Differentiation trajectories of the Hydra nervous system reveal transcriptional regulators of neuronal fate.

The small freshwater cnidarian polyp Hydra vulgaris uses adult stem cells (interstitial stem cells) to continually replace neurons throughout its life. This feature, combined with the ability to image the entire nervous system (Badhiwala et al., 2021; Dupre & Yuste, 2017) and availability of gene knockdown techniques (Juliano, Reich, et al., 2014; Lohmann et al., 1999; Vogg et al., 2022), makes Hydra a tractable model for studying nervous system development and regeneration at the whole-organism level. In this study, we use single-cell RNA sequencing and trajectory inference to provide a comprehensive molecular description of the adult nervous system. This includes the most detailed transcriptional characterization of the adult Hydra nervous system to date. We identified eleven unique neuron subtypes together with the transcriptional changes that occur as the interstitial stem cells differentiate into each subtype. Towards the goal of building gene regulatory networks to describe Hydra neuron differentiation, we identified 48 transcription factors expressed specifically in the Hydra nervous system, including many that are conserved regulators of neurogenesis in bilaterians. We also performed ATAC-seq on sorted neurons to uncover previously unidentified putative regulatory regions near neuron-specific genes. Finally, we provide evidence to support the existence of transdifferentiation between mature neuron subtypes and we identify previously unknown transition states in these pathways. All together, we provide a comprehensive transcriptional description of an entire adult nervous system, including differentiation and transdifferentiation pathways, which provides a significant advance towards understanding mechanisms that underlie nervous system regeneration.

A chromosome-scale epigenetic map of the Hydra genome reveals conserved regulators of cell state.

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 kilobase-scale chromatin interaction domains in the Hydra genome 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 of Hydra and the jellyfish Clytia hemisphaerica more than 400 million years ago.

Generic injuries are sufficient to induce ectopic Wnt organizers in Hydra.

During whole-body regeneration, a bisection injury can trigger two different types of regeneration. To understand the transcriptional regulation underlying this adaptive response, we characterized transcript abundance and chromatin accessibility during oral and aboral regeneration in the cnidarian Hydra vulgaris. We found that the initial response to amputation at both wound sites is identical and includes widespread apoptosis and the activation of the oral-specifying Wnt signaling pathway. By 8 hr post amputation, Wnt signaling became restricted to oral regeneration. Wnt pathway genes were also upregulated in puncture wounds, and these wounds induced the formation of ectopic oral structures if pre-existing organizers were simultaneously amputated. Our work suggests that oral patterning is activated as part of a generic injury response in Hydra, and that alternative injury outcomes are dependent on signals from the surrounding tissue. Furthermore, Wnt signaling is likely part of a conserved wound response predating the split of cnidarians and bilaterians.

PIWI-piRNA pathway-mediated transposable element repression in Hydra somatic stem cells.

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.

Stem cell differentiation trajectories in Hydra resolved at single-cell resolution.

The adult Hydra polyp continually renews all of its cells using three separate stem cell populations, but the genetic pathways enabling this homeostatic tissue maintenance are not well understood. We sequenced 24,985 Hydra single-cell transcriptomes 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 gene modules and putative regulators expressed along these trajectories, thus creating a comprehensive molecular map of all developmental lineages in the adult animal. In addition, we built a gene expression map of the Hydra nervous system. Our work constitutes a resource for addressing questions regarding the evolution of metazoan developmental processes and nervous system function.