for systematic investigation of regulators of limb regeneration. Project Number: 1R21HD119496-01 | Fiscal Year: 2025 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Joshua Currie | Institution: WAKE FOREST UNIVERSITY, WINSTON-SALEM, NC | Award Amount: $390,101 | Activity Code: R21 | Study Section: Development - 2 Study Section[DEV2] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21HD11949601