Altered heterochromatin binding by a hybrid sterility protein in Drosophila sibling species

Abstract

Hybrid sterility of the heterogametic sex is one of the first postzygotic reproductive barriers to evolve during speciation, yet the molecular basis of hybrid sterility is poorly understood. We show that the hybrid male sterility gene Odysseus-site homeobox (OdsH) encodes a protein that localizes to evolutionarily dynamic loci within heterochromatin and leads to their decondensation. In Drosophila mauritiana x Drosophila simulans male hybrids, OdsH from D. mauritiana (OdsHmau) acts as a sterilizing factor by associating with the heterochromatic Y chromosome of D. simulans, whereas D. simulans OdsH (OdsHsim) does not. Characterization of sterile hybrid testes revealed that OdsH abundance and localization in the premeiotic phases of spermatogenesis differ between species. These results reveal that rapid heterochromatin evolution affects the onset of hybrid sterility.

Fig. 1. OdsH proteins differ in their localization to D. simulans heterochromatin

We use D1 staining as (Fig. S1). (A) FLAG-OdsHsim or Venus-OdsHmau epitope-tagged proteins were expressed in transiently transfected D. simulans cultured cells (B–D) or in transgenic D. simulans larval neuroblasts (E–H) under the control of a heat-shock promoter. (B,C) D1 staining (red) is a cytological landmark for localization of OdsHsim (B) and OdsHmau (C) proteins (both shown in green) to D. simulans heterochromatin. DNA staining by DAPI shown in blue in merge. (D) Co-expression of OdsHsim (red) and OdsHmau (green). (E) OdsHsim (red) localizes to the X chromosome and adjacent to D1 staining (green) on the 4th chromosome but not to the Y chromosome. (F) OdsHmau (red) localization has additional localization to the Y chromosome. (G) Co-expression of OdsHsim (green) and OdsHmau (red) on male mitotic chromosomes versus (H) female mitotic chromosomes. Arrows in E–G highlight Y chromosomes. (I) GFP-Unc-4 (red) staining in interphase larval neuroblast cells of D. simulans is diffuse and does not overlap specifically with D1 (green). Scale bars represent 2 µm.

Fig. 2. OdsH-binding sites are evolutionary labile in sibling species

(A) A schematic phylogeny of the recently diverged D. simulans, D. sechellia and D. mauritiana species. (B) Summary of localization studies (C–F) highlighting the differences in genomic localization of OdsHsim and OdsHmau to the Y and 4^th chromosomes. (C) Localization of FLAG-OdsHsim fusion protein on neuroblast mitotic chromosomes from D. mauritiana/D. simulans and (D) D. sechellia/D. simulans hybrid larvae (Fig. S2). (E) Localization of Venus-OdsHmau fusion protein on neuroblast mitotic chromosomes from D. mauritiana/D. simulans and (F) D. sechellia/D. simulans hybrid larvae. In all cases OdsH staining is red and D1 staining green. Only relevant X, Y, and 4th chromosomes are shown.

Fig. 3. Endogenous OdsH localization to G2 spermatocyte nuclei

(A) Whole-mount immunohistochemistry with an OdsH antibody on adult male testes from D. simulans wildtype, (B) D. simulans fertile introgression, (C) D. simulans sterile introgression, and (D) D. mauritiana. Scale bars represent 50 µm. An 8X zoom on the regions identified by the red dashed boxes is shown in the insets along with arrows pointing to OdsH protein. (E) OdsH protein expression was significantly expanded in the sterile line, measured by the length of detectable protein expression within the testes (p < 0.001). (F) Western blot from D. simulans wildtype, fertile introgression, and sterile introgression testes immunoblotted for OdsH (top panel) and β-tubulin control (bottom panel). (G) OdsH (red) detected by immunofluorescence in G2 primary spermatocyte nuclei from the adult testes of D. simulans wildtype and (H) D. simulans sterile introgression. DNA staining in blue. Scale bars represent 5 µm.