Sakura expression pattern and mutant allele

(A) Drosophila Sakura protein (Sakura/CG14545) and its null mutant allele generated in this study. (B) sakura mRNA expression pattern. Data obtained from http://flybase.org/reports/FBgn0040602.htm. (C) Western blot of dissected fly tissues. (D) Western blot of ovary lysates. (E) Confocal images of the germarium from the sakura-EGFP transgenic fly. Sakura-EGFP (green), Hts (red), and DAPI (blue). Scale bar: 10 µm. (F) Confocal images of the egg chambers from the sakura-EGFP transgenic fly. Sakura-EGFP (green), Vasa (red), and DAPI (blue). Sakura-EGFP is expressed in nurse cells and enriched in the developing oocyte (yellow arrow). Scale bar: 20 µm.

sakura mutant flies are female-sterile and have rudimentary ovaries.

(A-B) Female fertility assays. (A) The number of eggs laid by test females crossed with OregonR wild-type males and (B) hatching rates of the eggs. Mean ± SD (n = 3). P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *. (C) Stereomicroscope images of dissected whole ovaries. Scale bar: 1 mm.

sakuranull ovaries are germless and tumorous

(A) Confocal images of the ovaries from control (sakuranull/+) and sakuranull/null harboring vasa-EGFP. Vasa-EGFP (green) and DAPI (blue). Yellow stars label the anterior tip of the ovaries. Scale bar: 100 µm. (B) Confocal images of sakuranull/null ovaries stained with anti-cleaved Caspase-3 antibody. Cleaved caspase-3 (green) and DAPI (blue). Control ovary is sakuranull/+. Yellow stars label the anterior tip of the ovaries. Scale bars: 20 µm for control and 100 µm for sakuranull/null. (C) Confocal images of control (sakuranull/+) and sakuranull/null ovaries stained with anti-Hts antibody to label spectrosomes and fusomes. Hts (red) and DAPI (blue). Yellow star labels the anterior tip in the control ovary. Red and green stars label the anterior tip of sakuranull/null tumorous and germless ovarioles, respectively. Scale bars: 20 µm for control and 50 µm for sakuranull/null. (D) Ratio (%) of normal, germless, and tumorous ovaries of indicated genotypes (n=33, 25, 74, 29 respectively). (E) Quantification of GSC-like cell number in germaria of indicated genotypes (n = 33, 30, 35, and 31 respectively).

Loss of sakura results in lower piRNA levels and loss of piRNA-mediated transposon silencing

(A) Scatter plots of normalized number of transposon piRNA reads from small RNA-seq of indicated genotypes compared with sakura+/+. Means of three biological replicates are plotted. Downregulated (fold-change < 0.5) and upregulated (fold-change > 2) transposon piRNAs are shown in cyan and magenta, respectively. (B) Volcano plots of transposon RNAs from RNA-seq of indicated genotypes compared with sakura+/+. Three biological replicates per genotype were analyzed. Downregulated (adjusted p-value < 0.001 and log2(fold-change) < -2) and upregulated (adjusted p-value < 0.001 and log2(fold-change) > 2) transposons are shown in cyan and magenta, respectively. (C) Normalized number of reads for Burdock piRNAs from small RNA-seq. Mean ± SD (n = 3.) P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *. (D) The Burdock sensor harbors a nanos promoter, a nuclear localization signal (NLS) appended to GFP and β-gal coding sequences, and a target sequence for Burdock piRNAs in the 3’UTR. Confocal images of ovaries from control (yRNAi) and sakuraRNAi flies harboring the Burdock sensor, where RNAi knockdown was specifically driven in the female germline with UAS-Dcr2 and NGT-Gal4. GFP (green), β-gal (red), and DAPI (blue). Scale bar: 50 µm.

sakura is important for oogenesis in later-stage egg chambers

(A) Stereomicroscope images of dissected whole ovaries. Scale bar: 500 µm. (B) Western blot of dissected ovary lysates. sakuraRNAi #1 and sakuraRNAi #2 are two independent RNAi lines. (C) Number of eggs laid by sakura RNAi knockdown driven by UAS-Dcr2 and TOsk-Gal4. Mean ± SD (n = 5). P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *. (D) Number of stage 14 oocytes produced by sakura RNAi knockdown flies driven by UAS-Dcr2 and TOsk-Gal4. P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *. (E) Confocal images of the ovaries from sakura RNAi knockdown flies driven by UAS-Dcr2 and TOsk-Gal4, stained with anti-Orb and anti-Vasa antibodies. Orb (green), Vasa (red), and DAPI (blue). Yellow arrows label the localization and enrichment of Orb in the developing oocytes. Scale bar: 100 µm. In A-E, y-RNAi was used as a control.

