Fig 1.
E(Pc) downregulates CySC-enriched transcription factors.
(A) A schematic diagram of Drosophila adult testes. CySCs: cyst stem cells; GSCs: germline stem cells. (B-B”) In Tj-Gal4 control testes, Zfh-1-positive early cyst cell zone (green dashed line) is separable spatially from the region with later stage cyst cells labeled by Eya (yellow arrows). (C-C”) In Tj>E(Pc) RNAi testes, Zfh-1-positive cell zone expands (green dashed line) with some cells co-expressing Eya (yellow arrows). (D) In Tj-Gal4 control testes, another marker Yan is highly enriched in CySCs and early cyst cells (yellow dashed line), a few cell-diameter away from the hub region (white outline). (E) In Tj>E(Pc) RNAi testes, Yan-positive cell zone (yellow dashed line) expands and is further away from hub (white outline). (F-G”) GFP-positive cells represent cells with E(Pc) knockdown. Compared with the GFP-negative and Eya-positive cyst cell (yellow arrowhead in F-F”), the neighboring GFP-positive and Eya-positive cyst cell (yellow arrow in F-F”) has higher Zfh-1 signal. Similarly, compared with GFP-negative and DAPI-positive cyst cell (yellow arrowhead in G-G”), the neighboring GFP-positive and DAPI-positive cyst cell (yellow arrow in G-G”) has higher Yan signal. Asterisk: hub. Scale bar: 20μm. See also S1 Fig.
Fig 2.
E(Pc) acts in cyst cells to promote germ cell differentiation and maintain germline identity.
(A-A”) In Tj-Gal4 control testes, DAPI bright region (yellow dashed line in A), and Notch positive cells (yellow dashed line in A’) represent GSCs and early-stage germ cells. (B-B”) Elongated DAPI bright region (yellow dashed line in B) and Notch-positive cell zone (yellow dashed line in B’) in Tj>E(Pc) dsRNA testes. (C) Immunostaining with germ cell marker Vasa (Green) and early cyst cell marker Zfh-1 in Tj>E(Pc) dsRNA testes: GSC- and GB-like germ cells intermingle with Zfh-1 positive cells. (D-D”) Immunostaining with antibodies against Armadillo and α–spectrin (D) in Tj>E(Pc) dsRNA testes show spermatogonial tumor cells interconnected with both round spectrosome (yellow arrowhead) and branched fusome (yellow arrow). EdU labeling in Tj>E(Pc) dsRNA testes (D’) show only a subset of overproliferating germ cells within one cyst are EdU-positive (yellow arrow). Scale bar: 10μm. (E-F”) In Tj>E(Pc) dsRNA testes, Vasa-positive germ cells (E, F, green in E” and F”) are also labeled with CySCs-enriched marker Zfh-1 (E’, red in E”) and Yan (F’, red in F”). Scale bar: 20μm. See also S2, S3, S4, S5, S6, S7 and S8 Figs.
Fig 3.
E(Pc) directly regulates multiple signaling pathway components and mainly represses gene expression in cyst cells.
(A) In Tj>E(Pc) cDNA-GFP, E(Pc) shRNA testes, GFP is only detectable in CySC lineage. White dotted line: hub. Scale bar: 20μm. (B) ChIP-seq was performed with the GFP antibody using Tj>E(Pc) cDNA-GFP, E(Pc) shRNA testes. Two independent ChIP experiments were performed. Average E(Pc) enrichment signal profile of 4698 genes over a -1-kb to +4-kb region with respect to the transcription start sites (TSSs). (C) GO term enrichment test to identify significant categories with distinct biological functions among E(Pc)-binding genes. Enrichment (N, B, n, b): N- total number of genes, B- total number of genes associated with a specific GO term, n- number of all E(Pc) target genes, b- number of E (Pc) target genes with this specific GO term. The scores mean overall enrichment of genes within annotated GO term. -Log 10 P-value annotates the significance of genes enrichment within this specific GO term. (D) Scatter plots of gene expression comparison between Tj-Gal4 control testes and Tj>E(Pc) shRNA testes. The two green lines outline differentially expressed genes with more than two-fold change. (E) Venn Diagram showing E(Pc) targets upregulated (overlap between red and green) and downregulated (overlap between blue and green) in Tj>E(Pc) shRNA testis. See also S9 Fig.
Fig 4.
E(Pc) directly regulates the CySC self-renewal factor Zfh-1.
(A) Expression levels of zfh-1, yan and eya in Tj-Gal4 control testis and Tj>E(Pc) shRNA testis. cRPKM: corrected RPKM (see EXPERIMENTAL PROCEDURES). Error bars, s.d. for N = 2 biological replicates. P value: two-tailed t test. (B) A genome browser snapshot of the zfh-1 gene region. Compared with the input control, anti-GFP immunoprecipitated chromatin from cyst cells show enrichment around zfh-1 promoter region, indicated by red lines below read density map and red box. The black line indicates average read density of chromosome 3R. (C) A genome browser snapshot of the eya gene region. No enrichment around promoter region is observed. The black line indicates average read density of chromosome 2L. (D) A genome browser snapshot of the yan (also known as aop) gene region. Local enrichment (labeled by red box with dashed line) could be detected around promoter region, although the enrichment did not pass the threshold using peak MACS2 calling algorithm. The black line indicates average read density of chromosome 2L. (E) Anti-GFP ChIPed DNA using Tj>E(Pc) cDNA-GFP, E(Pc) shRNA testes was analyzed by qPCR. Enrichment of E(Pc) at yan loci was presented as percentage of input. Error bars, s.d. for N = 2 biological replicates. See also S10 Fig.
Fig 5.
E(Pc) acts in synergy with EGF signaling pathway to promote cellular differentiation in both CySC and GSC lineages.
