Abstract:
Adult teleost fish and urodele amphibians can regenerate entire amputated appendages, whereas this ability is
restricted to the tip of the distal phalanx in humans and mice. Though modest in comparison to its zebrafish and
salamander counterparts, mouse digit tip (MDT) regeneration exhibits similar anatomic stages and proceeds
with intramembranous bone regrowth and mostly scarless healing, even in adults. By studying shared principles
from organisms with elevated regenerative potential and applying them to mammalian models, we can gain
valuable insight into strategies to augment human tissue repair and improve the care of limb loss patients. The
catalogue of defined molecular factors in tissue regeneration is expanding and it has become critical to determine
how genes involved in regenerative events are engaged upon injury. We previously identified a new class of
regulatory sequences we named tissue regeneration enhancer elements (TREEs) that contain sequence
information necessary for precise control of regeneration genes. We also reported that TREEs of zebrafish origin
can be engineered in viral gene therapy constructs to target expression of pro-regenerative factors to mammalian
injury sites and enhance tissue repair. In preliminary collaborative studies between the Poss and Brown groups,
we have identified a new set of candidate TREEs from chromatin samples of regenerating MDTs. We have also
performed comparative studies guided by single-cell transcriptome analysis of regenerating zebrafish fins and
complemented by molecular genetics in mice, to implicate a conserved family of transcription factors in control
of digit tip regeneration. Concurrently, we have identified TREEs of zebrafish origin and an effective adeno-
associated virus (AAV) capsid variant that can transduce limb tissue and enable selective expression of gene
cargoes at an injury site. Our preliminary studies thus unveil a pipeline of TREE control element identification,
novel candidate pro-regenerative factor identification, and application of gene therapy methodology. Here, we
propose to: 1) identify distal regulatory elements that control key programs in MDT regeneration; 2) define
transcriptional mechanisms by conserved factors; and 3) enhance MDT regeneration by spatiotemporal delivery
of pro-regenerative factors. This work will increase understanding of transcriptional regulation during mammalian
digit tip regeneration and provide important perspective for comprehending, and perhaps changing, existing
limitations in the regenerative capacity of human limbs.