Phenotype Scoring.

To score for lethality following ubiquitous CRISPRa, each sgRNA line was crossed to w; UAS:dCas9-VPR; tub-Gal4/SM5, TM6B at 27 °C, and the number of F1 were compared with siblings possessing balancers. The following genes were 100% lethal: Pvf1, Pvf2, bnl, ths, upd, upd2, upd3, scw, hh, Wnt2, Wnt4, Wnt6, WntD, Wnt10, aos, and jeb. The following lines had highly reduced survival (<20% of Mendelian expectation): pyr, egr, spz, and spz4. For wing and notum screens, sgRNA lines were crossed to w; nubbin-Gal4; UAS:dCas9-VPR/SM5, TMBb or w; UAS:dCas9-VPR/CyO; pannier-Gal4/TM6B. Wings (10–20 of each sex per cross) were mounted in Permount (Fisher) and imaged using brightfield optics on a Zeiss Axioskop 2 microscope. Nota and abdomens were photographed using a Spot RT3 Color Slider Camera Model 25.4.x (Diagnostic Instruments, Inc.) mounted on a Nikon SMZ1500 dissecting stereo microscope. A z-stack was captured and compiled into a single image using Helicon Focus software. The images represent the sex displaying the strongest phenotype. For wings, females are shown for nontargeting (n.t.), Wnt6, dpp, and pyr; males are shown for vn, upd1, upd2, upd3, ths, daw, and bnl. For nota, females are shown for n.t., hh, and Wnt6; males are shown for upd2, upd3, daw, and pyr.