Mechanisms of Ca2+/calcineurin-mediated organ size restoration during zebrafish fin regeneration - PROJECT SUMMARY This project provides the applicant with Ph.D. training in cell and developmental biology. Thesis research will further our understanding of how regenerating organs restrain outgrowth to return to their uninjured form. Zebrafish rapidly and robustly regenerate their fins to their original shape and size following injury. A fibroblast- derived, 'niche' blastemal sub-population promotes the progressive formation of replacement tissue. However, the niche proportionally depletes over regenerative outgrowth, exhausting as the original fin size is restored. Disrupted niche depletion in longfin mutants with ectopic expression of a potassium channel leads to disproportionately long fins. Small molecule inhibition of the Ca2+-dependent phosphatase calcineurin also prolongs regenerative outgrowth leading to long fins. Other long-finned models, including a new loss-of-function voltage-gated Ca2+ channel mutant, further tying ion signaling involving Ca2+/calcineurin to outgrowth control. However, the gene regulatory mechanisms and downstream cell behaviors by which Ca2+/calcineurin signaling promotes niche depletion and subsequently fin size are unresolved. This project aims to define how Ca2+/calcineurin and niche depletion gradually terminate fin outgrowth to help explain a classic mystery of robust organ size restoration during regeneration. Ca2+/calcineurin likely regulates a transcriptional pathway to drive niche depletion, possibly by favoring re-differentiation over niche cell renewal. However, the applicant’s preliminary data using long-term live imaging of a transgenic reporter line shows apoptotic niche cells within regenerating fins. This suggests Ca2+/calcineurin instead could drive depletion by programmed cell death. Together, these results support the applicant’s hypothesis that decelerating regenerative outgrowth is regulated by a Ca2+/calcineurin-dependent gene regulatory pathway facilitating niche depletion by re-differentiation and/or apoptosis. The applicant will explore this hypothesis with three aims. Aim 1 will Determine if ion signaling regulates calcineurin activity in niche cells to restrain fin outgrowth. Aim 2 will Define transcriptional targets downstream of voltage-gated Ca2+ channels and calcineurin signaling. Aim 3 will Determine if Ca2+/ calcineurin depletes regenerating fin niche cells by re-differentiation and/or apoptosis. Completing these aims will connect ion signaling (or “bioelectricity”) and organ size control to gene regulatory pathways and downstream cell behaviors in a compelling adult regeneration model. These insights will advance our mechanistic understanding of organ size control with potential biomedical significance for regenerative medicine and overgrowth-associated diseases including cancer. The applicant also will pursue activities supporting their professional development and acquisition of advanced imaging and bioinformatics, mentorship, and science communication skills to prepare them for a research leadership career.
