Germ Cell Specification Requires Zygotic Mechanisms Rather Than Germ Plasm in a Basally Branching Insect

Department of Organismic and Evolutionary Biology,Harvard University, 16 Divinity Avenue, Cambridge,MA 02138, USASummaryBackground:Primordialgermcell(PGC)speciﬁcationisauni-versalprocessacrossanimals,butthemolecularmechanismsspecifying PGCs are remarkably diverse. In Drosophila, PGCsarespeciﬁedbymaternallyprovided,asymmetricallylocalizedcytoplasmic factors (germ plasm). In contrast, historical litera-ture on most other arthropods reports that PGCs arise frommesoderm during midembryogenesis, suggesting that anarthropod last common ancestor may have speciﬁed PGCsvia zygotic mechanisms. However, there has been no directexperimental evidence to date for germ plasm-independentarthropod PGC speciﬁcation.Results: Here we show that in a basally branching insect, thecricketGryllusbimaculatus,conserved germplasm moleculesare ubiquitously, rather than asymmetrically, localized duringoogenesis and early embryogenesis. Molecular and cytolog-ical analyses suggest that Gryllus PGCs arise from abdominalmesoderm during segmentation, and twist RNAi embryos thatlack mesoderm fail to form PGCs. Using RNA interference weshow that vasa and piwi are not required maternally or zygoti-cally for PGC formation but rather are required for primaryspermatogonial divisions in adult males.Conclusions: TheseobservationssuggestthatGrylluslacksamaternally inherited germ plasm, in contrast with many holo-metabolous insects, including Drosophila. The mesodermalorigin of Gryllus PGCs and absence of instructive roles forvasa and piwi in PGC formation are reminiscent of mousePGC speciﬁcation and suggest that zygotic cell signaling maydirect PGC speciﬁcation in Gryllus and other Hemimetabola.IntroductionOf the many specialized cell types that comprise an animal’sbody, only one is capable of contributing genetic informationto the next generation: the germ cells. The restriction of repro-ductive potential to a small subset of cells is a universalprocess across sexually reproducing animals and representsa profound evolutionary novelty likely required for the evolu-tion of multicellularity [1]. The molecular mechanisms thatspecify these cells, however, are remarkably diverse betweentaxa [2–5] and only well understood in a handful of modelorganisms.Primordial germ cell (PGC) speciﬁcation mechanisms havebeen categorized into two modes: cytoplasmic inheritanceand zygotic induction [3, 4, 6]. Cytoplasmic inheritance (e.g.,in Drosophila melanogaster) involves the localization ofmaternalmRNAsandproteins(germplasm)toasubcellularre-gion of the oocyte. Germ plasm is necessary and sufﬁcient toinduce PGC fate. In zygotic induction (e.g., in Mus musculus),by contrast, there is no germ plasm, and PGCs instead formin response to inductive signals from neighboring somaticcells [7].Within insects, cytoplasmic inheritance appears to be aderivedcharacterconﬁnedprimarilytotheholometabolousin-sects [8](Figure 1A; see also Table S1 available online), wheregerm plasm has been demonstrated experimentally in manyspecies (Table S1). Histological studies of insects branchingbasally to Holometabola (the Hemimetabola), in contrast,have reported the absence of both germ plasm and pole cellsinnearlyallofthesetaxa[3,6](Figure1A;TableS1).Studies ofmolecular markers for PGCs in hemimetabolous insects havebeen limited to the highly atypical parthenogenetic embryosof the pea aphid Acyrthosiphon pisum, a milkweed bug, andseveral orthopteran species (Table S1), yet there is no con-served pattern of PGC origin across these taxa.In this study, we use multiple conserved molecular markersand RNAi to characterize PGC formation in the cricket Gryllusbimaculatus (Orthoptera), a hemimetabolous model speciesfor studying the development of basally branching insects[9]. We provide several lines of evidence that Gryllus PGCsform from the abdominal mesoderm via inductive signalingand discuss the implications of these results for the evolutionof germ plasm and the possibility of an ancient relationshipbetween bilaterian PGCs and mesoderm.ResultsGryllus Germ Cells Express a Suite of Conserved GenesWithin the Orthoptera, neither germ plasm nor pole cells havebeenreported(Figure1A;TableS1).Histologicalexaminationsof orthopteran embryos conducted by William Wheeler over acentury ago [10] suggested that PGCs arise from or amongabdominal mesoderm cells during abdominal segmentation(Figure 1C), consistent with reports of germline origin both inother Hemimetabola and in most arthropods [3, 6]. However,conservedmolecularmarkerscanrevealacryptic germplasmthat eludes histological examinations [11–13]. We thereforeexamined the expression of several conserved molecularPGC markers (vasa [14], piwi [14], tudor, boule, and germcell-less) and three additional PIWI family genes (Figures S1Aand S1B) in Gryllus ovaries and embryos. Because somegerm plasm components localize as proteins rather than tran-scripts (see for example [15, 16, 17]), we also examined theexpression of Vasa and Piwi proteins [14].Infullysegmented(stage9)Gryllusembryos(Figures1Band1C), we identiﬁed cells matching Wheeler’s description [10]that express both mRNA and protein of piwi and vasa,aswellasbolandgcltranscripts(Figures1D,1E,andS1C).Thesecell clusters were found in abdominal segments A2–A3 in allembryos, and in A4–A5 in 45% of embryos (Figures 1D and1E, arrowheads). Clusters were located on the dorsal medialface of mesodermal structures termed ‘‘coelomic pouches,’’which are present in every gnathal, thoracic, and abdominal