Fig. 1

Fig. 1

Purkinje cell-specific expression of a tamoxifen-inducible caspase. (A) Schematic diagram of the Caspase 8 activation. Caspase 8 fused to the mutant estrogen receptor ligand-binding domain (ER^T2) dimerizes upon addition of tamoxifen (4OHT; orange double-headed arrow). Subsequent proteolytic self-activation of Caspase 8 leads to activation of endogenous Caspase 3 and cell apoptosis. (B) Construct design for the generation of stable transgenic zebrafish PC-ATTAC^TM strain. Membrane-bound fluorescent FyntagRFP together with Caspase8ER^T2 linked by a self-cleaving T2A-peptide is co-expressed under the control of a Purkinje cell-specific regulatory element (PC) from carbonic anhydrase 8, which is combined with a CMV basal promoter (CMV). (C) F2 transgenic larvae were analyzed at 5 dpf by confocal microscopy to verify transgene encoding FyntagRFP expression exclusively in the cerebellum. (D,E) Purkinje cell (PC)-specific expression of the transgene was confirmed by double-immunostaining against (D) FyntagRFP (red) and ZebrinII (green), and (E) Caspase 8 (green, combined with FyntagRFP fluorescence), demonstrating that Caspase8ER^T2 expression is confined to FyntagRFP-fluorescent ZebrinII-expressing PCs. (F) Owing to membrane targeting of FyntagRFP axons, somata and dendrites of PCs, including the long axonal projections forming the cerebello-octavolateralis tract to vestibular nuclei (white arrowheads), could be monitored. (G) Concomitant Caspase8ER^T2 and FyntagRFP-T or FyntagRFP expression was verified by western blot analysis. FyntagRFP-T (bz4Tg, Matsui et al., 2014) alone was used as a control. PC, Purkinje cell; OC, otic vesicle. Scale bars: 10 μm (E) and 20 μm (F).