Supplementary Figure 7: Heme oxygenase (dHo) is essential for normal daily activity patterns in Drosophila: additional data.
From: Reciprocal regulation of carbon monoxide metabolism and the circadian clock
(a) Mean locomotor activity of Drosophila males analysed in 12 hr light: 12 hr dark conditions for 5-8 days. A different dHo-RNAi construct inserted either on chromosome 3 (UAS-dHo-RNAi:R1) or chromosome 2 (UAS-dHo-RNAi:R3) was either driven in all clock cells: timeless-gal4 (tim >); PDF-positive clock neurons: Pdf-gal4 (Pdf >); all neurons: elav-gal4 (elav >), or all glia cells: repo-gal4 (repo>). Note the phase advance caused by dHo knockdown in clock cells and neurons, whereas glia-specific knockdown or ‘driver-only’ controls results in normal behaviour. At least 16 flies were tested for each genotype. Bars show mean activity within in 30 min; dark bars: ‘lights-off’, white bars: ‘lights on’, dots indicate sem. (b) Real-time luciferase recordings of flies expressing different period-luciferase fusion genes. Male flies were recorded in LD and DD as indicated by the bars below the plots (white and black bars indicate ‘lights-on’ and ‘lights-off’ respectively). Left: Flies expressing luciferase under control of the period promoter in all clock cells (including peripheral clocks) encoded by the plo transgene29: grey circles—tim-gal4:27/+;plo:86-6/UAS-dHo-RNAi:21-1 (n=16); black circles—tim-gal4:27/UAS-GFP;plo:86:6/+ (n=12). Middle: Flies expressing a PERIOD-LUCIFERASE fusion protein in all clock cells (including peripheral clocks) encoded by the XLG-luc transgene30: grey circles—tim-gal4:27/+;XLG-luc/UAS-dHo-RNAi:21-1 (n=8); black circles—tim-gal4:27/UAS-GFP;XLG-luc/+ (n=8); open circles—tim-gal4:27/UAS-attP-51C;XLG-luc/+ (n=4). Note that in the left and middle panels no significant differences are observable between dHo-RNAi and control flies. Similar results were obtained in two independent experiments including the dHoRNAi:21-8 line. Right: Flies expressing a PERIOD-LUCIFERASE fusion protein in dorsal clock neurons encoded by the promoter-less 8.0-luc transgene30. Exact genotypes: grey circles—8.0-luc/+;tim-gal4:67/UAS-dHo-RNAi:21-8 (n=8); black circles—8.0-luc/UAS-GFP;tim-gal4:67/+ (n=10). Note the higher peak levels in LD and DD, and the increased amplitude of PER-LUC oscillations in dHo-RNAi flies.
