Chinmo prevents transformer alternative splicing to maintain male sex identity

Abstract

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, we show that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.

Fig 1. Chinmo is expressed dimorphically in Drosophila gonads.

(A) Schematic of the adult Drosophila testis (left) and ovary (right). In the testis, the niche (green) supports two populations of stem cells, germline stem cells (GSCs, dark pink) and somatic cyst stem cells (CySCs, dark blue). The GSC divides to produce differentiating daughter cells (light pink) that undergo four transit-amplifying divisions. The CySC divides to produce cyst daughter cells (light blue) that exit the cell cycle and ensheath the differentiating GSC daughter. Cyst cells continue to ensheath the associated spermatogonial cyst during transit-amplifying divisions. In the ovary, the niche (green) supports GSCs (dark pink), which divide to give rise to differentiating daughters (light pink) that undergo 4 mitotic divisions. The developing germline cyst is ensheathed by an epithelial layer of follicle cells (light blue). Follicle cells are proliferative descendants of follicle stem cells (FSCs, dark blue) located in the anterior part of the ovary. (B) Chinmo (green) is present in CySCs (B’, green arrowhead), GSCs (B’, magenta arrowhead), and niche cells (B’, cyan arrowhead) in a wild type testis. (C) Chinmo is not expressed in follicle cells of a wild type ovary. Vasa (red) marks the germline and Zfh1 (blue) marks somatic cells in the testis and ovary. Scale bars = 10 μm.

Fig 2. Chinmo is required in CySCs for male somatic sex identity and non-autonomously for germline maintenance.

(A) In a wild type testis, Dsx^M (green) is present in niche cells (A’, outlined with dotted green line), CySCs (A’, arrowheads) and cyst cells (A’, arrow). (B) Dsx^M is not expressed in a wild type ovary (B’, arrows indicate early follicle cells). (C) Dsx^M is lost in the CySC lineage in a tj>chinmo^RNAi testis (C’, arrows indicate early somatic cells). (D) Castor (Cas, green) is absent from a wild type testis (D’, arrows indicate early somatic cells). (E) In a wild type ovary, Cas (green) is expressed in early follicle cells (E’, arrowhead) and stalk cells (E’, arrow). (F) Cas (green) is ectopically expressed in feminizing somatic cells in the testis (F’, arrows) upon loss of Chinmo. (G) Fasciclin-3 (Fas3, green) is normally restricted to niche cells in a wild type testis. (H) In a wild type ovary, Fas3 (green) is high in early follicle cells (H’, arrowhead) and is lower in mature follicle cells (H’, arrow). (I) Fas3 (green) is ectopically expressed upon loss of Chinmo in CySC lineage. (J) Relative fertility of tj>chinmo^RNAi males (blue bars) is decreased at 9, 16 or 23 days (d) post eclosion compared with control tj>+ males (white bars). tj>chinmo^RNAi males are completely sterile by 23 days post eclosion (* denotes p<0.05; *** denotes p<0.001; **** denotes p<0.0001 as determined by single-factor ANOVA). Error bars represent SEM. In A-I, Vasa (red) marks the germline and Tj (blue) marks cyst cells. Scale bars = 20 μm. Time point in A-I is 7 days post-eclosion.

Fig 3. Dsx^F protein is synthesized in chinmo-mutant CySCs.

