bam or bgcn mutant germline tumors inhibit the differentiation of neighboring wild-type GSCs.

(A) Schematic cartoon for early oogenesis. The red dots and branches indicate spectrosomes and fusomes, respectively. GSC: germline stem cell. (B) Mosaic analysis strategy. The FLP recombinase triggers mitotic recombination by targeting FRT sequences. The nos>FLP method restricts FLP expression to the germline, while the hs-FLP method enables heatshock-inducible FLP expression. (C-F) Representative samples. The asterisks mark the niche cap cells, and the arrows indicate SGCs that have exited the niche and are surrounded by germline tumors mutant for bam or bgcn. Vasa, a germ cell marker, should label all germ cells. However, due to poor tumor permeability, staining often fails to detect tumorous germ cells in the central region (see Vasa panels in D-F). (G-I) Representative samples (z-stack projections). In (G), the arrowheads and arrow respectively mark two GSCs and one cystoblast, all containing dot-like spectrosomes, while the dotted lines delineate cystocytes with branched fusomes. In (H) and (I), the arrows denote SGCs that also contain dot-like spectrosomes, akin to GSCs and the adjacent GSC-like tumor cells. The online version of this article includes the following source data and figure supplement for figure 1: Source data 1. All genotypes. Figure 1—figure supplement 1 SGCs appear in egg chambers.

The inhibition of SGC differentiation depends on the lack of Bam expression.

(A) Representative sample. The arrowhead marks a BamC-positive 4-cystocyte germline cyst, while the arrows indicate BamC-negative SGCs. (B) Representative sample. The asterisk denotes niche cap cells, and the dotted circles outline bamP-GFP-negative GSCs. The solid circle marks a bamP-GFP-positive cystoblast. The arrow and arrowhead point to bamP-GFP-negative and -positive SGCs, respectively. (C) Quantification data. 14-day-old flies were used for the analyses. CBs: cystoblasts. (D) Schematic of the experimental strategy for (E-H). In “with hs-bam” flies (E and G), wild-type germ cells (both bam+/+ and bam+/-) carry the hs-bam transgene, while control “without hs-bam” flies (F) lack this element in their wild-type germ cells. (E-G) Representative samples. The arrows mark SGCs with dot-like spectrosomes, while the arrowhead indicates a 4-cystocyte germline cyst containing branched fusomes. (H) Quantification data. For each experiment, three independent replicates were performed, with over 100 SGCs and germline cysts quantified per replicate. Data represent mean ± SEM, and statistical significance was determined by t test. n.s. (P > 0.05). The online version of this article includes the following source data for figure 2: Source data 1. All genotypes. Source data 2. Raw quantification data.

SGCs maintain lower BMP signaling levels than GSCs within the niche.

(A and B) Representative samples. The asterisks mark niche cap cells, arrowheads indicate pMad-positive GSCs, and arrows point to pMad-negative SGCs. (C) Representative samples. The asterisks denote niche cap cells, arrowheads mark Dad-lacZ-positive GSCs, and arrows highlight Dad-lacZ-positive SGCs. The dotted cycles outline one Dad-lacZ-negative SGC. (D and E) Quantification data. 14-day-old flies were used for the analyses. In (E), data represent mean ± SEM, and statistical significance was determined by t test. (F) Representative sample. The asterisk marks the niche cap cell, while the arrows indicate a BrdU+ GSC within the niche. (G) Representative sample. The arrow indicates a BrdU+ SGC surrounded by germline tumors. (H) Quantification data. 14-day-old flies were used for the analyses. Statistical significance was determined by chi-squared test. The online version of this article includes the following source data for figure 3: Source data 1. All genotypes. Source data 2. Raw quantification data.

BMP signaling inhibits SGC differentiation.

(A) Schematic of the experimental strategy for (B-F). Genotypes were unambiguously distinguished using a triple-color system (red, yellow, and green). (B-D) Representative samples. The dotted cycles mark an SGC, while the solid lines outline germline cysts containing differentiating cystocytes. (E and F) Quantification data. 14-day-old flies were used for the analyses. (G) Schematic of the experimental strategy for (H-J). (H and I) Representative samples. The dotted lines mark an SGC, while the solid lines outline a germline cyst containing differentiating cystocytes. (J) Quantification data. 14-day-old flies were used for the analyses. For each experiment, three independent replicates were performed, with over 100 SGCs and germline cysts quantified per replicate. Data represent mean ± SEM, and statistical significance was determined by t test. The online version of this article includes the following source data for figure 4: Source data 1. All genotypes. Source data 2. Raw quantification data.