Germline clonal analysis of sakuranull

(A) Percentage of germaria with marked GSC clones indicated by the absence of GFP at 4, 7, and 14 days after clone induction at the adult stage. Arrows indicate the percent decrease of marked GSC clones compared to day 4. The genotype, actual percentage, and total number of germaria examined are shown in the adjacent table. (B) Confocal images of germ cell clones. sakuranull and control clones were marked by the absence of GFP. GFP (green), Hts (red), and DAPI (blue). Scale bar: 20 µm. (C) Number of marked (GFP-negative) GSC-like cells in germaria with marked GSCs of the indicated genotypes at 4, 7, and 14 days after clone induction. GSC-like cells containing round spectrosome were identified through immunostaining with anti-Hts antibody. P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *.

Loss of sakura inhibits Dpp/BMP signaling

(A) Confocal images of ovaries from control (yRNAi) and sakuraRNAi flies harboring the bam-GFP reporter, where RNAi knockdown was specifically driven in the female germline with UAS-Dcr2 and NGT-Gal4. Bam-GFP (green), Hts (red), and DAPI (blue). Scale bar: 20 µm. (B, C) Confocal images of germaria with germline clones of sakuranull stained with (B) anti-Bam antibody and (C) anti-pMad antibody. GFP (green), Bam or pMad (red), and DAPI (blue). Scale bar: 10 µm. (D) Mean pMad intensity in the germline clones of the indicated genotypes. Mean ± SD (n = 7). P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by * (E) Confocal images of germaria with germline clones of sakuranull stained with anti-CycA antibody. GFP (green), CycA (red), and DAPI (blue). Scale bar: 10 µm. (F) Mean CycA intensity in the germline clones of the indicated genotypes. Mean ± SD (n = 5 and 10 for FRT82B and FRT82B, sakuranull respectively). P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *. In B, C, and E, clones were marked with the absence of GFP.

The ratio of ovary germless phenotype from double RNAi knockdown of sakura with bam, cycA, or otu

Mean ratio (%) of ovaries with the germless phenotype observed in double RNAi knockdown of sakura with bam, cycA, or otu. UAS-Dcr2 and NGT-Gal4 was used to drive RNAi knockdown in the germline. n is the total number of ovaries examined for the indicated genotypes. Three independent crosses (biological replicates) were set up to generate the flies with genotypes of interest, and the ratio of ovaries with the germless phenotype in each replicate was calculated. Mean ± SD (three biological replicates). P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *.

Number of unique peptide counts detected by mass-spec.

Proteins with peptide signals present in all three biological replicates of the Sakura-EGFP samples without any signals in any of the three biological replicates of the negative control are shown.

Sakura interacts with Otu.

(A) Co-immunoprecipitation using anti-GFP magnetic beads followed by Western blotting. Ovary lysates expressing Sakura-EGFP and those from w1118negative control were tested. (B) Co-immunoprecipitation using beads bound with rabbit anti-Sakura followed by Western blotting. Ovary lysates from w1118 flies were used. Rabbit IgG was used as control IP. (C) Co-immunoprecipitation using beads bound with anti-FLAG antibody followed by Western blotting. S2 cell lysates expressing 3xFLAG-Otu and Sakura-mCherry-3xHA or mCherry-3xHA (negative control) were used.

N-terminal regions of Sakura and Otu are important for interaction.

(A) Predicted structure of the Sakura and Otu protein complex made by AlphaFold. Full-length Sakura and N-terminal Otu fragment (N1. 1-405aa) were used for prediction. (B) Full-length Otu and Otu fragments tested in co-immunoprecipitation assays. (C) Full-length Sakura and Sakura fragments tested in co-immunoprecipitation assays (N: N-terminal, M: middle, C: C-terminal). (D, E, F) Co-immunoprecipitation using anti-HA magnetic beads followed by Western blotting. S2 cell lysates expressing HA-tagged mCherry were used as negative controls.