(A) Schematic of dsx pre-mRNA splicing. dsx^F and dsx^M share the first three coding exons and differ at their C-termini. Exon 4 contains a non-canonical splice acceptor, and thus under default splicing conditions, exon 3 is adjoined with exon 5 to yield the male-specific dsx^M isoform (blue). In XX somatic cells, Tra^F is expressed and binds tandem Tra^F-binding sites (white dashed line) in exon 4. The Tra^F complex then recruits the spliceosome, leading to synthesis of the female-specific dsx^F isoform (pink). Pink and blue stars indicate female and male stop codons, respectively. (B) A knock-in transcriptional reporter for dsx (dsx-gal4) activates UAS-GFP expression (green) in the CySC lineage of a wild type testis. (C) dsx-gal4 activates GFP expression in both escort cells (C’, arrow) and follicle cells (C’, arrowheads) of wild type ovary. (D) dsx-gal4 activates GFP expression (D’, arrowheads) in the somatic lineage of a chinmo^ST/chinmo^ST testis. Note that the feminized soma in this testis is organized at the periphery, adjacent to the muscle sheath. (E) Semi-quantitative RT-PCR on homogenized wild type testes (left lane), wild type ovaries (middle lane), and tj>chinmo^RNAi testes (right lane). As determined by primers that recognize both dsx mRNA isoforms (dsx^COMMON, or dsx^C), dsx transcripts are present in both wild type testes and wild type ovaries (left and middle lanes). dsx mRNA is also expressed in tj>chinmo^RNAi testes (right lane). α-tub (tub) was used as a loading control. Flies were aged 9–20 days prior to dissection. (F) Semi-quantitative RT-PCR on RNA extracts from FACS-purified cyst cells from control tj>+ or tj>chinmo^RNAi testes. Whole adult ovaries from yw females (labeled “WT ovary”) were homogenized as a positive control for dsx^F mRNA detection. dsx^F is detected in wild type ovaries (left lane) and in tj>chinmo^RNAi cyst cells (right lane) but not in tj>+ cyst cells (middle lane). β-tubulin (tub) was used as a loading control. (G,H) Dsx (green), as detected by the Dsx^C antibody that recognizes both Dsx^F and Dsx^M, is present in somatic cells of a wild type testis (G) and a tj>chinmo^RNAi testis (H). Tj (magenta) marks cyst cells. In B-D, Vasa (red) marks germ cells and Tj (blue) marks cyst cells. Scale bars = 20 μm.

Fig 4. Dsx^F is required for feminization in chinmo-mutant CySCs.

(A-C) Representative confocal images of testes from chinmo^ST/chinmo^ST; dsx^D/dsx¹ flies. In a testis from a chinmo^ST/CyO; dsx^D/dsx¹ sibling male, only niche cells express Fas3 (A’). By contrast, in a chinmo^ST/chinmo^ST testis most of the somatic lineage is positive for Fas3 (B’). In a testis from a “rescued” chinmo^ST/chinmo^ST; dsx^D/dsx¹ male, Fas3 is again restricted to niche cells (C’). Results are quantified in Fig 4D and S1 Table. Vasa (red) marks germ cells and Tj (blue) marks cyst cells. Scale bars = 20 μm.(D) Quantification of Fas3-positive aggregates as a readout of feminization. 100% of chinmo^ST/chinmo^ST; TM2/TM6B testes contain Fas3-positive aggregates (dark blue bar) at 7 days post-eclosion. By contrast, none of the testes from chinmo^ST/CyO; dsx^D/dsx¹ sibling males contain aggregates (second bar). There is a significant reduction in the percentage of testes from chinmo^ST/chinmo^ST; dsx^D/dsx¹ (purple bar) or chinmo^ST/chinmo^ST; tra¹/Df(3L)st-j7 (green bar) containing Fas3-positive aggregates, indicating a partial rescue of the feminization. However, testes from the various sibling controls are still feminized (light blue and yellow bars). Sample sizes are indicated within bars. *** denotes p<0.001 and **** denotes p<0.0001 as determined by two-tailed Student’s t-test (for qRT-PCR, compared with tj>+ controls) or Fisher’s Exact Test (for quantifications, compared with chinmo^ST/chinmo^ST). n.s. means not significant. See S1 Table for percentage values.

Fig 5. The female sex determinant Tra^F is required for feminization of chinmo-mutant CySCs.

