Fig. 1. Characterization of twin mutants and function in the germline. (A) Schematic representation of twin locus and mutants. Black boxes indicates exons. Intron 5 is 19.6 kb long according to genomic and EST sequences in FlyBase. The arrow indicates the transcription start site. P elements (not drawn to scale) inserted in the twin locus in the three alleles are shown. In twin¹²²⁰⁹ and twin⁸¹¹⁵, insertions are P-UAS-GFP and P-UAS, respectively. Insertion sites were verified by DNA sequencing. Coordinates of the insertion sites, according to the AE003746 sequence in NCBI, are 189597 and 173089 for twin^KG877, 189529 for twin¹²²⁰⁹ and 174697 for twin⁸¹¹⁵. (B-D) Immunostaining of wild-type and twin mutant ovaries with anti-CCR4 antibody. (B) Wild-type, (C) twin¹²²⁰⁹ and (D) twin⁸¹¹⁵ stage 10 egg chambers, stained with anti-CCR4 (left) and DAPI to visualize DNA (right). (E) twin mutant females show maternal effect embryonic lethality. Females of the indicated genotypes were crossed with wild-type males and hatched and unhatched embryos were scored. A proportion of these embryos have a thin chorion. When possible, both twin homozygous females and twin alleles in combination with Df(3R)Exel6198 were analysed. twin^KG877 homozygous are lethal at larval stage, due to an independent mutation on the chromosome (Temme et al., 2004). Df(3R)Exel6198 is a deficiency overlapping the twin locus, which is independent and shorter than Df(3R)crb-F89-4. The phenotypes of twin¹²²⁰⁹/Df(3R)Exel6198 and twin⁸¹¹⁵/Df(3R)Exel6198 are stronger than that of homozygous twin¹²²⁰⁹ and twin⁸¹¹⁵ homozygous females, respectively, indicating that none of these alleles is null. (F-K) Ovarian phenotypes of twin mutant females were analysed by DAPI staining. (F) Stage 3 to 10 egg chambers were scored according to their numbers of cells. Df is Df(3R)crb-F89-4. Similar results were obtained when Df(3R)Exel6198 was used (wt, wild type; other: phenotypes including no oocytes, two oocytes or one mislocalized oocyte). (G) Wild-type egg chambers. (H,I) twin¹²²⁰⁹/Df(3R)crb-F89-4. An example of 32 germline cell phenotype and of an apoptotic egg chamber are shown in H and I, respectively. (J,K) Staining with DAPI and anti-HtsRC antibody that recognizes ring canals. The oocyte is linked to four nurse cells by four ring canals in wild-type egg chambers (J, arrow). An example of twin¹²²⁰⁹/Df(3R)crb-F89-4 egg chamber, where the oocyte is linked by five ring canals (K, arrow). (L,M) Mitosis defects in embryos from twin mutant females visualized by DAPI staining. Nuclear cleavages are synchronous in wild-type syncytial blastoderm embryos (L), whereas nuclei in all the phases of the cell cycle are found in a same embryo from twin^KG877/Df(3R)crb-F89-4 female (M). Bottom panels show high magnifications of images in L,M. Anterior is oriented toward the left in all panels.