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. 2023 Jul 10;120(29):e2303779120. doi: 10.1073/pnas.2303779120

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Copyright © 2023 the Author(s). Published by PNAS.

This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

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Fig. 4. — Adult-specific loss of PDF using an inducible Cas9 system results in loss of rhythmicity and increased sleep. (A) Representative images of sLNvs stained for GFP, mRFP and PDF. 2 wk post heat shock, there is loss of PDF staining in cells expressing both UAS-Cas9 and UAS-Pdf-g. In about 60% of the hemibrains analyzed, PDF staining was lost from all sLNvs, whereas in the remaining 40% hemibrains, some mosaicism was observed as PDF staining was unchanged in 1 to 2 sLNvs. (The scale bar represents 10 µm.) (B) Quantification of PDF staining intensity from sLNvs represented with boxplots, n ≥ 39 cells from at least 12 hemibrains per genotype/condition. There is a significant effect of both variables on the staining intensity and a significant interaction when CRISPR reagents for inducible CRISPR for Pdf were combined with 3× heat shock (Two-way ANOVA; *** Interaction P < 0.001). (C) Actograms represent double-plotted average activity of flies from an experiment across multiple days. Yellow panel indicates lights ON. Panels in dotted boxes indicate heat-shocked flies. (D) Rhythmicity index for individual flies quantified (from DD2-7) represented by a boxplot, n ≥ 29 per genotype. There is a significant effect of both variables on rhythmicity and a significant interaction when CRISPR reagents for inducible CRISPR for Pdf were combined with 3× heat shock (Two-way ANOVA; *** Interaction P < 0.001). (E) Total sleep duration in a day for individual flies quantified for DD1-4 represented by a boxplot. For the group expressing both UAS-Cas9 and UAS-Pdf-g with heat-shock, only data from the arrhythmic flies are shown and used for statistics (indicated by AR). Also see SI Appendix, Fig. S5D. There is a significant effect of both variables on sleep duration and a significant interaction when CRISPR reagents for inducible CRISPR for Pdf were combined with 3× heat shock (Two-way ANOVA; *** Interaction P < 0.001). (F) p-wake over a 24-h period for individual flies quantified (from DD1-4) represented by a boxplot. For the group expressing both UAS-Cas9 and UAS-Pdf-g with heat-shock, only data from the arrhythmic flies are shown and used for statistics (indicated by AR). Also see SI Appendix, Fig. S5D. Only heat-shock had a significant effect on p-wake and there was no interaction between the variables (Two-way ANOVA; N.I. = No Interaction, p = 0.31).