Fig. 3. Wild-type function of stet is required in the male germline. (A) Apical tip of a testis expressing nuclear ubiquitin-GFP. Nuclei of early germ cells at the apical tip and spermatocytes are seen as round GFP-signals (arrowheads), while nuclei of somatic cyst cells appear triangular (arrows). (B) Wild-type control clones (outlined) marked by lack of nuclear targeted GFP. Somatic cyst cell nuclei were detected associated with these clones out of focus in the image shown. (C) Same testes as in (B) double labeled for GFP and DAPI (red). GFP-negative cells in control clones differentiate normally and have large size DAPI-stained spermatocyte nuclei (arrowheads). (D) stet mutant germline clone (outlined) marked by lack of nuclear targeted GFP. (E) Same clone as in D double labeled for GFP and DAPI (red), GFP-negative cells contain small bright DAPI-stained nuclei. (F) stet mutant germline clone. No triangular GFP-positive nuclei were detected on top, under or beside the clone; dying cells in stet mutant testes autofluoresce (arrowhead). (G) Cells in a stet mutant clone (arrowheads) express piwi-RNA, a marker usually expressed only in early germ cells at the apical tip (arrow). (H-L) Immunofluorescence staining with anti-Tj. (H) Cyst cell nuclei (arrows) at the tip of a stet/+ testes. (I) Clone of stet mutant germ cells located further basally in the testes shown in H; stet mutant germ cells appear slightly brighter than the surrounding stet/+ spermatogonia because of background staining. No Tj-positive cyst cell nuclei were detected associated with the stet mutant germ cells. (J,K) Examples of stet mutant germ cell clones associated with (J) one and (K) two Tj-positive nuclei (arrows). (L) Cyst cell nuclei (arrows) stained for the late cyst cell marker Eya were associated with neighboring stet/+ spermatocytes, but not with the clone of stet mutant germ cells in the center of the image. (M-P) DAPI images of clones corresponding to images in I-L, respectively. Note the many small brightly stained nuclei. Scale bars: in A, 0.1 mm for A-F; in G, 0.1 mm; in H, 0.1 mm; in I, 0.1 mm for I-P.