Multiple Transgenic Models for Degenerative & Regenerative Research in Zebrafish

DESCRIPTION (provided by applicant): Zebrafish have a remarkable capacity for cellular regeneration and are amenable to mutational genetic screens. Accordingly, a transgenic zebrafish system that facilitates the removal of specific cell types and the detection of regenerative "replacement" cells has been developed. This inducible ablation platform, termed ZAP (zebrafish ablation-reporter protein), is based on the principle of cytotoxic pro-drug conversion and can be targeted to any genetically definable cell type. Transgenic expression of ZAPs can therefore be used to model any degenerative condition linked to the loss of specific cells or tissues. ZAPs consist of a pro-drug converting enzyme fused to a fluorescent reporter. The pro-drug converting enzyme converts otherwise innocuous substances into cellular toxins, facilitating inducible elimination of ZAP-expressing cells. The fluorescent reporter allows the presence or absence of targeted cell types to be tracked in living fish. Thus, the ZAP platform provides a means to discovering genetic pathways that regulate the regeneration of specific cell types from discrete adult stem cell populations. Proposed is the creation of a set of transgenic zebrafish lines designed to promote maximum flexibility regarding where, when, how much, and what color ZAPs are expressed. The Gal4-UAS system is employed as a modular component that separates transgene expression into elements conferring timing and location (Gal4-expressing "activator" lines) from elements specifying amount and type of transgene product produced (UAS:reporter "effector" lines). By mating different Gal4 and UAS lines, any expression pattern can be conferred to any reporter/effector gene. An enhancer trap approach, based on Tol2-mediated transposition, will be used to create a series of ~500 Gal4-expressing transgenic lines that allow specific cell types to be targeted. A complimentary series of UAS lines will facilitate control over ZAP expression levels and choice of fluorescent reporter color. Collectively then, these models will promote insight into the development, function, and regeneration of specific cell types. Accordingly, the transgenic lines generated will be characterized in detail and distributed to the larger research community. The short-term goal is to foster elucidation of the molecular mechanisms regulating adult stem cell niches within the regenerative biology research community. The long-term goal of this project is to promote the development of regenerative therapies capable of reversing the debilitating effects of degenerative conditions that plague humankind.