NRY domain, but not NPIIY domain, is required for germ cell bilateral sorting. (A) Experimental design. Embryos carrying the mCherry::Vasa fusion protein, which marks all germ cells (red), as well as the nanosGal4::VP16 driving UAS-αTub::photoactivatable GFP (PAGFP), were oriented dorsal side down on a coverslip and activated with a 405-nm laser laterally just after germ cell formation at the posterior pole (stage 5) and allowed to develop until germ cells had laterally sorted and entered the mesoderm (stage 12 or later). (B) Quantification of photoactivated germ cells of the indicated genotype that followed midline cues and migrated to the same side gonad with respect to their formation (“same side”) or moved to opposite sides (“opposite side”). N represents the number of activated germ cells analyzed. Data are summarized in Table S3; for crossing schemes and genotypes, see Table S1. Wild-type embryos (C) and embryos lacking wun and wun 2 zygotically (wun Z⁻; E) were used as controls for germ cells responding and unresponsive to midline cues, respectively. (C, E, G, I, K, and M) Representative images of germ cells with PAGFP (green) in lateral germ cells at stage 5 in embryos of indicated genotypes. (D, F, H, J, L, and N) Visualization of the same photoactivated cells in embryo of respective genotypes. Activated cells are denoted with yellow arrows in D′, F′, H′, J′, L′, and N′. Bars, 20 µm.