General outline of the fly crosses and the workflow used in the screen. Crosses are shown for the second chromosome. EMS-treated males were mated to hs-hid balancer virgin females, and F₁ single males from this cross were again mated to hs-hid balancer virgin females. Heat-shocking the progeny directly produced balanced stocks where each single mutated chromosome could be further propagated. Living embryos and larvae where thereafter screened for eGFP expressions under a fluorescence microscope. A similar crossing scheme was used for the third chromosome, using hs-hid/TMB6B, Dfd-YFP. Outlined below is the subsequent flow of the screen.