Determining the role of cell proliferation in mediating circadian effects on regeneration - SUMMARY: Circadian clocks affect regeneration and wound healing, and the possibility of manipulating circadian state to improve physiological outcomes has great promise for regenerative medicine. Clocks regulate cell cycle checkpoints and cell proliferation, and this is a likely mechanism for circadian effects on regeneration. However, this mechanism has not been explored in the context of regeneration of entire structures—such as limbs, organs, or whole bodies. Regeneration has complex but stereotyped temporal dynamics with an initial wound healing phase followed by coordinated programs of cell death and proliferation. In general, we do not understand which phases of regeneration are specifically subject to circadian control, nor the molecular and cellular mechanisms mediating these effects. We hypothesize that circadian clocks affect regeneration efficiency by controlling cell proliferation, and that circadian defects will thus primarily affect proliferation-dependent phases of regeneration. We will test this hypothesis using an invertebrate model, the highly-regenerative sea anemone Nematostella vectensis, because this is the only model system that is well-established for both whole-body regeneration and circadian rhythms. We will perform a series of physiological, molecular, and bioinformatic experiments to determine the role of circadian rhythms in modulating wound healing and regeneration, with a focus on identifying the temporal windows of regeneration most susceptible to circadian control and the cellular mechanisms mediating these effects. First, we will determine the effects of circadian rhythms on the temporal progression of whole-body regeneration, including effects on cell proliferation (Aim 1). We will accomplish this using a series of circadian manipulations (e.g., genetic clock defects and different environmental regimes), and assays of morphological endpoints, cell death, and proliferation with high temporal resolution. We will also determine how circadian state affects the temporal dynamics of gene expression during regeneration, which will identify genes and molecular processes that may mediate circadian-regeneration interactions (Aim 2). Finally, we will quantify changes in cell cycle dynamics and cell populations across circadian states using single-cell RNA-sequencing and analyses of RNA velocity (Aim 3). This will test whether circadian rhythms affect regeneration-induced cell proliferation and identify cell populations with key roles in regeneration. Collectively, these aims will advance our knowledge of how regeneration programs operate within the dynamic temporal environment of an organism and clarify the role of circadian-mediated cell proliferation on regeneration outcomes. This research will be performed at the University of California, Santa Barbara under the co-mentorship of Dr. Todd Oakley and Dr. Joel Rothman. The proposed training plan will prepare the fellow for a future career as an independent principal investigator by acquiring new research skills in regeneration biology, microscopy, and single-cell sequencing methods, and developing key professional skills such as mentorship, pedagogy, and grant writing.
