NRSE-delimited transgene expression is REST dependent. A-L show representative time series confocal images at 3, 6 and 9 dpf of NRCK; 14xNTR-Ch; 5xMY-HMY triple transgenic larvae ± rest morpholino (MO) injection. (A-D) Uninjected control larvae showed the delayed repression phenotype of NRCK line gmc607, where early skeletal muscle expression (A, B, arrows) fades over time (C, D). Note that loss of the YFP reporter is delayed relative to the mCherry reporter. (E-H) Control MO-injected larvae display the same phenotype as uninjected controls with muscle expression (E, F, arrows) fading over time (G, H). (I-L) Conversely, rest MO-injected larvae have an expansion of skeletal muscle expression at day 3 (I, K, arrows) that fails to be repressed with time (K, L, arrows). (M-O) Quantification of phenotypes between uninjected, control MO, and rest MO-injected larvae; the number of larvae with detectable mCherry expression in muscle cells at 6 dpf was increased for rest morphants (green bar) relative to controls (M, asterisk P < 0.01). The number of mCherry (N) and YFP expressing (O) muscle cells also increased for rest morphants (green bars) relative to uninjected (blue bars) and MO controls (red bars) at all time points (N, asterisks P ≤ 0.01, number symbols P < 0.05; O, asterisks P < 0.001, number symbols P < 0.05). Morpholinos were injected at 0.25, 0.5, 1 and 2 μM with similar results (data from representative 0.5-μM injections is shown, experiment was repeated six times). Statistical comparisons were performed using an independent sample t-test to compare across treatment conditions per time point (asterisks), and a repeated measures t-test to compare individual samples to their initial values at 3 dpf (number signs).