The proposed career development award is designed to support the transition of David A. Brown, M.D., Ph.D., to an independent investigator in the field of wound healing. Dr. Brown is a plastic and reconstructive surgeon at Duke University with a Ph.D. in tissue engineering. His long-term career goal is to utilize principles of bioengineering and regeneration biology to develop novel regenerative therapies for chronic wounds. Advanced training in regeneration biology is proposed, comprised of laboratory work under the mentorship of Kenneth Poss, Ph.D., a leader in the field of zebrafish models for tissue regeneration, as well as structured activities, coursework, and mentorship from faculty in related fields. The project will focus on re- epithelialization, which is mediated by migration of basal epidermal keratinocytes and stands as a central process in wound healing required to prevent progression to chronic wounds. Zebrafish harbor an innate ability to re-epithelialize wounds and regenerate skin more rapidly and completely than mammals, although the underlying mechanisms of this ability remain unclear. Based on prior work that demonstrates the existence of tissue regeneration enhancer elements (TREEs) involved in zebrafish heart and appendage regeneration, the central hypothesis of this project is that re-epithelialization is governed by regeneration-linked regulatory elements that orchestrate coordinated basal keratinocyte behaviors and gene expression. This proposal represents a novel approach to wound healing research and will provide the candidate with high-level training in live imaging, transgenesis, and genomics that will supplement his background in tissue engineering.
In Aim 1, live clonal analysis and transgenic zebrafish lines will be used to map dynamic basal keratinocytes behaviors in re-epithelialization. Experiments will track cell fate decisions and morphologic changes during re- epithelialization and identify subpopulations of basal cells and their relative contributions to the neoepidermis. A quantitative imaging practical course will be pursued along with mentorship from experts in wound healing and live imaging.
In Aim 2, the candidate will determine the role of the 103runx1 TREE in basal keratinocyte fate decisions, then use transcriptome sequencing, chromatin profiling, and transgenic assays to identify other key cis-acting regulatory sequences involved in skin regeneration. University courses in transcriptional regulation and computational genomics will be completed as well. Altogether, these experiments are expected to link critical cell fate decisions of basal keratinocytes to activation of specific regulatory elements. Future studies will test the hypothesis that genetic manipulation of these elements can enhance re-epithelialization by promoting basal keratinocyte migration and proliferation, thus leading to improved wound healing. This project will position the candidate to submit an R01 application in the final years of the award period, which will be focused on the next translational step in a mammalian model.
Zebrafish exhibit a remarkable ability to regenerate complex tissues such as heart and spinal cord. This project examines the molecular mechanisms of skin regeneration in zebrafish, particularly the central process of re- epithelialization, in order to develop novel regenerative strategies for human wound healing. A career development plan is proposed that supports the transition of the applicant, a plastic and reconstructive surgeon with a background in biomedical engineering, to an independent investigator in this field.
