The phylogenetic origin of oskar coincided with the origin of maternally provisioned germ plasm and pole cells at the base of the Holometabola

Abstract

The establishment of the germline is a critical, yet surprisingly evolutionarily labile, event in the development of sexually reproducing animals. In the fly Drosophila, germ cells acquire their fate early during development through the inheritance of the germ plasm, a specialized maternal cytoplasm localized at the posterior pole of the oocyte. The gene oskar (osk) is both necessary and sufficient for assembling this substance. Both maternal germ plasm and oskar are evolutionary novelties within the insects, as the germline is specified by zygotic induction in basally branching insects, and osk has until now only been detected in dipterans. In order to understand the origin of these evolutionary novelties, we used comparative genomics, parental RNAi, and gene expression analyses in multiple insect species. We have found that the origin of osk and its role in specifying the germline coincided with the innovation of maternal germ plasm and pole cells at the base of the holometabolous insects and that losses of osk are correlated with changes in germline determination strategies within the Holometabola. Our results indicate that the invention of the novel gene osk was a key innovation that allowed the transition from the ancestral late zygotic mode of germline induction to a maternally controlled establishment of the germline found in many holometabolous insect species. We propose that the ancestral role of osk was to connect an upstream network ancestrally involved in mRNA localization and translational control to a downstream regulatory network ancestrally involved in executing the germ cell program.

Figure 1. Current understanding of the distribution of maternal germ plasm, pole cells, and oskar orthologs in the insects.

Genus names in blue are those in which maternal germ plasm and pole cells have been described. Asterisks indicate a sequenced genome. Green boxes indicate confirmed presence of osk. Red boxes indicate apparent absence of osk in the genome. Dashed green box indicates the hypothesis that species with posteriorly localized maternal germ plasm and pole cells require a factor with Osk-like function and regulation.

Figure 2. Sequence features of Nv-Osk protein.

CLUSTALW generated alignment of D. melanogaster and N. vitripennis Osk proteins. Red text is the fly long-Osk specific region. Blue indicates the putative LASP binding domain of fly Osk. Pink text indicates the Lotus/Tejas homology domain. The characterized missense mutations in fly osk were mapped on the alignment, and were categorized as follows: green shaded residues are those that are conserved between wasp and fly Osk, but are not conserved in the mosquito sequences (osk2). Red shaded residues are conserved in wasp, mosquito, and fly (osk6B10 and osk 5+6). Pink shading indicates residues that are conserved between wasp and mosquito Osk, but not in Drosophila (osk8). Finally, light blue shaded residues are conserved between mosquitoes and fly, but not in the wasp (osk3 and osk7). Orange boxes delineate the putative hydrolase homology domains in Drosophila and Nasonia Osk.

Figure 3. Expression of Nv-osk during oogenesis and embryogenesis.

During oogenesis (A–C) and embryogenesis (D–G). A, A′: Expression of Nv-osk (green) and Nv-nos (red) in early oogenesis. Arrows mark oocyte. B: Later stage of oogenesis, after completion of encapsulation of the oocyte by follicle cells. nc = nurse cells. C: Toward the end of oogenesis, most Nv-osk mRNA is rapidly dumped from the nurse cells into the oocyte (compare lower egg chamber to the upper). D: Embryo in division cycle 2–3 stained for Nv-osk. E: Embryo just before syncytial blasotoderm formation. F: Embryo in early syncytial blastoderm stage. G: Embryo just before cellularization of the blastoderm. Scale bars = 100 micrometers.

Figure 4. Effects of Nv-osk pRNAi during oogenesis.

A: Wild type Nasonia ovariole stained with Nv-otd1 (green) Nv-nos (red), and DAPI (blue). B: Strong Nv-osk pRNAi knockdown , very few mature egg chambers are formed. C,D: In weaker Nv-osk pRNAi knockdowns the linear arrangement of egg-chambers is severely disrupted. Egg chambers in reverse orientation (arrowhead) or perpendicular to the AP axis of the ovariole (arrows) are observed. Within the oocytes, axial polarity (asterisks) and mRNA localization (arrowhead in C) defects occur. E, E′: In wild type, Nv-Vas protein is not localized in young oocytes (E′) even though high levels of Nv-osk mRNA are localized at the posterior pole (E). Nv-Vas protein appears to be concentrated on the surface of the most anterior nurse cell nuclei. F, F′: Nv-osk mRNA (F) and Nv-Vas (F′) accumulation late in oogenesis. G, G′: Expression of Nv-osk (G) and Nv-Vas (G′) after Nv-osk pRNAi.

