Quantitative dissection of size memory during zebrafish appendage regeneration

ABSTRACT The ability of mammals to regenerate injured appendages is limited to healing bone fractures and regenerating digit tips. For any future therapies to be useful they must pattern regrowth so that it restores tissue size and function and avoids hypertrophy and dysmorphology. In contrast to mammals, zebrafish regenerate entire appendages following an injury, such as amputation. Notably, regenerated fins reproduce the original size, shape, and function of the injured appendage. While previous work has identified molecules and cellular events required for promoting zebrafish fin regeneration, we still do not fully understand how cells within a regenerating appendage encode size memory or how cells dynamically monitor the progression of regenerative outgrowth to ensure accurate tissue size. This is partly due to the difficulty of rigorously documenting cellular events, such as signaling levels, in vivo in complex tissues at single-cell resolution. This proposal will overcome these difficulties by combining quantitative live imaging approaches, computational analysis, and theoretical modeling to map cell signaling activity with single cell resolution. Specifically, Aim 1 will dissect how the initial conditions encoding size memory are established for fin rays of different lengths. Aim 2 will uncover how size memory is processed in fibroblast tissue, which comprises connective tissue-secreting cells that lie inside and between bony hemirays. Aim 2 will also test the hypothesis that size memory is differentially regulated in fibroblasts and osteoblasts (bone-matrix secreting cells) by distinct upstream Fibroblast Growth Factor (Fgf) ligand expression. The research outlined here will form the intellectual basis of my own independent research program. Together with my advisors, Dr. Stefano Di Talia and Dr. Ken Poss, I have developed a training plan that will enable me to master skills in theoretical approaches and transgenic zebrafish generation as well as gain hands-on lab management experience during the K99 award phase. Furthermore, I have established an exceptional committee of advisors, including expert theorists and experimental biologists, who are committed to helping me develop my independent research program. The research and training described here will uniquely position me to apply this interdisciplinary, quantitative approach to additional signaling pathways, such as Wnt, and other regenerating tissues, including the vasculature and the nervous system. Long-term, I will lead my own research group in generating a wholistic understanding of how size memory is established and dynamically processed during fin regeneration. I expect the insights gained from this research will inform the development of regenerative therapies that promote tissue regrowth in humans without inducing pathogenic overgrowth or dysmorphology.