Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila

Abstract

Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. We also find that mitosis/cytokinesis proteins are especially important for male GSC maintenance. Our findings provide valuable insights and resources for studying stem cell regulation at the organismal level.

Figure 1. Transgenic RNAi screen.

(a) Workflow of the GSC RNAi screen. (b) Pie chart summarizing the screen results. Fly lines shown in the table are those that gave a lethal phenotype when crossed with Act-Gal4. (c) Pie chart summarizing phenotype observed in all the 720 lines. Act^ts>RNAi male flies were dissected after 7 days at 29 °C. Their testes were stained with antibodies (vasa, 1B1 and Arm) and analysed by confocal microscopy. The phenotypes were divided into two major categories (GSC decrease and increase). Percentage of fly lines showing no phenotypes are also shown in the chart. (d) Confidence of 530 genes identified from the screen. High-confidence genes were identified by two or more independent RNAi lines. Medium-high-confidence genes were identified by one RNAi line, but they shared a complex with high-confidence hits. Medium-low-confidence genes were identified by one RNAi line, but they shared a complex with other low-confidence hits. Low-confidence hits were identified by one RNAi only.

Figure 2. Representative RNAi screen phenotypes using Act^ts-Gal4.

(a) Diagram of GSC lineage and marker expression. GSCs and GBs have round spectrosomes (green dots) and spermatogonia contain branched fusomes (green branched lines). GSCs are enclosed by two CySCs (pink). (b) Wild-type (WT) control. (c) Knockdown of tum resulted in GSC loss and premature differentiation into spermatogonial cells (white arrow). (d) Knockdown of Nedd8 resulted in GSC loss and death of differentiated cells. (e) Knockdown of bab2 (bric a brac 2) resulted in GSC loss and GSCs that directly differentiated into spermatogonial cells (yellow dotted line) and spermatocytes (light blue arrows). (f) Knockdown of Taf8 resulted in GSC differentiation defects. (g) Knockdown of Cka (Connector of kinase to AP-1) resulted in GSC overproliferation at the expense of terminal differentiation. (h) Knockdown of Vein (Vn) resulted in GSC differentiation defects. Testes of the indicated flies were dissected, stained with antibodies to Vasa (red, marks all germ cells including GSCs), Arm (green, hub cells at the apex), 1B1 (green, marks round spectrosomes and branched fusomes) and DAPI (blue, marks the nuclei), and analysed by confocal microscopy. Asterisks indicate hub cells. White arrows near hub cells indicate GSC and green arrows indicate GBs (in b,d,e), and light blue arrows (b,c,e,h) indicate spermatocytes. White dotted lines indicate GSCs (near hub in a) and GSC-like cells (in f–h). Scale bars, 10 μm.

Figure 3. Cell-type-specific knockdowns of mitotic/cytokinesis genes.

(a) A schematic diagram showing expression pattern of Nos-Gal4 in adult testis. Knockdown of Smc2 (b), APC10 (c), Cenp-C (Centromeric protein-C) (d) and tum (e) in germ cells (Nos-Gal4) resulted in dramatic GSC loss. (f) A schematic diagram showing expression pattern of c587-Gal4 in adult testis. Knockdown of Smc2 (g), Cenp-C (i) and tum (j) in CySC (c587-Gal4) resulted in GSC differentiation defects (white dotted lines, g,i,j); however, knockdown of APC10 (h) in CySCs resulted in GSC loss. (k) A schematic diagram showing expression pattern of Bam-Gal4 in adult testis. Knockdown of Smc2 (l) and APC10 (m) Cenp-C (n) and tum (o) in spermatogonial cells (Bam-Gal4) resulted in normal (l,m) or polyploid (n,o, yellow arrows) cells. (p,q) FRT^42D-SMC2^f06842 mosaic clones of GSCs 2 days (p, white dotted lines with white arrows, β-galactosidase (green) negative) and 7 days (q, yellow dotted lines with black arrows, β-galactosidase (green) negative) after clonal induction (ACI). Arm-negative GSC clones were detected in 4 out of 8 testes 2 days ACI but in 0 out of 20 testes 7 days ACI. Therefore, SMC2 is intrinsically required for GSC maintenance. Testes of the indicated flies were dissected, stained with antibodies to Vasa (red, marks all germ cells including GSCs), Arm (green, hub cells at the apex), 1B1 (green, marks round spectrosomes and branched fusomes) and DAPI (blue, marks the nuclei) in b–o, and analysed by confocal microscopy. White arrows near hub cells indicate GSC and green arrows indicate GBs (in l–o). Asterisks indicate hub cells. The flies were cultured at 29 °C for 7 days in b–o. Scale bars, 10 μm.