Figure 5. Effects of Nv-osk pRNAi during embryogenesis.

A: Wild type localization of Nv-nos (red) and Nv-otd1 (green) mRNA in early embryogenesis. B, C: Expression of Nv-nos and Nv-otd1 in early embryos after Nv-osk pRNAi. D: Wild type expression of Nv-nos and Nv-otd1 just after pole cell formation. E: Expression of Nv-nos and Nv-otd1 in an Nv-osk pRNAi embryo at a stage similar to D. F: Expression of Nv-nos and Nv-otd1 in Nv-osk pRNAi embryo just before cellularization.

Figure 6. Function of Nv-vas and Nv-tud in oosome formation and Nv-osk localization.

A, A′: After Nv-vas pRNAi, late oocytes show a looser localization of Nv-osk mRNA at the posterior pole, and no accumulation of Nv-Vas is seen in the oocyte (compare to wild type in Figure 4F, 4F′). B, B′: After Nv-tud pRNAi, the polarity of the egg chambers within the ovariole can often be disturbed. In spite of this, Nv-Vas still accumulates at the posterior pole, and Nv-osk mRNA localization appears normal. C: Wild type expression of Nv-osk during early syncytial divisions. D: Wild type Nv-osk expression just after pole cell formation. E: Nv-osk expression in early Nv-vas pRNAi embryo. F: Nv-vas pRNAi embryo at stage similar to D. G: Early cleavage stage Nv-tud pRNAi embryo. H: Nv-tud pRNAi early blastoderm embryo.

Figure 7. Function of RNA binding proteins in oosome assembly in Nasonia.

A: Wild type ovarian expression of Nv-otd1 (green) and Nv-osk (red). A′: DIC optical cross section of same egg chambers in A (red = Nv-osk). B, B′: Large, dense particles containing Nv-otd1 and Nv-osk mRNA often observed within the nurse cells after Nv-bruno pRNAi. C, C′: Nv-osk and Nv-otd1 mRNAs are sometimes concentrated in smaller particles on the surface of the posteriormost nurse cells. D, D′: Ectopic co-localization of Nv-osk and Nv-Vas in nurse cells after Nv-bruno RNAi. E, F: Nv-hrp48 pRNAi disrupts the normally tight localization of posteriorly localized mRNAs of Nv-otd1 and Nv-osk. In extreme cases (arrows in F) these mRNAs are completely delocalized. G, G′: Nv-hrp48 pRNAi only weakly affects Nv-Vas accumulation in Nasonia oocytes, despite the looser localization of oosome to the posterior.

Figure 8. Oskar and oosomes in other Holometabolan species.

A, B: An oskar ortholog is present in the ant Messor pergandi, and is localized posteriorly in an oosome-like structure during oogenesis, and is localized posteriorly during embryogenesis. C: Vas expression in early Acanthoscelides oogenesis. nc = nurse cells, tc = trophic cords oo = oocyte. D: Vas localization in a late Acanthoscelides oocyte. E: Vas expression in early Tribolium oogenesis. F: Vas expression in a late Tribolium oocyte.

Figure 9. Phylogenetic pattern of losses and gains of maternal germ plasm, pole cells, and oskar among the insects.

Genus names in blue are those in which maternal germ plasm and pole cells have been described. Asterisks indicate a sequenced genome. Green boxes indicate confirmed presence of osk. Red boxes indicate apparent absence of osk in the genome. Orange arrow indicates the ancestral use of zygotic induction of germline fate among insects. Green circles and squares indicate the proposed evolutionary origin of osk and maternally synthesized germ plasm, while red circles and squares indicate the proposed loss of these features, respectively. Tree was drawn based on the phylogenetic relationships described in , , .