Fig. 3. Bam promotes differentiation of TkvAct ovarioles in part by downregulating Dpp signaling prior to overt GSC differentiation. (A-C) Fluorescence (A) and confocal (A inset, B,C) projections of ovarioles from females of genotype P{hs-bam.O}/+; P{UAS.p-TkvAct)/P{Gal4::VP16-nos.UTR}. Anti-Orb (red). (A) DAPI staining (green). Ovariole of female subject to daily heat shock from day 10 of development. (Inset) Abnormal egg chamber from an ovariole of a similarly treated female. (B,C) Yopro-1 staining (green) of (B) ovarioles and (C) germarium of 10-day-old female subject to daily heat-shock from day 9-10 of development until 7 days post-eclosion. Note complete rescue of the tumorous phenotype (B), with differentiation of wild-type appearing egg chambers with 15 polyploid nurse cells and localization of Orb to the presumptive oocyte, and wild-type appearance of the germarium (C). (D-K) Confocal projections (D-G) or confocal sections (H-K) of germaria or ovarioles from females of genotype P{hs-bam.O}/+; P{nos-myc.V}/+; P{UAS.p-TkvAct} P{lacZ}Dad^P1883/P{GAL4::VP16-nos.UTR}. (D,E) Anti-spectrin (red), anti-Myc (green). In a non-heat shocked control germarium (D), all germ cells express Nos (circle, one example). In a germarium from a 2-day-old adult female heat shocked daily from day 9-10 of development (E), all spectrosome-containing cells express Nos (circle, one example). Nos is downregulated in all germline cysts that have between two and eight cells (asterisks), but is re-expressed at high levels in 16 cell cysts (arrowhead). (F,G) Anti-Myc (red), anti-lacZ (green). Ovarioles from 2-day-old female without heat shock (F), or with daily heat shocks starting at day 10 of development (G). In heat-shocked ovarioles, the level of Nos-Myc is unchanged in germ cells, while the level of Dad-lacZ expression is significantly reduced in germ cells, but not in follicle cells. (H) Anti-lacZ (green). A confocal section from part of the projection in G, showing lacZ expression is reduced but not eliminated in the germline. (I-K) Germaria from control (J) and heat shocked (I,K) females (anti-lacZ, green; anti-Hts, red). Compared with control females (J), heat-shock causes reduction, but not elimination of lacZ expression in spectrosome containing cells (K, asterisk), and complete elimination of lacZ expression in fusome-containing cysts (I, asterisk). (L,M) Confocal projections of ovarioles from females of genotype P{UAS-lacZ.p}/P{hs-bam.O}; bam^D86/P{GAL4::VP16-nos.UTR} bam^D86 (anti-Hts, red; anti-lacZ, green). Germarium of non-heat shocked control (L) and germarium of 2-day-old female heat shocked daily from day 9-10 of development (M), showing rescue of tumorous phenotype as evidenced by differentiating egg chambers. Levels of lacZ are not reduced after heat shock. (A-E,I-M) Arrow indicates terminal filament.