Both NRY and NPIIY domains are required for proper midgut exit timing. (A–C) Representative images of fixed embryos at stage 10 stained for Vasa (green, germ cells), Hindsight (midgut, red), and actin (phalloidin, blue). All embryos shown lack maternal and zygotic contribution of endogenous tre1 gene and inherited maternally provided Tre1 wild-type (tre1⁺; A) or mutated forms of the receptor expressed from the respective tre1 transgene under nanos promoter, nanos 5′ and 3′ UTRs (B and C). (A′–C′) Outline of the midgut was generated using fluorescent maximum projection of Hindsight staining of midgut nuclei and phalloidin staining of cell cortices. The resulting mask (in yellow) outlines the extent of the midgut primordium. (A) In tre1⁺, germ cells exit at stage 10 as the midgut (red) begins delaminating (A′). (B and C) In absence of either domain, germ cells remain in the midgut even after the midgut has undergone epithelial-to-mesenchymal transition (B′ and C′). (D) Bars represent mean percentage of germ cells in each location at stage 10. (E) Bars represent average percent of germ cells in the given location at stage 13 or later. Error bars are standard deviation. Position of germ cells was determined relative to midgut and somatic gonad. At least 17 embryos were analyzed per stage and genotype. N indicates number of embryos analyzed. Bars, 20 µm. For crossing schemes and genotypes, see Table S1.