Figure 6.
NaChBac expression in LNV pacemaker neurons alters the phase of PDP1 clock protein accumulation in clock neurons on circadian day 2 in constant darkness. pdf-GAL4 virgin female flies were crossed to UAS-NaChBac1 or UAS-dORK Δ-NC1 flies. After entrainment in diurnal 12 h light/dark conditions, pdf>dORKΔ-NC1 and pdf>NaChBac1 progeny were released into constant darkness and then processed for anti-PDP1 immunofluorescence at the indicated CT on the second day in constant darkness. Bar graphs show mean ±SEM normalized integrated anti-PDP1-staining intensities, except for the DN2s, in which case the bar graph shows the percentage of hemispheres in which an anatomically distinguishable pair of stained DN2s was detectable above background (see Materials and Methods). Representative pseudocolored photomicrographs of clock neurons of the indicated cell groups and genotypes at the indicated circadian times are shown for those time points and genotypes where staining was detectable above background or, in the case of the DN2s, where >25% of hemispheres exhibited detectable DN2 staining. Whereas control pdf>dORKΔ-NC1 flies exhibit a similar temporal pattern of PDP1 accumulation in the sLNV, LND, and DN1 groups, with peak levels at CT22 (p < 0.05; ANOVA with Tukey–Kramer multiple comparison test), late in subjective night, the DN2s exhibit a peak of accumulation centered at CT14 (p < 0.05;χ2), 8 h earlier. pdf>NaChBac1 flies expressing NaChBac in the LNVs exhibit temporal patterns of PDP1 accumulation in the sLNV,LND, and DN2 cell groups that are similar to control, except that peak accumulation is significantly less than in controls for the sLNV and LND cell groups (p < 0.05; ANOVA with Tukey–Kramer multiple comparison test). In contrast, pdf>NaChBac1 flies exhibit a significantly different temporal profile of PDP1accumulation from controls in the DN1 group of clock neurons, with peak accumulation 8 h earlier at CT14 (p < 0.05; ANOVA with Tukey–Kramer multiple comparison test). In addition, the amplitude of PDP1 oscillation in the sLNVs, LNDs, and DN1s is damped in the pdf>NaChBac1 flies, with peak accumulation at CT22 significantly less than in controls (p < 0.05; ANOVA with Tukey–Kramer multiple comparison test). The value n > 12 hemispheres for each experimental group.