FIG. 3.

Improved MTZ-induced ablation of interneurons by mutNTR. (A–F’) Comparison of ablation efficiency between complementary wtNTR (A, C, E: isl1-wtNTR-05 line) and mutNTR (B, D, F: isl1-mutNTR-06 line) expressing transgenic lines using confocal time series imaging. Pretreatment images of labeled spinal interneuron subpopulations were captured at 4 dpf (A–F) and at 6 dpf, following the indicated MTZ treatments (A’–F’). (A, A’, B, B’) Control larvae exposed to 0.1% DMSO for 24 h showed no decreases in YFP expression. (C, C’, D, D’) Larvae treated with 10 mM MTZ for 24 h (with 1 day of recovery) yielded a loss of fluorescence in labeled cell types, again demonstrating that both NTR types are competent to induce ablation under these conditions. (E, E’, F, F’) Larvae treated with 10 mM MTZ for 4 h revealed little to no effect on labeled interneurons expressing wtNTR (E, E’); however, ablation of mutNTR expressing interneurons (F, F’) occurred equivalently to 24 h MTZ-treated larvae (compare C’ and D’ with F’). (G, H) Acridine Orange labeling of apoptotic cells after 4 h MTZ exposure and 24 h of recovery, spinal cord regions indicated by dashed lines; (G) isl1-wtNTR-05, (H) isl1-mutNTR-06. (I) Quantification of Acridine Orange labeled apoptotic cells. (J) Quantification of TagYFP fluorescence by plate reader after MTZ treatment of isl1-wtNTR-05 and isl1-mutNTR-06 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: *=3.2×10⁻⁴, ^#=4.9×10⁻⁴, a,b≤0.05, c,d>0.05. (K) Quantification of TagYFP fluorescence by plate reader (as in panel J) after MTZ titration; S:B ratios were plotted as the fraction of post-treatment (6 dpf) over pretreatment (4 dpf) values, at indicated MTZ concentrations. Data were analyzed by Student's t-tests: all 4 h treatment data points between wtNTR and mutNTR were significantly different; within wild-type and mutant datasets, data points that are not significantly (n.s.) different from the 4 h/10 mM MTZ treatment data are indicated with brackets. (L–Q) Representative images of 6 dpf larvae after the plate reader assay in panel (K). (L, N, P) isl1-wtNTR-05, (M, O, Q) isl1-mutNTR-06; (L, M) No MTZ, (N, O) treated 4 h with 4 mM MTZ, (P, Q) treated 4 h with 10 mM MTZ. All error bars denote SEM. Color images available online at www.liebertpub.com/zeb