Figure 5.
Acute blockade of the BMP pathway in adults dramatically reduced FMRFa peptide. Genotypes were as follows: w1118 control flies (A, E) (apGAL4/+; tub-GAL80TS,UAS-nEGFP/+); tkvDN;witDN flies (B, F) (apGAL4/UAS-tkvDN; tub-GAL80TS, UAS-nEGFP/UAS-witDN); MadDN flies (C, G) (apGAL4/UAS-MadDN; tub-GAL80TS, UAS-nEGFP/UAS-MadDN). Flies were raised at 18°C until A1. They were then kept at 18°C for up to 30 d (A–D), or switched to 29°C for up to 15 d (E–H). D, H, Experimental results comparing relative pixel intensity of FMRFa immunoreactivity at each time point, for each genotype. Each datum point was normalized to the percentage of the mean of the w1118 control at each time point. Data for each genotype at each time point are presented as mean ± SEM. For details, see supplemental Table 2 (available at www.jneurosci.org as supplemental material). A–C, In noninduced flies maintained at 18°C for 30 d, GAL4 activity was repressed, as shown by lack of nEGFP (green) expression in Tv cluster neurons (anti-Eyes absent; blue). High-level FMRFa immunoreactivity (red; anti-FMRFa) was observed in control w1118 flies (A), tkvDN;witDN flies (B), and MadDN flies (C). D, Quantification of FMRFa immunoreactivity intensity for flies kept at 18°C, at A1 (time 0), and at 10, 20, and 30 d later. No significant difference was observed between genotypes at any time point. E–G, In induced flies maintained at 29°C for 15 d, GAL4 activity was induced, as shown by nEGFP expression (green) in Tv cluster neurons (anti-Eyes absent; blue). FMRFa immunoreactivity (red) in Tv neurons (arrows) was observed in w1118 control flies (E), but was severely reduced in tkvDN;witDN (F) and MadDN flies (G). H, Quantification of FMRFa immunoreactivity intensity for flies induced at 29°C, at A1 (time 0), and at 5, 10, and 15 d later. Throughout the induction period, there was a progressive, dramatic reduction of FMRFa peptide levels after BMP pathway blockade until it was almost entirely absent. *p < 0.0001 compared with control at each time point.