FIG. 2.
Improved MTZ-induced ablation of spinal motor neurons by mutNTR. (A–F’) Comparison of ablation efficiency between complementary wtNTR (A, C, E: mnx1:wtNTR line) and mutNTR (B, D, F: mnx1:mutNTR line) expressing transgenic lines using confocal time series imaging. Pretreatment images of labeled spinal motor neuron and uncharacterized brain neuron subpopulations were captured at 4 dpf (A–F), post-treatment images at 6 dpf, thus at ≥1 day after the indicated MTZ treatments (A’–F’). (A, A’, B, B’) Control larvae treated in 0.1% DMSO for 24 h yielded no decrease in YFP expression. (C, C’, D, D’) Larvae treated in 10 mM MTZ for 24 h (with 1 day of recovery) yielded 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 treated in 10 mM MTZ for 4 h showed little, if any, effect on spinal motor neurons expressing wtNTR (E, E’). In contrast, appreciable ablation of labeled cells was evident in mutNTR expressing fish (F, F’). (G, H) Acridine Orange labeling of apoptotic cells after 4 h of MTZ and 6 h of recovery, spinal cord regions labeled by dashed lines; (G) mnx1:wtNTR, (H) mnx1:mutNTR showed increased numbers of dying cells (bright dots) in mutNTR expressing larvae. (I) Quantification of Acridine Orange-labeled apoptotic cells in spinal cord regions after 4 h of MTZ or vehicle control, and 6 h recovery. (J) Quantification of TagYFP fluorescence by plate reader after MTZ treatment of mnx1:wtNTR and mnx1:mutNTR 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 differences (p≤0.05) 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, or mutant 4 h MTZ data at 5 and 6 days; p-values between data pairs: *=3.4×10−3, #=3.8×10−4, a,b,d ≤0.05, c>0.05. All error bars denote SEM. NTR, nitroreductase; mutNTR, mutant NTR; S:B, signal to background; wtNTR, wild-type NTR. Color images available online at www.liebertpub.com/zeb