Characterization of Gγ1^N159 mutant. (A and B) Zygotic phenotype of Gγ1^N159 mutants. Stage-15 embryos of wild-type (A) and Gγ1^N159 zygotic mutant (B) were stained for Miranda (green), phosphohistone H3 (red), a mitosis marker, and DNA (TOTO-3, blue). At late embryonic stages, wild-type mitotic NBs (A) still localize Miranda in a well-defined crescent, whereas NBs tends to localize Miranda uniformly along the cell cortex or in the cytoplasm in the Gγ1^N159 zygotic mutant (B, arrow). (C and D) Cuticles of wild-type (C) and Gγ1^N159 mutant (D) embryos. Gγ1^N159 embryos showed characteristic anterior and posterior holes (arrowheads). (E–G) Wild-type (E) and Gγ1^N159 mutant (F and G) embryos stained for Miranda (green), DNA (TOTO-3; blue) and phosphohistone H3 (red). Although wild-type NBs (E) divide perpendicular to the embryonic surface, the division axis in Gγ1^N159 mutant NBs (F and G) becomes randomized, causing divisions at an oblique angle (F) or parallel (G) to the surface. Apical is up. (H–O) Wild-type (H–K) and Gγ1^N159 mutant (L–O) NBs were stained for Miranda (green) and phosphohistone H3 (red); prophase (H and L), metaphase (I and M), anaphase (J and N), and telophase (K and O). In contrast to the wild-type, the Gγ1^N159 mutant NBs cleave into nearly equal-sized daughter cells with one daughter inheriting Miranda. (P–S) Wild-type (P and Q) and Gγ1^N159 mutant (R and S) NBs stained for DNA (TOTO-3; blue) and α-tubulin (red) at metaphase (P and R), and telophase (Q and S). The Miranda crescents are oriented toward the bottom in H–S, although NBs in Gγ1^N159 mutant embryos divide in random orientations. Miranda staining is not depicted for P–S for better visualization of microtubule organization. The ratios of the distances of the centrosome from the cleavage plane (NB/GMC) are: wild-type, 2.19 ± 0.47 (n = 30); Gγ1^N159, 1.16 ± 0.13 (n = 23). Bars, 5 μm.

Evidence that N159 is a mutation of the Gγ1 gene. (A) The N159 mutation is a single C to T nucleotide change that inserts a stop codon at the position of amino acid 56 of the Gγ1 subunit of G protein. The truncated sequence includes the amino acid that is isoprenylated (underlined) and which is needed to target the Gβγ complex to the cell membrane. (B) In situ hybridization with a Gγ1 probe. Gγ1 transcripts are abundant in early embryonic stages (stage 5). Gγ1 is ubiquitously expressed throughout embryogenesis (as observed at stage 10). (C) Localization of Gβ13F in the Gγ1^N159 mutant. Stage-9 embryos were stained with anti-Cen190 antibody (red), to reveal nuclei and centrosomes, and with anti-Gβ13F antibody (green). In wild-type NBs and epithelial cells, Gβ13F is mainly distributed throughout the cell cortex (top). In Gγ1^N159 embryos, Gβ13F fails to localize to the cell cortex (bottom). Arrows indicate the centrosomes of metaphase NBs, which are stained for Cen190. Apical is toward the top. Bar, 5 μm. (D) Coimmunoprecipitation of Gγ1 and Gβ13F. Gβ13F is immunoprecipitated from an extract of wild-type embryos by anti-Gβ13F antibody (lane 1). Gβ13F is also immunoprecipitated by anti-FLAG antibody from an extract of embryos expressing FLAG-tagged Gγ1 (lane 3) or FLAG-tagged Gγ1C67S (lane 4, a Gγ1 mutant protein defective in COOH-terminal isoprenylation) or FLAG-tagged Gγ1^N159 (lane 5). Gβ13F is not immunoprecipitated from wild-type embryos (lane 2) or from embryos expressing FLAG-tagged Miranda (lane 6). Anti-Gβ13F antibody was used for the Western blot. (E) Western blots of protein extracts prepared from wild-type embryos (wt) or embryos from Gγ1^N159 germline clones (Gγ1 N159). In the mutant embryos, Gαi levels are strongly decreased (top), but Gβ13F (middle) and α-tubulin (bottom) levels are unchanged.

Effect of baz overexpression on localization of Miranda. (A–D) baz-overexpressing NBs were double-stained: red for Baz (A–D) and green for Miranda (A′), DaPKC (B′), Insc (C′), or Pins (D′). Merged images for each double staining are shown in A′′, B′′, C′′, and D′′. The overproduced Baz is not restricted to the apical cortex but is found more broadly in the lateral and basal cortex (A–C) or throughout the cortex (D). In these NBs, Miranda and Baz distribute in a complementary pattern (A and A′); DaPKC (B′) and Insc (C′) codistribute with Baz (B and C). However, Pins distribution (D′) is restricted to the apical cortex regardless of the uniformly cortical localization of Baz (D). (E) baz-overexpressing NBs produce unequal-sized daughters even when Baz is distributed all over the cortex of both the prospective NB and GMC during cytokinesis. (F) Miranda distributes uniformly in the NB cortex in wild-type embryos injected with baz dsRNA. (G and H) Mitotic pins mutant NBs stained for Insc (G, red), DaPKC (H, red), and Miranda (G and H, green); 70% of NBs localize Miranda asymmetrically along the division axis, which is often misorientated. In pins mutant NBs, Insc localizes asymmetrically in the cytoplasm (G), and DaPKC forms a weak cortical crescent (H). Apical is up. DNA was stained with TOTO-3 (blue). Bar (A–H), 5 μm.

