Sex-converted testis somatic cells acquire female-specific behaviors and alter XY germline identity

Abstract

Establishment and maintenance of cellular sex identity is essential for reproduction. Sex identity of somatic and germline cells must correspond for sperm or oocytes to be produced, with mismatched identity causing infertility in all organisms from flies to humans. In adult Drosophila testes, Chronologically inappropriate morphogenesis (Chinmo) is required for maintenance of male somatic identity. Loss of chinmo leads to feminization of the male soma, including adoption of female-specific cell morphologies and gene expression. However, the degree to which feminized somatic cells initiate female-specific cellular behaviors or influence the associated XY germline is unknown. Using extended live imaging, we find that chinmo-depleted somatic cells acquire cell behaviors characteristic of ovarian follicle cells, including incomplete cytokinesis and rotational migration. Importantly, migration in both contexts require the basement membrane protein Perlecan and the adhesion protein E-cadherin. Finally, we find that sex-converted somatic cells non-autonomously induce expression of an early oocyte specification protein in XY germ cells. Taken together, our work reveals a dramatic transformation of somatic cell behavior during sex conversion and provides a powerful model in which to study soma-derived induction of oocyte identity.

Keywords: Chinmo; Germline; Sex determination; Somatic cell.

Fig. 1.

chinmo-deficient somatic cells in the testis acquire a female-specific cytokinesis program. (A-D) Diagrams showing soma-germline organization in a wild-type testis (A), a 0-1D chinmo^RNAi testis (B) and a 5D chinmo^RNAi testis (C) showing progressive feminization of somatic cell morphology, and a wild-type ovary (D). (E-H) Stills from time-lapse imaging of somatic cells expressing UAS tubulin::GFP (green) and UAS anillin::RFP (magenta) in the anterior region of a control testis (E; arrowhead indicates a quiescent cyst cell), a control ovary (F), a 0-1D chinmo^RNAi testis (G) and a 5D chinmo^RNAi testis (H) (arrows indicate dividing cells). Asterisks indicate the niche. Dotted lines delineate the tissue boundary. Images are representative of 10/10 testes. (I) Quantification of the distance of all dividing somatic cells from the niche (n≥113 cells in 10 testes). (J-L) Time-lapse of somatic tubulin (green) and Anillin (magenta) during division and cytokinesis in male CySCs (J), female FCs (K) and 0-1D chinmo^RNAi soma (L) (arrows point to midbodies). Asterisks indicate niches. White lines outline somatic cells. (M) Quantification of cytokinesis timing through abscission (n≥113 cells in 10 testes). ****P<0.0001 (non-parametric Mann–Whitney U-test). (N) Data from I shown in false color to indicate somatic cells that completed cytokinesis (blue) or exhibited incomplete cytokinesis (red). All experiments represent n≥2 trials. Scale bars: 20 µm (E-H); 5 µm (J-L). Each image is composed of one to five z-slices.

Fig. 2.

The sex-converted soma performs collective rotation similar to female follicle cells. (A-C) Time-lapse imaging of somatic expression of UAS tubulin::GFP (green) and UAS anillin::RFP (magenta) in a control testis (A), a control ovary (B) and a 5D chinmo^RNAi testis (C). White outlines circle groups of cells that are either non-migratory or migratory. Curved arrows indicate directional migration of cells. (A′-C′) Time-lapse imaging of somatic nuclei (gray) in a control testis (A′), a control ovary (B′) and a 5D chinmo^RNAi testis (C′). Yellow transparent dots indicate cell migration over time. Asterisks in A-C′ indicate stem cell niches. Each image is composed of one or two z-slices. Images are representative of 10/10 testes (A), 10/10 ovarioles (B) and 7/8 testes with complete somatic epithelium. (D-F) Stills from live imaging displaying colored tracks that indicate the movement of nuclei over time in a control testis (D), a control ovary (E) and a 5D chinmo^RNAi testis (F). (G) Quantification of migration speed in µm/min (n≥50 cells in 10 samples). (H-J) Live stills displaying total x-axis displacement of somatic nuclei over 5 h in a control testis (H), a control ovary (I) and a 5D chinmo^RNAi testis (J). (K) Quantification of total x-axis displacement measured in µm/5 h (n≥60 cells in at least four samples). ****P<0.0001 (one-way ANOVA). ns, not significant. Error bars represent s.d. All experiments n≥2 trials. Each Imaris image is composed of 40 z-slices. Scale bars: 20 µm (A-C); 5 µm (A′-C′;D-F,H-J).

Fig. 3.

