Stellate Genes and the piRNA Pathway in Speciation and Reproductive Isolation of Drosophila melanogaster

Abstract

One of the main conditions of the species splitting from a common precursor lineage is the prevention of a gene flow between diverging populations. The study of Drosophila interspecific hybrids allows to reconstruct the speciation mechanisms and to identify hybrid incompatibility factors that maintain post-zygotic reproductive isolation between closely related species. The regulation, evolution, and maintenance of the testis-specific Ste-Su(Ste) genetic system in Drosophila melanogaster is the subject of investigation worldwide. X-linked tandem testis-specific Stellate genes encode proteins homologous to the regulatory β-subunit of protein kinase CK2, but they are permanently repressed in wild-type flies by the piRNA pathway via piRNAs originating from the homologous Y-linked Su(Ste) locus. Derepression of Stellate genes caused by Su(Ste) piRNA biogenesis disruption leads to the accumulation of crystalline aggregates in spermatocytes, meiotic defects and male sterility. In this review we summarize current data about the origin, organization, evolution of the Ste-Su(Ste) system, and piRNA-dependent regulation of Stellate expression. The Ste-Su(Ste) system is fixed only in the D. melanogaster genome. According to our hypothesis, the acquisition of the Ste-Su(Ste) system by a part of the ancient fly population appears to be the causative factor of hybrid sterility in crosses of female flies with males that do not carry Y-linked Su(Ste) repeats. To support this scenario, we have directly demonstrated Stellate derepression and the corresponding meiotic disorders in the testes of interspecies hybrids between D. melanogaster and D. mauritiana. This finding embraces our hypothesis about the contribution of the Ste-Su(Ste) system and the piRNA pathway to the emergence of reproductive isolation of D. melanogaster lineage from initial species.

Keywords: Drosophila; Stellate genes; hybrid sterility; piRNA pathway; reproductive isolation.

Graphical Abstract

The Stellate-Su(Ste) genetic system is maintained only in the D. melanogaster genome. X-linked testis-specific Stellate genes are repressed in the testes of wild-type flies with the aid of piRNAs derived from the homologous Y-linked Su(Ste) locus that is essential for male fertility. Derepression of Stellate genes in the testes of hybrids of D. melanogaster with closely related D. mauritiana leads to the crystalline aggregate formation and causes strong meiotic failures and male hybrid sterility. Thus, the Stellate-Su(Ste) system and the piRNA pathway contribute to the emergence of the reproductive isolation of D. melanogaster from initial species.

Figure 1

General scheme of Stellate and Su(Ste) repeats. Promoters are indicated by a blue color bar, introns are depicted by green lines, intergenic spacers are depicted by gray lines. Stellate gene contains an ORF (brown color bar) and two introns (green lines). An individual Su(Ste) repeat carries the region homologous to the Stellate ORF (brown color bar), Y-specific region (orange bar) and an insertion of the defective hoppel transposon (violet bar) flanked by inverted repeats (not shown) in the promoter. Start sites of sense transcription of Stellates and Su(Ste) and multiple starts of antisense Su(Ste) transcription within the body of hoppel are indicated by black arrows [modified from Aravin et al. (2001)].

Figure 2

Distribution of derepressed Stellate protein in the testes of D. melanogaster. (A–C) Internal confocal slices of stained testis preparation of cry¹ males (A,C) and wild-type control (B). Testes were immunofluorescently stained with anti-Stellate (green) and anti-lamin (red) antibodies, chromatin was stained with DAPI (cyan). Anti-lamin staining indicates nuclear membrane position. (A,C) Diffuse Stellate signals in the nuclei (arrows in A) and bright needle-like and dot-like crystalline Stellate aggregates mainly in the cytoplasm are seen in spermatocytes of cry¹ males. (C) The nuclei of mature spermatocytes. (D) 3D reconstruction of the stained testis preparation of cry¹ males. (A–C) are reproduced from Figure 2 in Egorova et al. (2009). (D) is reproduced from Figure 2 in Kibanov et al. (2013) by permission of Elsevier (Licenses ## 4913121387410 and 4913131090753).

Figure 3

Reconstruction of the basic steps of the origin and evolution of Ste-Su(Ste) repeats in the D. melanogaster genome. Non-homologous recombination between the promoter of βNACtes genes and Y-linked βCK2tes copy led to the formation of a chimeric intermediate. Subsequently, the ancestral copies with the acquired promoters were amplified in the sex chromosomes. Insertion of the hoppel transposon in the Su(Ste) promoter region allowed the acquisition of Stellate repressor functions. The homologous sequences are marked by the same color. The model is developed with the usage and modification of ones from Usakin et al. (2005), Chang and Larracuente (2019).

Figure 4

The piRNA biogenesis in the nuage granules of Drosophila spermatocytes. (A) Long transcripts of Su(Ste) piRNA precursors are exported from the nucleus and are presumably cleaved by endonuclease Zucchini located on the outer membrane of mitochondria, forming the 5′-end of the future piRNA. The cleaved transcript is loaded into Aubergine, and then trimmed from the 3′-end by an unknown trimmer nuclease. Aubergine loaded with guide antisense piRNAs recognizes and cleaves the complementary Stellate mRNAs producing the 5′-end of a new sense piRNA. The new piRNA is loaded into AGO3 and in turn performs cleavage of the complementary Su(Ste) transcript. This step generates a new antisense piRNA that is identical or very similar to the initiating piRNA (ping-pong cycle). (B) Vasa and Aubergine are colocalized in the periphery lobes of the piNG-body. (C) AGO3 is located in the central lobe of the piNG-body and does not colocalized with Vasa. (B,C) Confocal slices (left) and 3D images (right) of premeiotic spermatocytes in testis preparations are reproduced from Figure 3 in Kibanov et al. (2011) by permission of MBoC. Under the License and Publishing Agreement, authors grant to the general public, the non-exclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons-Non-commercial-Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0). Scale bars are 3 μm.

Figure 5

Derepression of Stellate genes in the testes of interspecies hybrids D. melanogaster with D. mauritiana. (A–C) Hybrid testes were stained with anti-Vasa (red) and anti-Stellate (green) antibodies, chromatin was stained with DAPI (cyan). 3D images are present for apical (A) and distal (B) testes ends; an internal confocal slice is present for the distal testis end stained with DAPI (C). Scale bars are 30 μm for (A) and (B), 15 μm for C. Stellate genes are derepressed in spermatocytes of hybrid testes and generate abundant crystals (A,B). Spermatids are absent at the distal end of hybrid testes (B). Giant conglomerations of decondensed chromatin (yellow arrows) are found at the distal ends of hybrid testis (C) indicating a severe meiotic catastrophe. (D) Derepession of Stellate genes in the testes of hybrids causes meiotic failures and male hybrid sterility. (A–D) Images are reproduced from Figure 6B in Kotov et al. (2019), and the scheme is adapted from Figure 7B in Kotov et al. (2019) by permission of Oxford University Press (http://global.oup.com/academic) without the need to obtain written permission from OUP and payment as authors of this publication.