Figure 4. TCTP differentially regulate GSCs from different cells.

(a) Knockdown of Tctp using Act-Gal4 resulted in GSC differentiation defects. (b) Knockdown of Tctp using Nos-Gal4 resulted in GSC loss. (c) Knockdown of Tctp using c587-Gal4 resulted in GSC differentiation defects. FRT^82B-piM mosaic clones of GSCs 2 days (d) and 7 days (e) ACI. FRT^82B-Tctp^h5p mosaic clones of GSCs 2 days (f) and 7 days (g) ACI. In control flies, Arm-lacZ-negative GSC clones were detected in 33 out of 37 testes 2 days ACI and in 51 out of 64 testes 7 days ACI; in Tctp^h5p flies, Arm-lacZ-negative GSC clones were detected in 37 out of 53 testes 2 days ACI and in 8 out of 63 testes 7 days ACI. Thus, Tctp is intrinsically required for GSC maintenance. Testes of the indicated flies were dissected, stained with antibodies to Vasa (red, germ cells including GSCS), β-galactosidase (green) and DAPI (blue, marks the nuclei) in d–g, and Vasa (red), Arm (green, hub cells at the apex), 1B1 (green, marks round spectrosomes and branched fusomes) and DAPI (blue, marks the nuclei) in a–c, and analysed by confocal microscopy. The flies were cultured at 29 °C for 7 days in a–c. White arrows near hub cells indicate a GSC clone (d–f) and yellow dotted lines in d–g indicate GSC clones. White dotted lines in a,c indicate GSC-like cells. A yellow arrow in b indicates a remaining germ cell. Asterisks indicate hub cells. Scale bars, 10 μm.

Figure 5. Vers interacts with EGFR and Dpp to regulate GSC and CySC.

(a,b) Knockdown of vers in CySC lineage c587-Gal4) caused GSC differentiation defects. (c) Wild-type (control) testis. (d) Knockdown of vers in CySC lineage (c587-Gal4) caused CySC expansion. (e) Wild-type (control) testis. (f) Knockdown of vers in CySC lineage (c587-Gal4) activates both EGFR and Dpp signalling. Testes of the indicated flies were dissected, stained with antibodies to Vasa (red), Arm (green, hub cells at the apex) and 1B1 (green, marks round spectrosomes and branched fusomes) in a,b, Vasa (red) and Zfh1 (green, marks CySCs) in c,d, Smad3 (red) and Cic (green) in e,f, and DAPI (blue, marks the nuclei), and analysed by confocal microscopy. White dotted lines in a,b indicate GSC-like cells. White arrow near hub cells indicates a GSC and green arrow indicates a CySC (c). Yellow dotted lines in f indicate Smad3-positive cells. Asterisks indicate hub cells. The flies were cultured at 29 °C for 7 days. Scale bars, 10 μm.

Figure 6. Cell proliferation in vers knockdown and Vers expression.

(a) Wild-type (control) testis. (b) Knockdown of vers in the CySC lineage (c587-Gal4) resulted in an expansion of mitotic cells. Vers expression in the testis (c) and in the midgut (d). (e) Comparison of male GSC, ISC, neuroblast and female GSC RNAi screens. Number of genes identified in the male GSC, ISC, neuroblast and female GSC screens. Seven genes were found in all four screens; 9 genes were shared by male GSC, female GSC and ISC screens; and 38 genes were shared by male GSC, female GSC and neuroblast screens. Venn diagram was prepared from: http://bioinfogp.cnb.csic.es/tools/venny/index.html. Testes of the indicated flies were dissected, stained with the antibodies to phospho-Histone 3 (pH3) (red, marks mitotic cells) (a,b), Vers (red, all type of cells in the testis and midgut), Arm (green, hub cells in testis (c) and epithelial membrane in the midgut (d)) and DAPI (blue, marks the nuclei), and analysed by confocal microscopy. Asterisks indicate hub cells. Yellow arrows in d indicate ISC/EB (enteroblast) cells and white arrows indicate enterocyte (EC) cells. The flies were cultured at 29 °C for 7 days in a–d. Scale bars, 10 μm.