FIG. 4.
Improved MTZ-induced ablation of cranial motor neurons by mutNTR. (A–F’) Comparison of ablation efficiency between complementary wtNTR (A, C, E: isl1-wtNTR-03 line) and mutNTR (B, D, F: isl1-mutNTR-04 line) expressing transgenic lines by confocal time series imaging. Pretreatment images of cranial motor neuron subpopulations in individual larvae were captured at 4 dpf (A–F) and at 6 dpf, after MTZ treatment (A’–F’). (A, A’, B, B’) Control larvae treated with 0.1% DMSO for 24 h yielded no decrease in YFP expression. (C, C’, D, D’) Larvae treated with 10 mM MTZ for 24 h (with 1 day of recovery) showed a loss of fluorescence in labeled cell types, demonstrating that both NTR types are competent to induce ablation under these conditions. (E, E’, F, F’) Larvae exposed to 10 mM MTZ for 4 h revealed little to no effect on labeled motor neurons expressing wtNTR (E, E’); in contrast, this treatment induces ablation of a subset of neurons expressing mutNTR (F, F’; orange boxes). (G) Quantification of TagYFP fluorescence by plate reader after MTZ treatment of isl1-wtNTR-03 and isl1-mutNTR-04 lines; S:B ratios were calculated from individual larvae at 4 dpf (pretreatment), 5 dpf and 6 dpf, then plotted as the fraction of individual pretreatment values. Data were analyzed by Student's t-tests: All MTZ-treated samples yielded significant (p≤0.05) differences when compared with corresponding untreated (No MTZ) controls, and there was no significant difference between wild-type and mutant 24 h MTZ treatment data at 6 days; p-values between data pairs: *=3.9×10−4, #=8.6×10−5, a,b,c,d<0.05. All error bars denote SEM. Color images available online at www.liebertpub.com/zeb