Figure 3. Cell-specific disruption of per or tim in Mai179⁺ neurons causes complete loss of behavioral rhythmicity and efficient loss of the targeted protein.

(A) Diagram of the circadian neuron subset marked by Mai179-Gal4. (B) Disruption of per or tim in Mai179⁺ neurons caused complete loss of behavioral rhythmicity. (C) Scatter plot showing the period of rhythmic flies with Mai179-Gal4-driven disruption of acp, per, or tim. (D) Average actograms showing the activity of flies in constant darkness with Mai179-Gal4-driven disruption of acp, per, or tim. Six days of activity are displayed, double-plotted. Dark gray rectangles = subjective day, black rectangles = subjective night. (E and F) Background-subtracted nuclear fluorescence intensity of Per (E) or Tim (F) at ZT0 in GFP⁺ neurons, grouped by brain with mean ± SEM shown. Individual brains are shown in the same order in both E and F. (G and H) Mean nuclear fluorescence intensity of Per (G) or Tim (H) at ZT0 in GFP⁺ neurons, averaged per brain (acp n = 18; per n = 16; tim n = 15). **: p<0.01; ****: p<0.0001; n.s.: not significant, p>0.05. Significance determined by Kruskal-Wallis nonparametric ANOVA (to account for non-normality of samples) followed by Dunn’s multiple comparisons test; reported p-values are multiplicity adjusted to account for multiple comparisons.

Figure 3—figure supplement 1. Cell-specific disruption of per or tim in Mai179⁺ cells causes loss of rhythm strength.

χ² peak height values (rhythm strength) for Mai179-Gal4 targeting of acp (gray), per (orange), or tim (blue). Significance determined by Kruskal-Wallis nonparametric ANOVA (to account for non-normality of samples) followed by Dunn’s multiple comparisons test; reported p-values are multiplicity adjusted to account for multiple comparisons. ****: p<0.0001; n.s.: not significant, p>0.05.

Figure 3—figure supplement 2. Period and Timeless immunohistochemistry in Mai179-Gal4-driven CRISPR flies.

Maximum intensity projections of Mai179-Gal4-driven GFP⁺ dorsal lateral (LNd, top) and ventral lateral (LNv, bottom) neurons with immunohistochemistry for GFP (yellow), Period (green) and Timeless (magenta); scale bar = 10 μm. Arrowheads indicate GFP⁺ (CRISPR-targeted) cells.

Figure 3—figure supplement 3. Cell-specific disruption of per or tim in Mai179⁺ neurons is most efficient in dorsal lateral (LNd) and small ventral lateral (s-LNv) neurons.

Background-subtracted nuclear fluorescence intensity of Per (A, C, E) or Tim (B, D, F) at ZT0 in GFP⁺ neurons, grouped by brain and separated by cell type (A, B: dorsal lateral neurons; C, D: small ventral lateral neurons; E, F: large ventral lateral neurons) with mean ± SEM shown. Individual brains are shown in the same order in all graphs (acp n = 18; per n = 16; tim n = 15 brains).

Figure 3—figure supplement 4. Average actograms for Mai179-Gal4 targeted flies under 12 hr:12 hr light:dark conditions.

Average actograms for the first day in LD for Mai179-Gal4 targeting of acp (gray), per (orange), or tim (blue).