Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons

Abstract

The zebrafish is a well-established model organism to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive thereby creating additional tissue damage and potential infection sites, or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell-death-inducing compounds. We have recently established a novel inducible genetic cell ablation system that is based on Tamoxifen-inducible Caspase8-activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here we prove its suitability in vivo by the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish, which coexpress the inducible Caspase and a fluorescent reporter to monitor ablation processes. Incubation of larvae in Tamoxifen for 8 hrs activated endogenous Caspase3 and cell death, while incubation for 16 hrs led to the nearly complete loss of PCs by apoptosis. Using live confocal imaging, we observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of Apoptosis Through Targeted Activation of Caspase by Tamoxifen (ATTACTM) further expands the repertoire of genetic tools in zebrafish for conditional interrogation of cellular functions.