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. 2017 Dec 2;14(12):1727-1741.
doi: 10.1080/15476286.2017.1349048. Epub 2017 Sep 13.

Characterization of the piRNA Pathway During Development of the Sea Anemone Nematostella Vectensis

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Free PMC article

Characterization of the piRNA Pathway During Development of the Sea Anemone Nematostella Vectensis

Daniela Praher et al. RNA Biol. .
Free PMC article

Abstract

PIWI-interacting RNAs (piRNAs) and associated proteins comprise a conserved pathway for silencing transposons in metazoan germlines. piRNA pathway components are also expressed in multipotent somatic stem cells in various organisms. piRNA functions have been extensively explored in bilaterian model systems, however, comprehensive studies in non-bilaterian phyla remain limited. Here we investigate the piRNA pathway during the development of Nematostella vectensis, a well-established model system belonging to Cnidaria, the sister group to Bilateria. To date, no population of somatic stem cells has been identified in this organism, despite its long life-span and regenerative capacities that require a constant cell-renewal. We show that Nematostella piRNA pathway components are broadly expressed in early developmental stages, while piRNAs themselves show differential expression, suggesting specific developmental roles of distinct piRNA families. In adults, piRNA associated proteins are enriched in the germline but also expressed in somatic cells, indicating putative stem cell properties. Furthermore, we provide experimental evidence that Nematostella piRNAs cleave transposable elements as well as protein-coding genes. Our results demonstrate that somatic expression of piRNA associated proteins as well as the roles of piRNAs in transposon repression and gene regulation are likely ancestral features that evolved before the split between Cnidaria and Bilateria.

Keywords: Cnidaria; Nematostella vectensis; Piwi; Vasa; evolution; piRNA.

Figures

Figure 1.
Figure 1.
Phylogenetic relationship of Nematostella PIWI proteins. Phylogenetic tree of full sequences of metazoan PIWI proteins. Maximum likelihood was calculated with the LG model (+I, +G, +F). Bootstrap support values above 50% appear next to the respective node. Bayesian analysis was performed by the WAG model. Posterior probabilities (PP) are indicated by green (PP = 1), blue (0.95 ≤ PP < 1.0) or red (0.7 ≤ PP < 0.95) asterisks. Species abbreviations: Porifera (sponges): Aqu, Amphimedon queenslandica; Efl, Ephydatia fluviatilis; Ctenophora (comb jellies): Pba, Pleurobrachia bachei (sea gooseberry); Cnidaria: Ami, Acropora millepora (staghorn coral); Apa, Aiptasia pallida (brown anemone); Aau, Aurelia aurita (moon jelly); Che; Clytia hemisphaerica; Hvu; Hydra vulgaris (fresh water polyp); Nbi, Nanomia bijuga; Nve, Nematostella vectensis (starlet sea anemone); Pca, Podocoryne carnea. Lophotrochozoa: Lgi, Lottia gigantea (owl limpet); Vli, Villosa lienosa (little spectaclecase). Ecdysozoa: Bmo, Bombyx mori (silkworm); Dme, Drosophila melanogaster (fruit fly); Ptr, Portunus trituberculatus (horse crab). Vertebrata: Hsa, Homo sapiens (human); Dre, Danio rerio (zebrafish); Mmu, Mus musculus (mouse).
Figure 2.
Figure 2.
Spatiotemporal expression of transcripts of piRNA pathway proteins. Heat map displaying normalized read counts of homologs of bilaterian genes involved in the piRNA pathway created by direct quantification of transcripts from different developmental stages (A) and various body parts of adult animals (B) by the nCounter platform. Investigated stages include unfertilized egg (UE), blastula (BL), gastrula (GA), 2 d old (early) planula (2d), 4 d old (late) planula (4d), 6 d old metamorphosing planula (6d), 10 d old primary polyp (10d), 2 and 4 months old polyp (2m, 4m) and male and female adult (male and female). Body parts contained mesenteries, pharynx, tentacles and physa (B). Argonaute1 (AGO1), Argonaute2 (AGO2), Dicer1 (Dcr1), Dicer2 (Dcr2) and GW182 comprise the outgroup. Scale bar depicts fold change of expression.
Figure 3.
Figure 3.
piwi genes and Vasa2 exhibit broad expression domains in early developmental stages. Spatial expression patterns of Piwi1 (A-E), Piwi2 (F-J), ExPiwi1 (K-O) and Vasa2 (P-T) in early developmental stages ranging from blastula to primary polyp obtained by in situ hybridization. Lateral views, oral pole is oriented to the left. Scale bars represent 100 µm.
Figure 4.
Figure 4.
piwi and vasa genes are expressed in germ cells in adult animals. Spatial expression patterns of Piwi1, Piwi2, ExPiwi1, Vasa1 and Vasa2 in female (A, C, E, G, I) and male gonads (B, D, F, H, J) determined by in situ hybridization. Shown are mesenteries with the gonadal compartments. All genes except for ExPiwi1 are expressed in the early germ cells (early oocytes and cells at the margin of spermaries). Scale bars represent 100 µm.
Figure 5.
Figure 5.
Vasa2 protein accumulates around nuclei. (A), (E), (I) and (M) show schematic representations of a blastula (A), gastrula (E), planula (I) and a mesentery (M). Vasa2 antibody staining of a blastula (B-D), gastrula (F-H; oral view), planula (J-L; lateral view), cross sections of female (N-P) and male mesenteries with gonads (Q-S) are shown. The staining in blastulae (B) and gastrulae (F) is ubiquitously distributed throughout the embryos, whereas the planula (J) displays a stronger staining in the endoderm in the position of the future mesenteries. In female gonads only the early oocytes are Vasa2 positive (N). Male gonads show a strong staining in the marginal zone of the spermaries (Q). Counterstaining with DAPI reveals a perinuclear staining of Vasa2 (D, H, L, and S). The arrowhead in (D) indicates an M-phase chromosome with no detectable Vasa2 staining. White: Vasa2; red: DAPI. Abbreviations: ec, ectoderm; en, endoderm; g, gonad; m, mesentery; pm, parietal muscle; rm, ring muscle; sf, septal filament. Asterisks in (E), (F), (I) and (J) mark the oral pole. All pictures are single optical sections except for (N) displaying a z-projection. Scale bars in (B), (F), (J), (N) represent 50 µm, in (C), (G), (K), (R) 10 µm and in (Q) and (O) 100 µm.
Figure 6.
Figure 6.
Developmental regulation of Nematostella piRNA expression. (A) Heat map of piRNA cluster expression across developmental stages. Each row denotes one piRNA cluster, and the blue-red scale represents the standardized total reads that were assigned across developmental stages. (B) Plots of the first 3 principal components of individual piRNA enrichment across developmental stages, which account for 90.5% of the variation. Points cluster by developmental stage in all components, indicating the inter-stage variability is replicated. Principal components 1 and 2 separate early-adult and mid stages, respectively, whereas principal component 3 accounts for the differences between adult male and female piRNAs.
Figure 7.
Figure 7.
Targeting potential of Nematostella piRNAs. (A) Groups of Nematostella piRNAs significantly enriched for targeting TEs have distinct enrichment profiles during development. The number of associated piRNA loci contributing to the respective groups is depicted with an “n.” Group 3, group 6 and group 11 are characterized by enrichment of TE-targeting piRNAs in the primary polyp stage, with only group 10 reaching an average of more than 2-fold greater enrichment in the adult male stage. (B) Almost half of Nematostella piRNAs (45%) potentially target transposable elements. piRNA targets were classified into 4 classes: “intergenic regions,” “genes,” “transposons” and “both,” combining genes and transposons.

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