Germline tumors secret the BMP ligands.

(A and B) Representative samples. In (A), the asterisks denote dpp-lacZ-high niche cap cells, and solid lines outline GFP-negative bam mutant germline tumor cells. In (B), the dotted lines highlight GFP-positive wild-type cystocytes. (C) Quantification data. 14-day-old flies were used for the analyses. (D and E) Quantification data for RT-qPCR assays. 14-day-old flies were used for the analyses. For each experiment, three independent replicates were performed. Statistical significance in (C) was determined by one-way ANOVA and in (D and E) by t test. The online version of this article includes the following source data for figure 5: Source data 1. All genotypes. Source data 2. Raw quantification data.

Dpp and Gbb secreted by germline tumors are required to inhibit SGC differentiation.

(A) Schematic of the experimental strategy for (C-F). (B) Schematic of the experimental strategy for (G-I). (C-E, G, and H) Representative samples. The arrows mark SGCs containing dot-like spectrosomes, while the arrowheads denote germline cysts with differentiating cystocytes that possess branched fusomes. (F and I) Quantification data for the SGC phenotype. 14-day-old flies were used for the analyses. For each experiment, three independent replicates were performed, with over 100 SGCs and germline cysts quantified per replicate. Data represent mean ± SEM. Statistical significance in (F) was determined by one-way ANOVA and in (I) by t test. The online version of this article includes the following source data and figure supplement for figure 6: Source data 1. All genotypes. Source data 2. Raw quantification data. Figure 6—figure supplement 1 Monoallelic deletion of dpp or gbb does not affect GSC maintenance, germ cell differentiation, and female fly fertility. Figure 6—figure supplement 2 dpp bam or gbb bam double-mutant germline tumor cells divide more slowly than bam single-mutant ones. Figure 6—figure supplement 3 The SGC phenotype is unchanged irrespective of the number of surrounding germline tumors.

A working model.

bam or bgcn mutant germline tumors secrete the BMP ligands Dpp and Gbb to activate BMP signaling in out-of-niche GSCs (called SGCs in this study) to inhibit their differentiation (left panel). In contrast, dpp bam and gbb bam double-mutant germline tumors exhibit a significant loss of this differentiation-inhibiting ability (right panel).

SGCs appear in egg chambers.

(A, C) Representative samples. The arrows indicate SGCs enclosed within egg chambers. (B) Schematic of the experimental strategy for (C). See also Source data 1..

Monoallelic deletion of dpp or gbb does not affect GSC maintenance, germ cell differentiation, and female fly fertility.

(A-D) Representative samples. (E) Quantification data. 14-day-old flies were used for the analyses. GSCs are germ cells that are located within the niche and contain dot-like spectrosomes. Cystoblasts are germ cells that have exited the niche, remain in contact with GSCs, and maintain dot-like spectrosomes. Cystocytes in germline cysts are germ cells that are characterized by branched fusomes. (F) Fertility test. 3-day-old flies were used for the analyses. For each genotype, three independent replicates were performed. Data represent mean ± SEM, and statistical significance was determined by one-way ANOVA. n.s. (P > 0.05). See also Source data 1 and 2.

dpp bam or gbb bam double-mutant germline tumor cells divide more slowly than bam single-mutant ones.

(A, B, D, and E) Representative samples. The arrows indicate BrdU+ germline tumor cells mutant for bam, dpp bam, or gbb bam. (C and F) Quantification data. 14-day-old flies were used for the analyses. Statistical significance was determined by chi-squared test. See also Source data 1 and 2.

The SGC phenotype is unchanged irrespective of the number of surrounding germline tumors.

(A) Representative samples. The arrows denote SGCs. Both images are of the same magnification. (B) Quantification data for tumor size. Data represent mean ± SEM. (C) Quantification data for the SGC phenotype. In (B and C), 14-day-old flies were used for the analyses. Statistical significance in (B) was determined by t test and in (C) by chi-squared test. n.s. (P > 0.05). See also Source data 1 and 2.