Stefan Siebert

The epithelial and interstitial stem cells of the freshwater polypHydraare the best-characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation inHydrais needed. To this end, we generated extensive new resources for characterizing transcriptional regulation inHydra, including new genome assemblies forHydra oligactisand the AEP strain ofHydra vulgaris, an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large kilobase-scale chromatin interaction domains in theHydragenome that contain transcriptionally coregulated genes. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types: isorhiza-type nematocytes and somatic gonad ectoderm. Finally, we identified novel candidate regulators of cell type–specific transcription, several of which have likely been conserved at least since the divergence ofHydraand the jellyfishClytia hemisphaericamore than 400 million years ago.

The epithelial and interstitial stem cells of the freshwater polyp Hydra are the best characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation in Hydra is needed. To this end, we generated extensive new resources for characterizing transcriptional regulation in Hydra , including new genome assemblies for Hydra oligactis and the AEP strain of Hydra vulgaris , an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large chromatin interaction domains in the Hydra genome that likely influence transcriptional regulation in a manner distinct from topologically associating domains in bilaterians. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types, isorhiza-containing nematocytes and somatic gonad ectoderm. We identified novel candidate regulators of cell-type-specific transcription, several of which have likely been conserved at least since the divergence of Hydra and the jellyfish Clytia hemisphaerica over 200 million years ago. The resources generated in this study, which collectively represent the most comprehensive characterization of transcriptional regulation in a cnidarian to date, are accessible through a newly created genome portal, available at research.nhgri.nih.gov/HydraAEP/ .

Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage (≥5 dpf). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA sequencing studies which identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells and interstitial stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, for select examples, we used gene knockout experiments to determine the role of newly identified genes. Our results reveal novel insights into ovarian development and function and the sequencing information will provide a valuable resource for future studies.

Background Siphonophores are complex colonial animals, consisting of asexually-produced bodies (called zooids) that are functionally specialized for specific tasks, including feeding, swimming, and sexual reproduction. Though this extreme functional specialization has captivated biologists for generations, its genomic underpinnings remain unknown. We use RNA-seq to investigate gene expression patterns in five zooids and one specialized tissue (pneumatophore) across seven siphonophore species. Analyses of gene expression across species present several challenges, including identification of comparable expression changes on gene trees with complex histories of speciation, duplication, and loss. Here, we conduct three analyses of expression. First, we examine gene expression within species. Then, we conduct classical analyses examining expression patterns between species. Lastly, we introduce Speciation Branch Filtering, which allows us to examine the evolution of expression in a phylogenetic framework. Results Within and across species, we identified hundreds of zooid-specific and species-specific genes, as well as a number of putative transcription factors showing differential expression in particular zooids and developmental stages. We found that gene expression patterns tended to be largely consistent in zooids with the same function across species, but also some large lineage-specific shifts in gene expression. Conclusions Our findings show that patterns of gene expression have the potential to define zooids in colonial organisms. We also show that traditional analyses of the evolution of gene expression focus on the tips of gene phylogenies, identifying large-scale expression patterns that are zooid or species variable. The new explicit phylogenetic approach we propose here focuses on branches (not tips) offering a deeper evolutionary perspective into specific changes in gene expression within zooids along all branches of the gene (and species) trees.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI–piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI–piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI–piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI–piRNA pathway.

Transposable elements (TEs) can damage genomes, thus organisms employ a variety of mechanisms to repress TE expression. However, these mechanisms often fail over time leading to de-repression of TEs in aging tissues. The PIWI-piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the epithelial stem cells of Hydra , a long-lived freshwater cnidarian. Hydra have three stem cell populations; endodermal and ectodermal epithelial stem cells are strictly somatic, whereas the interstitial stem cells retain germline competence. In our previous study, we found that the PIWI proteins are expressed in all three Hydra stem cell types. In this study, we focus on the ectodermal and endodermal epithelial stem cells to understand the somatic function of the pathway. We isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI-piRNA pathway represses TEs in Hydra epithelial stem cells. First, epithelial knockdown of the Hydra PIWI protein hywi resulted in upregulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Interestingly, we found that RNAi knockdown of hywi leads to an upregulation of genes involved in innate immunity, which may be in response to TE upregulation; this is consistent with recent studies on TE expression in mammalian cells. Altogether, this study suggests a function for the PIWI-piRNA pathway in maintaining the long-lived somatic cell lineages of Hydra and may point to a broader role for this pathway in protecting somatic tissue from TE-induced damage.

Mapping Hydra development cell by cell: Hydra continually renews all cells in its body using three stem cell populations. This feature of Hydra allowed Siebert et al. to identify the transcriptional signatures of stem cells, progenitors, and terminally differentiated cells using single-cell RNA sequencing of adult Hydra (see the Perspective by Reddien). From these data, they built differentiation trajectories for all cell lineages, identified gene modules expressed along these trajectories, and identified putative regulators of genes within these modules. In addition, they identified candidate markers for elusive cell populations (such as multipotent stem cells and germline stem cells) and built a molecular map of the nervous system. Science , this issue p. eaav9314 ; see also p. 314

The adult Hydra polyp continuously renews all of its cells using three separate stem cell populations, but the genetic pathways enabling homeostatic tissue maintenance are not well understood. We used Drop-seq to sequence transcriptomes of 24,985 single Hydra cells and identified the molecular signatures of a broad spectrum of cell states, from stem cells to terminally differentiated cells. We constructed differentiation trajectories for each cell lineage and identified the transcription factors expressed along these trajectories, thus creating a comprehensive molecular map of all developmental lineages in the adult animal. We unexpectedly found that neuron and gland cell differentiation transits through a common progenitor state, suggesting a shared evolutionary history for these secretory cell types. Finally, we have built the first gene expression map of the Hydra nervous system. By producing a comprehensive molecular description of the adult Hydra polyp, we have generated a resource for addressing fundamental questions regarding the evolution of developmental processes and nervous system function.