Summary and model. (A) Division orientation of epithelial cells and mitotic domain 9 cells (Fig. 6) is summarized together with that of NBs. Note that the spindle is always pointed to the localization of Pins. However, spindles orient randomly when Pins is uniformly distributed or absent. See Results and Discussion for details. (B) The two apical signaling pathways are differentially regulated by the Gβ/Gγ signal and play overlapping but distinct roles in the asymmetric division of NBs. See Discussion for details.

Sizes of daughter cells resulting from division of Gγ1^N159 mutant and wild-type NBs. (A) Relative sizes of GMC and sibling NB at the first cell division in wild-type (blue), Gγ1^N159 mutant (red), and Gγ1^N159 mutant embryos injected with baz dsRNA (green). (B–D) Telophase NBs in wild-type (B), Gγ1^N159 embryos (C), and Gγ1^N159 embryos injected with baz dsRNA (D), which were stained for Miranda (green), γ-tubulin (red), and DNA (TOTO-3; blue). In the Gγ1^N159 mutant, NBs localize Miranda in the crescent to segregate it into the GMC, which is slightly smaller than the sibling NB (C). Inhibition of baz activity (by RNAi) in Gγ1^N159 mutant embryos results in complete loss of cell-size asymmetry and uniform Miranda distribution (D). The cell size ratios (GMC/NB) for dividing NBs in B–D are 50%, 81%, and 97%, respectively, and are marked in A. Bar (B–D), 5 μm.

Localization of apical components in metaphase NBs in Gγ1^N159 and Gβ13F mutants. Localization of the apical components in mitotic NBs in the wild-type (A, D, G, J, M, and P), Gγ1^N159 mutant (B, E, H, K, N, and Q), and Gβ13F mutant (C, F, I, L, O, and R). Stage-9 embryos were stained for Pins (A–C), Gαi (D–F), Insc (G–I), Baz (J–L), DmPar-6 (M–O), DaPKC (P–R, red), and Miranda (green), as well as for DNA (blue). The Miranda crescents are oriented toward the bottom but are shown only in P–R (green). In Gγ1^N159 and Gβ13F mutant NBs, Pins distributes all around the cell cortex, as well as in the cytoplasm (B and C), Gαi is not detectable (E and F), Insc distribution is less polarized in the cytoplasm and the cell cortex (H and I) than in the wild type; and Baz (K and L), DmPar-6 (N and O), and DaPKC (Q and R) form cortical crescents, although the intensity of antibody staining is less than in the wild type. Bar (A–R), 5 μm.

Effect of pins and G protein mutations on spindle orientation. (A–D) Wild-type (A), pins (B and C), and Gαi (D) mutant epithelial cells were stained for the membrane marker BP106 (red) and for phosphohistone H3 (green). BP106 is found in the lateral and basal membrane domains in epithelial cells but stains the entire cell contour in NBs. The apical surface of epithelial cells does not stain for BP106. The epithelial cells of pins and Gαi mutants often divide perpendicular (B) or at an oblique angle (C and D) to the embryo's surface, but wild-type cells divide parallel to the surface (A). Apical is up. (E–H) Wild-type (E and F) and Gγ1^N159 mutant (G and H) epithelial cells were stained for BP106 (E and G, red), Pins (F and H, green), and DNA (blue). In both wild-type and Gγ1^N159 mutant epithelial cells Pins is localized in the lateral cortex and the cells divide parallel to the surface (F and H). Apical is up. (I–L) Mitotic domain 9 cells in wild-type (I), pins (J), Gαi (K), and Gγ1^N159 (L) mutants were stained for Miranda (green) and phosphohistone H3 (red) in I–K or for Miranda (green), Pins (red), and DNA (blue) in L. Wild-type cells (I) in mitotic domain 9 divide perpendicular to the embryonic surface. Cells in the pins (J) and Gαi (K) mutants divide not only perpendicular but also parallel or at an oblique angle to the surface. Mitotic domain 9 cells in the Gγ1^N159 mutant (L) localize Pins at the lateral cortex and divide parallel to the surface. Arrows show the orientations of divisions. Apical is up. (M–P) Surface views of mitotic domain 9 in wild-type (M), pins (N), Gαi (O), and Gγ1^N159 (P) mutant embryos stained with anti-α-tubulin. Mitotic spindles are oriented perpendicular to the surface in the wild-type (M), parallel or perpendicular to the surface in the pins (N) and Gαi (O) mutants, and parallel in the Gγ1^N159 mutant (P). Arrowheads indicate divisions perpendicular to the embryonic surface; arrows indicate divisions parallel to the surface. (Q) Quantitative measurements of the orientations of epithelial cell divisions in the wild-type, pins, Gαi, and Gγ1^N159 mutants. n = number of telophase epithelial cells scored. Bars: (A–L) 5 μm; (M–P) 10 μm.