PROJECT SUMMARY / ABSTRACT Tissue regeneration is the process through which damaged tissue is restored to its original structure and function. There is wide variation across species in their regenerative ability. For example, zebrafish can regenerate all retinal neurons after injury while humans and mice cannot. Understanding the genetic basis and molecular underpinnings of complex tissue regeneration in model species holds the promise to enhance human regenerative medicine. Here I am using zebrafish to test the novel hypothesis that the control of transposable elements (TEs) is a necessary checkpoint for complex tissue regeneration. TEs are mobile DNA elements capable of self-replication that are ubiquitous and abundant in eukaryotes. Uncontrolled TE activity leads to accumulation of TE-encoded nucleic acids and proteins that interfere with cell homeostasis and can result in DNA damage, disrupting genome integrity. TE upregulation has been reported during tissue regeneration in salamanders, sea cucumbers, and worms. I hypothesize that TE activation is a hallmark of tissue injury that must be suppressed for successful regeneration, and an inability to suppress TEs will stall regeneration. Supporting this hypothesis, my preliminary analyses of bulk RNA-seq data reveal TE upregulation during early stages of eye regeneration that are later restored to control levels prior to tissue repair. I predict that zebrafish and other organisms with a strong regenerative capacity deploy specific control systems to suppress TE activity during regeneration. Here I will directly test the role of the Piwi pathway in suppressing TE activity during zebrafish eye regeneration. The Piwi pathway is known to repress TEs in animal gonads, including zebrafish, but there is growing evidence that the pathway is active in somatic tissues and required for regeneration in planarians. Furthermore, I have detected piwil1 expression in the zebrafish eye, raising the testable hypothesis that it functions during eye regeneration. I will utilize a model of zebrafish retinal regeneration and a 2-pronged approach combining multimodal genomics and manipulative experimentation. First, I will further establish that TE upregulation is a hallmark of tissue injury by profiling TE expression changes across five regenerating tissues using publicly available single- cell transcriptomic data. Second, I will generate a multi-omic single-cell dataset to assess TE expression changes during cone regeneration from the onset of injury through to functional recovery. These data will provide the most comprehensive and precise view of TE expression dynamics during regeneration for any species. Lastly, I will directly test whether TE repression is required for regeneration by modulating TE activity using Piwi pathway mutants and chemical inhibitors of TE activity. Together the outcomes of this project will be the first to directly assess the role of TE activity and regulation during complex tissue regeneration. Moreover, these studies will lay the foundation for new testable hypotheses surrounding differences between regeneratively competent versus incompetent organisms and lead to the development of novel regenerative therapies.

PROJECT NARRATIVE Transposable elements are differentially regulated upon injury; however, a functional assessment of their contribution or hinderance to tissue regeneration has yet to be established. Expanding our understanding of how transposons respond to and are regulated within the regeneration environment will greatly expand our understanding of how transposons affect the regeneration outcome. The work proposed herein will provide foundational insights regarding the transposon response to injury and define transposon control as a critical checkpoint for successful tissue regeneration.