(A-A”) In Vein-LacZ, Tj-Gal4 control testes, Vein is not expressed in Zfh-1 positive CySCs (yellow arrowhead), but becomes detectable in differentiated cyst cells (yellow arrows). (B-B”) In Vein-LacZ, Tj> E(Pc) shRNA testes, Vein is not expressed in CySCs (yellow arrowhead), almost undetectable in later stage cells (yellow arrows) several cell diameter away from hub (Asterisk). (C) Quantification of Vein expression in later stage cyst cells (labeled by yellow arrows in A” and B”). ****P<0.0001. Two-tailed t test. (D) Quantification of percentage of testes with expanded DAPI bright region (severe, medium and normal, refer to S6A–S6C Fig) in Tj>E(Pc) dsRNA, Tj>E(Pc) dsRNA, Egfrf2/+ and Tj-Gal4, Egfrf2/+ testes 3 days after shifting to 29°C. Removing one copy of Egfr using loss-of-function Egfrf2 allele enhanced the phenotype. ****P<0.0001, chi-square test. (E-E”) Ectopic expression of consistent active (CA) Yan in cyst cells caused overpopulation of Zfh-1 cells (red in E, E’) accompanying GSC- and GB-like tumor (yellow dashed line) and spermatogonial tumor (white dashed line) within spermatogonial cyst shown by anti-Armadillo immunostaining (E”). (F) Quantification of percentage of testes with expanded DAPI bright region in Tj>E(Pc) shRNA, Tj>E(Pc) shRNA, YanIP/+ and Tj-Gal4, YanIP/+ testes 5 days after shifting to 29°C, using the same criterion as in (D). Removing one copy of yan using a null allele yanIP suppressed the phenotype. ****P<0.0001, chi-square test. (G-G’) Level and localization of phosphorylated ERK (dpERK) in GFP-positive (yellow arrow) and GFP-negative wild-type cells (yellow arrowhead). (H-H’) Level and localization of dpERK had no detectable difference between E(Pc) knockdown cyst cells (GFP-positive, yellow arrow) and control cyst cells with normal E(Pc) (GFP- negative, yellow arrowhead). Asterisk: hub. Scale bar: 20μm.
Fig 6.
E(Pc) represses JAK-STAT signaling in the CySC lineage.
(A) Genome browser snapshots of E(Pc) enrichment at domeless (dome), hopscotch (hop), stat92E gene loci. One replicate of ChIP experiment is shown here and the other replicate shows similar pattern. Peak calling is labeled by red lines below read density map and also red box. The black line indicates average read density of relative chromosome. (B-B”) In Stat-GFP, Tj-Gal4 control testes, GFP signal is enriched in CySCs which are one cell diameter away from hub (asterisk). GFP positive cells (yellow arrowhead) are separable from Eya-positive later stage cyst cells (yellow arrow). (C-C”) In Stat-GFP, Tj>E(Pc) shRNA testes, GFP-positive cells expand from apical CySCs (yellow arrowhead) to Eya-positive cells (yellow arrows). Asterisk: hub. Scale bar: 20μm.
Fig 7.
E(Pc) acts in synergy with Tip60 whose function in cyst cells depend on its histone acetyl-transferase activity.
(A-A”‘) Tj>Tip60 RNAi testes show expansion of cells with DAPI bright nuclei (yellow dashed line in A) and Zfh-1-positive cyst cells (green dashed line in A’ and A”‘). Zfh-1 is co-expressed with late-stage cyst marker Eya (yellow arrows). (B-B”‘) Tip60 HAT deficient dominant active form (Tip60E431Q) expressed using Tj-Gal4. Tj>Tip60E431Q testes show expansion of cells with DAPI bright nuclei (yellow dashed line in B), including cells co-expressing Zfh-1 and Eya (yellow arrows in B’-B”‘). (C) In Tj>Tip60 RNAi testes, GSC- and GB-like cells are intermingled with Zfh-1-positive cells (cells from the hub region to yellow dashed line). (D) Spermatogonial tumor with more than 16 germ cells (Vasa positive) within one cyst (white dashed line) interconnected by both branched fusome (yellow arrow) and round spectrosome (yellow arrowhead) in Tj>Tip60 RNAi testes. (E-F) Similar GSC- and GB-like tumor (cells from hub region to yellow dashed line in E) and spermatogonial tumor (white dashed line in F) are also detected in Tj>Tip60E431Q testes.(G-H”) In Tj>Tip60 RNAi testes, Vasa-positive cells (G, G”, H, H”) also have expression of CySC-enriched marker Zfh-1 (G’,G”) and Yan (H’, H”). (I-J”) In Tj>Tip60E431Q testes, Vasa-positive cells (I, I”, J, J”) also have expression of Zfh-1 (I’,I”) and Yan (J’,J”). Asterisk: hub. Scale bar: 20μm. See also S11 and S12 Figs.
Fig 8.
A model to describe both the cell-autonomous functions of E(Pc) in CySC lineage and its non-cell-autonomous roles in regulating germ cell differentiation and maintaining germline identity.
(A) In cyst cells of wild-type testes, E(Pc) represses CySC-enriched factors (for example Zfh-1, Yan) and JAK-STAT signaling pathway to promote cyst cell differentiation cell-autonomously. E(Pc) also acts in synergy with the EGF signaling pathway. Under this condition, both cyst cell and germ cell differentiate properly. (B) When E(Pc) is knocked down in cyst cells, ectopic expression of Zfh-1 and Yan, as well as hyperactivation of the JAK-STAT signaling but compromised EGF signaling activity lead to germ cell tumors including both GSC- or GB-like tumor and spermatogonial tumor. In addition, the excess germ cells turn on early-stage cyst cell markers.