Loss of otu phenocopies loss of sakura

(A) Confocal images of ovaries from otu-EGFP and otu(ΔTudor)-EGFP transgenic flies. Otu-EGFP and Otu(ΔTudor)-EGFP (green), DAPI (blue). Yellow arrows show the enrichment of Otu-EGFP and Otu(ΔTudor)-EGFP in developing oocytes. Scale bar: 20 µm. (B) Stereomicroscope images of dissected whole ovaries from wRNAi (control) and otuRNAi flies where RNAi knockdown was driven in the female germline with nos-Gal4-VP16 or TOsk-Gal4. Scale bar: 500 µm. (C) Number of eggs laid by TOsk-Gal4 > wRNAi and TOsk-Gal4 > otuRNAi flies. Mean ± SD (n = 5). P-value < 0.05 (Student’s t-test, unpaired, two-tailed) are indicated by *. (D) Violin plots of the number of stage 14 oocytes produced in TOsk-Gal4 > wRNAi and TOsk-Gal4 > otuRNAi flies. n = 30. P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *. (E) Confocal images of the ovaries from wRNAi (control) and otuRNAiflies harboring the Burdock sensor where RNAi knockdown was driven in the female germline with UAS-Dcr2 and NGT-Gal4. GFP (green), β-gal (red), and DAPI (blue). Scale bar: 50 µm. (F) Confocal images of the ovaries from nos-Gal4-VP16 > wRNAi and nos-Gal4-VP16 > otuRNAi flies stained with anti-cleaved Caspase 3 antibody. Cleaved Caspase-3 (green) and DAPI (blue). Scale bar: 20 µm

Loss of otu results in low levels of pMad and derepression of bam in the germaria

(A) Confocal images of the ovaries from control (wRNAi) and otuRNAi flies harboring the bam-GFP reporter, where RNAi knockdown was driven in the female germline with nos-Gal4-VP16. Bam-GFP (green), Hts (red), and DAPI (blue). Scale bar: 20 µm. (B) Confocal images of germaria from control (otu14/+) and otu14/14 mutant flies stained with anti-pMad (green) and anti-Hts (red) antibodies. Scale bar: 10 µm.

Schematic illustration of Drosophila ovary and germarium

(A) Drosophila female has a pair of ovaries, each composed of 12-16 ovarioles. The germarium is located at the anterior tip of the ovarioles and consists of germ cells and somatic cells. Germline stem cells (GSCs), cystoblasts, cysts, and more differentiate oocytes are germ cells, while terminal filament cells, cap cells, escort cells, and follicle cells are somatic cells. GSCs and cystoblasts have round-shaped and unbranched spectrosomes, while cysts have branched fusomes. (B) Cap cells secrete diffusible Decapentaplegic (Dpp), which is received by its receptor, a heterodimer of Thick vein (Tkv) and Punt (Put), in GSCs. The activated Dpp signaling eventually phosphorylates Mother-against-dpp (Mad). The phosphorylated Mad (pMad) represses the transcription of bag-of-marbles (bam). The repression of bam in GSCs is crucial for maintaining their stemness. Cystoblasts do not receive Dpp, and Bam expression is crucial for promoting cystoblast differentiation from GSCs.

Male fertility assay.

The numbers of the progeny flies obtained from crosses between test males and wild-type (OregonR) virgin females are shown. Mean ± SD (n = 5).

sakuranull ovaries are tumorous

Violin plots of GSC-like cell numbers in germaria of indicated genotypes. Mean ± SD and the biological replicate number n are also shown. P-value < 0.05 (Student’s t-test, unpaired, two-tailed) is indicated by *

sakuranull clone germline cells intrinsically cause tumorous phenotype

Violin plots of GSC-like cell number in germaria of indicated genotypes. Mean ± SD, and the biological replicated number n are also shown. P-value < 0.05 (Student’s t-test, unpaired, two-tailed) are indicated by *

Co-immunoprecipitations assay to test interaction between N-terminal fragments of Sakura and Otu in S2 cells

Co-immnuprocepitation assay using beads bound with anti-FLAG antibody followed by Western blotting. S2 cell lysates expressing mCherry-3xHA were used as controls. This is the reciprocal co-immunoprecipitation of Fig 10F.

In vitro deubiquitination assay.

The mean fluorescence intensity of three replicates was plotted. Error bars (+/-) are standard deviations. Firefly Luciferase was used as a negative control.