(A) Semi-quantitative RT-PCR of total tra and tra^F mRNA in control tj>+ testes (first lane), control tj>+ ovaries (second lane) and tj>chinmo^RNAi testes (third lane). All three samples contain a band for total tra (first row, blue arrowhead). However, a tra^F band is detected in ovaries, as expected, and in tj>chinmo^RNAi testes (second row, red arrowhead) but not in control tj>+ testes. rpl15 was used as a loading control (third row). Flies were aged 9–20 days prior to dissection. (B-C) qRT-PCR of total tra (B) or tra^F (C) in control tj>+ testes (white bars), control tj>+ ovaries (gray bars) and tj>chinmo^RNAi testes (blue bars). There is significantly more total tra mRNA (B) and tra^F (C) in tj>chinmo^RNAi testes compared to control tj>+ testes. Values represent the average of three biological replicates. * denotes p<0.05; *** denotes p<0.001 as determined by two-tailed Student’s t-test (compared with tj>+ testes). Error bars represent SEM. Flies were aged 9–20 days prior to dissection. (D-F) GFP caused by alternative splicing of traF^ΔT2AGFP pre-mRNA is not observed in control tj>traF^ΔT2AGFP testes (D’). By contrast, GFP indicative of tra^F splicing is robustly observed in follicle cells of a control tj>traF^ΔT2AGFP ovary (E’, arrowheads) and in somatic cells of a tj>traF^ΔT2AGFP; chinmo^RNAi testis (F’, arrowheads). Tj (blue) marks somatic cells. Fas3 (red) marks hub cells in wild type testes, follicle cells in wild type ovaries and feminized cyst cells in tj>chinmo^RNAi testes. (G-I) In a control tj>+ testis, only niche cells express Fas3 (G’). By contrast, in a tj>chinmo^RNAi testis, most of the somatic lineage is positive for Fas3 (H’). In a “rescued” tj>tra^RNAi; chinmo^RNAi testis, Fas3 is again restricted to niche cells (I’). Vasa (red) marks germ cells, Tj (blue) marks cyst cells, and Fas3 (green) marks niche cells and feminizing somatic cells. Results are quantified in Fig 4J and S1 Table. (J) Quantification of CySC feminization in tj>GFP; chinmo^RNAi testes and various genotypes. 100% of tj>GFP; chinmo^RNAi testes contain Fas3-positive aggregates (dark blue bar). There is a significant reduction in the percentage of feminized testes when tra is concomitantly depleted from tj>chinmo^RNAi testes (purple bar). There is also a significant reduction when vir or fl(2)d is depleted from tj>chinmo^RNAi testes (green and yellow bars, respectively). However, there is no rescue of male sex identity in tj>chinmo^RNAi testes when Sxl^, or nito is concomitantly depleted (light blue and red bars, respectively). Sample sizes are indicated within bars. *** denotes p<0.001; **** denotes p<0.0001 as determined by Fisher’s Exact Test (for rescue quantifications, compared with tj>GFP; chinmo^RNAi). n.s. means not significant. See S1 Table for percentage values. (K-M) Fas3 is not ectopically expressed in CySCs from control (tj^TS>+) testes (K’), testes with somatic mis-expression of tra^F (tj^TS>tra^F) (L’), or testes with somatic mis-expression of fl(2)d (tj^TS>fl(2)d) (M’). (N-P) Cas is not expressed in control (tj^TS>+) (N’), tj^TS>tra^F testes (O’), or tj^TS>fl(2)d testes (P’). In K-P, Vasa (red) marks germ cells, and Zfh1 or Tj (blue) mark cyst cells. Scale bars = 20 μm.

Fig 6. Sxl is not required for feminization of chinmo-mutant CySCs.

(A) Schematic of tra pre-mRNA splicing. The poly(U) tract upstream of exon 2 is bound by the RRM domain of Sxl in females, causing skipping of exon 2. In wild type males, exons 1–4 comprise tra mRNA and translation terminates at the early stop codon in exon 2 (red star). Pink dashed lines indicate female-specific alternative splicing and blue dashed lines indicate non-sex-specific default splicing. (B-D) Sxl is not expressed in a control tj>+ testis (B’) but is expressed in follicle cells (C’, arrowheads) and in an early germ cell (C’, arrow) of a control tj>+ ovary. Sxl protein is not detected in a tj>chinmo^RNAi testis (D’). (E) Semi-quantitative RT-PCR on Sxl in homogenized control tj>+ testes (left lane), control tj>+ ovaries (middle lane), and tj>chinmo^RNAi testes (right lane). Control tj>+ testes express Sxl^M transcripts (blue arrowhead), while control tj>+ ovaries express Sxl^F transcripts (red arrowhead). Sxl^M is still present and Sxl^F is undetectable in tj>chinmo^RNAi testes (right lane). Sxl^JYR primers were used to differentiate between Sxl^M and Sxl^F mRNA isoforms in this experiment. α-tubulin (tub) was used as a loading control. (F) Quantification of CySC feminization in Sxl; chinmo^ST backgrounds. Sample sizes are indicated within bars. **** denotes p<0.0001 as determined by Fisher’s Exact Test (compared to FM7/Y; chinmo^ST/chinmo^ST). See S1 Table for percentage values. (G-K) Representative images for Sxl; chinmo^ST epistasis experiments. Genetic loss of Sxl by 3 different alleles–Sxl ^f1 (I), Sxl ^f2 (J), or Sxl ^f18 (K)–does not prevent feminization of chinmo^ST/chinmo^ST cyst cells (G’), defined by the accumulation of Fas3-positive aggregates. Control FM7/Y; chinmo^ST/CyO cyst cells do not feminize (H’). Scale bars = 20 μm.

