Figure 3.
Figure 3. NPIIY domain, but not NRY domain, is required for Rho1 localization and germ cell polarization. (A–D) Representative images of embryos at stage 9 stained for Vasa (green, germ cells), Rho1 (red), and actin (phalloidin, blue). Bar, 20 µm. All embryos shown lack maternal and zygotic contribution of endogenous tre1 gene. Embryos in A, C, and D inherited maternally provided Tre1 wild-type (tre1+; A) or mutated forms of the receptor NRY− (C) and NPIIY− (D) expressed from the respective tre1 transgene under nanos promoter, nanos 5′ and 3′ UTRs. (A′–D′) Rho1 intensity measurement portrayed in fire pseudocolor from black/blue (low) to yellow/white (highest). Rho1 concentrates in tail region and germ cells polarize in embryos with Tre1+ and Tre1 NRY− receptors. Polarity is lost, and Rho1 distributes along germ cell cortex in embryos lacking Tre1 (B) or Tre1 NPIIY− (D) mutant receptor. Bars, 20 µm. For crossing schemes and genotypes, see Table S1. (E–H) Quantification of cortical concentration and polarization. (E) To measure cortical concentration of Rho1, intensities of at least five ROIs; 72 pixels each) of germ cell cortices (pink) were measured using ImageJ and compared with anterior midgut cortices (blue) as internal controls. Intensity of averaged germ cell ROI measurements was normalized to averaged midgut ROI measurements (Kunwar et al., 2008). (F) Localization quantification is graphed using a minimum of seven embryos for each maternal genotype described in A–D. Relation of germ cell cortex/midgut cortex measurement of wild type was arbitrarily set to 1. ***, P < 0.0001. Error bars represent standard deviation. (G) Polarization quantification. The fluorescent intensity of a 10-µm, one germ cell diameter rectangle encompassing the entire width of the germ cell cluster (35–55 µm) was measured. Fluorescent intensities were normalized to the mean intensity from each embryo and plotted. Bar, 10 µm. (H) At least five embryos for each maternal genotype described in A–D were measured and the intensities aligned to the center.

NPIIY domain, but not NRY domain, is required for Rho1 localization and germ cell polarization. (A–D) Representative images of embryos at stage 9 stained for Vasa (green, germ cells), Rho1 (red), and actin (phalloidin, blue). Bar, 20 µm. All embryos shown lack maternal and zygotic contribution of endogenous tre1 gene. Embryos in A, C, and D inherited maternally provided Tre1 wild-type (tre1+; A) or mutated forms of the receptor NRY− (C) and NPIIY− (D) expressed from the respective tre1 transgene under nanos promoter, nanos 5′ and 3′ UTRs. (A′–D′) Rho1 intensity measurement portrayed in fire pseudocolor from black/blue (low) to yellow/white (highest). Rho1 concentrates in tail region and germ cells polarize in embryos with Tre1+ and Tre1 NRY− receptors. Polarity is lost, and Rho1 distributes along germ cell cortex in embryos lacking Tre1 (B) or Tre1 NPIIY− (D) mutant receptor. Bars, 20 µm. For crossing schemes and genotypes, see Table S1. (E–H) Quantification of cortical concentration and polarization. (E) To measure cortical concentration of Rho1, intensities of at least five ROIs; 72 pixels each) of germ cell cortices (pink) were measured using ImageJ and compared with anterior midgut cortices (blue) as internal controls. Intensity of averaged germ cell ROI measurements was normalized to averaged midgut ROI measurements (Kunwar et al., 2008). (F) Localization quantification is graphed using a minimum of seven embryos for each maternal genotype described in A–D. Relation of germ cell cortex/midgut cortex measurement of wild type was arbitrarily set to 1. ***, P < 0.0001. Error bars represent standard deviation. (G) Polarization quantification. The fluorescent intensity of a 10-µm, one germ cell diameter rectangle encompassing the entire width of the germ cell cluster (35–55 µm) was measured. Fluorescent intensities were normalized to the mean intensity from each embryo and plotted. Bar, 10 µm. (H) At least five embryos for each maternal genotype described in A–D were measured and the intensities aligned to the center.

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