Somatic expression of adherens junction and ECM proteins is required for rotational migration. (A-C) Immunofluorescence staining of Traffic jam (magenta) and Fas3 (gray) in a control testis (A), a 10-12D chinmo^RNAi testis (B) and 10-12D chinmo^RNAi+Ecad^RNAi testes (C). Each image is one z-slice. Images are representative of 20 (A), 25 (B), 11 (C, left), 36 (C, right) testes. (D) Graph of the percentage of feminized testes as measured by Fas3 expression in non-niche cells (n≥15 testes). **P<0.0086, ****P<0.0001 (chi-squared test). (E-J) Stills from live imaging displaying colored tracks that indicate the movement of nuclei over time in a control ovary (E), an Ecad^RNAi ovary (F), a Pcan^RNAi ovary (G), a 5D chinmo^RNAi testis (H), a 5D chinmo^RNAi+Ecad^RNAi (I) and a 5D chinmo^RNAi+Pcan^RNAi testis (J). (K) Quantification of migration speed in µm/min (n≥40 cells in at least eight samples). Data for control ovary and 5D chinmo^RNAi repeated from Fig. 2G. (L-Q) Live stills displaying x-axis displacement of somatic nuclei over 3 h in a control ovary (L), Ecad^RNAi ovary (M), Pcan^RNAi ovary (N), 5D chinmo^RNAi testis (O), 5D chinmo^RNAi+Ecad^RNAi (P) and a 5D chinmo^RNAi+Pcan^RNAi testis (Q). (R) Quantification of total x-axis displacement measured in µm/3 h. **P<0.0047, ***P<0.0006, ****P<0.0001 (one-way ANOVA). ns, not significant. Error bars represent s.d. All experiments n≥2 trials. Each Imaris image is composed of 40 z-slices. Scale bars: 20 µm (A-C); 5 µm (E-J,L-Q).

Fig. 4.

Sex-converted somatic cells induce female-like behaviors and fate changes in the germline. (A) Diagrams of feminized somatic epithelium (magenta dots mark nuclei) rotating around GCs (yellow dots mark nuclei). Blue dashed line indicates imaging planes. Black arrows indicate the direction of collective migration. Black xy axis indicates orientation of tissue. Part of this figure was created in BioRender by Roach, T. 2025. https://BioRender.com/nlsa4nx. This figure was sublicensed under CC BY 4.0 terms. (B) Time-lapse imaging of somatic anillin::RFP (gray) migrating in the x direction. (C) Time-lapse imaging of GCs expressing nos-lifeact::tdTomato (gray) migrating in the x direction. Images are representative of 7/8 testes. (D-G) Immunofluorescent staining of somatic tubulin::GFP (green), germline Vasa (magenta) and Orb (gray) in a control ovary (D), a control testis (E), a partial epithelium 5D chinmo^RNAi testis (F) and a complete epithelium 5D chinmo^RNAi testis (G). (H) Quantification of Orb fluorescence intensity relative to Vasa (n≥20 cells in at least eight samples). (I-L) Immunofluorescence staining of somatic tubulin::GFP (green), germline Vasa (magenta) and BicD (gray) in a control ovary (I), a control testis (J), a partial epithelium 5D chinmo^RNAi testis (K) and a complete epithelium 5D chinmo^RNAi testis (L). (M) Quantification of BicD fluorescence intensity relative to Vasa (n≥14 cells in at least eight samples). ***P<0.0006, ****P<0.0001 (one-way ANOVA). ns, not significant. Error bars represent s.d. All experiments n≥2 trials. Each image is composed of one to three z-slices. Scale bars: 5 µm (B,C); 20 µm (D-G,I-L). AU, arbitrary units.

Fig. 5.

Model for gain of female-specific characteristics following loss of chinmo from testis somatic cells. (A) Wild-type testis somatic cells divide with complete cytokinesis within the niche and produce quiescent daughter cells that encyst the germline cells. (B) Wild-type ovarian somatic cells divide outside of the niche with frequent incomplete cytokinesis, producing mitotically active daughter cells that form a rotating epithelium around germline cells. (C) 0-1D chinmo^RNAi testis somatic cells initiate frequent incomplete cytokinesis within and outside the niche. (D) 5D chinmo^RNAi testis somatic cells form epithelia that initiate rotational migration around GCs. (E) 5D chinmo^RNAi testis somatic cells non-autonomously induce early oocyte specification in associated XY GCs. (F) Apical and basal expression of Ecad and Pcan, respectively, results in functional migration (black arrow) along the basement membrane (BM). (G) Somatic depletion of Ecad in chinmo^RNAi testes prevents functional migration (black arrow) along the BM. (H) Somatic depletion of Pcan in chinmo^RNAi testes prevents functional migration (black arrow) along the BM.