Fig 7. Vir and Fl(2)d, but not Nito, are required for feminization of chinmo-deficient CySCs.

(A) Semi-quantitative RT-PCR of vir, fl(2)d, and nito in homogenized control tj>+ testes (left lane), control tj>+ ovaries (middle lane) and tj>chinmo^RNAi testes (right lane). vir, fl(2)d, and nito are expressed at higher levels in ovaries (middle lane) than in tj>+ testes (left lane). vir, fl(2)d, and nito are expressed at higher levels in tj>chinmo^RNAi testes (right lane) than in control tj>+ testes (left lane). α-tubulin (tub) was used as a loading control. Flies were aged 9–20 days prior to dissection. (B) qRT-PCR analysis of vir, fl(2)d, and nito in homogenized control tj>+ testes (white bars), control tj>+ ovaries (gray bars) and tj>chinmo^RNAi testes (blue bars). vir, fl(2)d, and nito are expressed at significantly higher levels in ovaries and tj>chinmo^RNAi testes compared to control testes. * denotes p<0.05; ** denotes p<0.01; *** denotes p<0.001; **** denotes p<0.0001 as determined by two-tailed Student’s t-test. Error bars represent SEM. Flies were aged 9–20 days prior to dissection. (C) Quantification of GFP levels (synthesized from UAS-traF^ΔT2AGFP transgene) in tj>+, tj>vir^RNAi, and tj>fl(2)d^RNAi ovaries represented in D-F. Errors represent SEM. **** denotes p<0.0001 as determined by Student’s t-test. (D-F) Representative images of control tj>+ (D’), tj>vir^RNAi (E’), and tj>fl(2)d^RNAi (F’) ovaries in a UAS-traF^ΔT2AGFP background. GFP expressed from UAS-traF^ΔT2AGFP is detectable in escort cells (D’, arrow) and follicle cells (D’, arrowhead). GFP levels are dramatically reduced upon vir or fl(2)d depletion (E’ and F’, respectively) compared with wild type follicle cells (D’). Tj (blue) marks escort/follicle cells and Fas3 (red) marks follicle cells. (G-I) Depletion of nito (I’) does not reduce Fas3-positive (green) aggregates (a readout for feminization) in tj>chinmo^RNAi testes. By contrast, depletion of vir (G’) or fl(2)d (H’) in tj>chinmo^RNAi reduces the percentage of testes with these aggregates. Results are quantified in Fig 5J and S1 Table. Vasa (red) marks germ cells and Tj (blue) marks somatic cells. Scale bars = 20 μm.

Fig 8. Model for adult somatic sex maintenance in the Drosophila somatic gonad.

Left: In XX animals, the production of Sxl leads to alternative splicing of tra pre-mRNA into tra^F. Tra^F protein then alternatively splices dsx pre-mRNA into dsx^F. The Dsx^F protein promotes female-specific transcriptional changes. Right: In XY animals, Sxl is not produced. Neither tra nor dsx pre-mRNA are alternatively spliced, resulting in the production of Dsx^M protein, which ensures male-specific transcription of target genes. In addition to the absence of Sxl in XY cells, adult somatic stem cells of the Drosophila testis have an extra level of insurance of male sex identity. Chinmo, which is expressed only in male but not female somatic gonadal cells, represses expression of tra, vir and fl(2)d in CySCs. This safeguards male identity by reducing the availability of tra pre-mRNA and of factors (i.e., Vir and Fl(2)d)) that can splice it into tra^F. Thus, in addition to the canonical sex determination pathway that establishes male and female programs from early development, adult male, sexually dimorphic cells protect their sexual identity by transcriptional repression of tra and its splice factors.