Halocarbon oil 27

All reporter plasmids were integrated into a unique landing site on the third chromosome using strain 9750 (Bloomington Drosophila Stock Center). In Figure S6, PP7-lacZ reporter plasmid was integrated into a unique landing site on the third chromosome using strain 9748 (Bloomington Drosophila Stock Center). Microinjection was performed as described (Ringrose, 2009 (link)). Zero to 1-hour embryos were collected and dechorionated with bleach. Aligned embryos were dried with silica gel for 12 min and covered with Halocarbon oil 27 (Sigma). Subsequently, microinjection was performed using Picospritzer III (Parker) and Narishige M-152 Micromanipulator (Narishige). Injection mixture contains 500 ng/μl plasmid DNA, 5 mM KCl ,0.1 mM phosphate buffer, pH 6.8. mini-White marker was used for subsequent screening.

One- to three-day-old females were aged on yeast with males for 1–2 days (control and Lar mutants) or overnight (fat2 mutants) before dissection. Ovaries were dissected as previously described (Prasad et al., 2007 (link)) in live imaging media (Schneider’s Drosophila medium containing 15% FBS and 200 μg/mL insulin) containing CellMask Deep Red Plasma Membrane Stain (ThermoFisher; 1:1000). Ovarioles were transferred to an agar pad (live imaging media with 0.4% NuSieve GTG low-melt agarose) formed on a gas permeable membrane slide (Sarstedt x-well) (Cetera et al., 2016 ). A 30 mm coverslip was placed on top of the sample, stabilized with silicon vacuum grease at each corner, and gently compressed. Halocarbon oil 27 (Sigma) was added around the coverslip to prevent evaporation. Egg chambers were imaged with a 40× 1.3 NA PlanApo objective on a laser-scanning confocal miscroscope (Zeiss LSM 880) controlled by LSM acquisition software. Time-lapse movies were performed for 10–20 minutes for control epithelia and 20–40 minutes for mutant epithelia. Single confocal slices were acquired near the basal epithelial surface every 20–30 seconds. To measure epithelial migration rates, kymographs were generated from time-lapse image stacks in Fiji (ImageJ) using the KymoResliceWide plugin (see Figure S1A for details). At least 5 egg chambers of each stage were measured.

MCP-GFP, His2Av-mRFP virgins were mated with homozygous males carrying the MS2 reporter gene. Resulting trans-heterozygote virgins were collected and mated with homozygous males carrying snail shadow enhancer. In Figure S1A and B, heterozygote MCP-GFP, His2Av-mRFP virgins were mated with homozygous males carrying snail shadow enhancer-evePr-MS2-lacZ reporter gene. The resulting embryos were dechorinated and mounted between a semipermeable membrane (In Vitro Systems & Services) and a coverslip (18 mm × 18 mm), and embedded in Halocarbon oil 27 (Sigma). Embryos were imaged using a Zeiss LSM 880 at room temperature. Plan-Apochromat 40x / 1.3N.A. oil immersion objective was used. At each time point, a stack of 21 images separated by 0.5 μm was acquired and the final time resolution is 13 sec. Images were captured in 16 bit. For each cross, three biological replicates were taken using the same setting of the microscope. The same laser power and microscope setting were used throughout this study.

Embryos were reared in collection cups at 18°C or 32°C, following the scheme shown in Table S1. Late cellularization-stage embryos were hand-selected under a dissecting microscope, transferred to fresh apple juice plates, covered in Halocarbon oil 27 (Sigma Aldrich, St. Louis, MO), and incubated at 22°C in humidifying chambers (80×15 mm glass Petri dish lined with moist paper towels). After 48 hours plates were scored for the percent of hatched larvae.