Hydractinia symbiolongicarpus is a powerful model for stem cell research and maintains a population of pluripotent adult stem cells throughout its lifetime. Here we describe a gene expression-agnostic FACS technique to isolate a live cell population from Hydractinia feeding polyps that appear to be stem cells. This technique utilizes only the general cellular component stains DAPI, DRAQ5, Calcein AM, and Pyronin Y. The stem cell population was identified via subtractive gating based on samples whose stem cell populations had been selectively depleted with the DNA-alkylating agent Mitomycin C. To validate the identity of the isolated population, a colorimetric cytological assay capable of simultaneously discriminating between all major Hydractinia cell types in a live-dissociated cell solution was developed using May-Grünwald and Giemsa stains. The isolated cell population was significantly depleted by Mitomycin C administration, had a high RNA content, was proliferative, had a cytological profile that matched that of Piwi1+ stem cells, and was ~10x enriched with Piwi1+ stem cells compared to whole cell suspension, all of which support the conclusion that the isolated population is indeed comprised of stem cells. This gene-agnostic FACS technique will serve future research into Hydractinia stem cell biology by enabling the use of isolated populations of live stem cells in transplantation, cell culture, and spheroid experimentation, and may serve as a reference for the development of new methods in other cnidarian species.

Motivation:The colonial hydroid Hydractinia exhibits several unique biological properties, including its remarkable regenerative capacity and the ability to distinguish self from non-self, characteristics that make them valuable models for studying human disease and aging. The availability of well-annotated multi-omic data, as well as tools to visualize these data, is essential for advancing the use of these model organisms to enhance our understanding of the relationship between genomic and morphological complexity, the evolution of multicellularity, and the emergence of novel cell types. Results:We present the Hydractinia Genome Project Portal, a comprehensive resource providing genomic, transcriptomic, and proteomic datasets for two widely studied Hydractinia species. The portal provides extensive sequence, structure, and functional annotation resources that are not available elsewhere, including genome browsers, a single-cell gene expression atlas, a protein structure viewer, and a custom BLAST implementation. We demonstrate the portal's utility for biological discovery and have used a subset of Hydractinia-specific stem cell gene markers to explore known gaps in annotation transfer methods, illustrating how structure-based deep learning methods such as DeepFRI can significantly improve the functional annotation of heretofore unannotated i-cell markers. Availability and implementation:The Hydractinia Genome Project Portal is freely available at https://research.nhgri.nih.gov/hydractinia.

The third iteration of the Cnidarian Model Systems Meeting (Cnidofest) was held August 14–17th, 2024 at Lehigh University in Bethlehem, PA. The meeting featured presentations from laboratories representing 11 countries, covering a broad range of topics related to cnidarian species. The research highlighted diverse topics, with sessions focused on regeneration, evo-devo, genomics, symbiosis, cell biology, physiology, neurobiology, and development. A notable shift at this meeting was the extent to which established cnidarian model systems have caught up with the classical laboratory models such as Drosophila and vertebrates, with modern genomic, genetic, and molecular tools now routinely applied. In addition, more cnidarian systems are now being developed for functional studies by the community, enhancing our ability to gain fundamental insights into animal biology that are otherwise difficult in the complex bilaterian model systems. Together, the integration of cnidarian and bilaterian model systems provides researchers with a broader toolkit for selecting animal models best suited to address their specific biological questions.

Single-cell RNA sequencing (scRNA-seq) has revolutionized transcriptomic research, enabling the creation of detailed tissue, organ, and species-level atlases for model organisms. In Hydractinia, a cnidarian model for stem cell and regeneration studies, recent atlases have revealed key insights into cell types and developmental processes. However, these atlases remain limited in cell numbers and transcriptomic depth and cell type assignments were largely made in silico. Here, we present an updated Hydractinia single-cell atlas by integrating new datasets from fixed cells with previously published live-cell data. This expanded atlas captures over 47,000 cells from feeding polyps and stolon tissue, recovering and refining major somatic cell lineages including cnidocytes, neurons, gland cells, epithelial cells, and stem cells (i-cells), as well as identifying a novel population of putative immune cells. We investigated the spatial expression patterns of selected marker genes and validated all major cell types and several cell states. Our analyses uncovered a previously undescribed neural subtype, two spatially distinct gland cell populations, a stolon-specific cell type, and a putative immune cell cluster. Additionally, we recovered and explored a complete Hydractinia cnidocyte trajectory with two distinct endpoints, supported by spatial marker gene expression that reflects the developmental progression of cnidoblasts as they mature and migrate towards the tentacles. Subclustering of somatic i-cells revealed putative progenitor states and a potential population of true stem cells. Together, this atlas significantly advances our understanding of Hydractinia cellular diversity and dynamics, allowing us to generate new hypotheses and provide a valuable resource for the cnidarian research community and beyond.

For over a century, the colonial cnidarian Hydractinia has been employed as a research organism to study stem cells, germ cells, regeneration, and coloniality. For the last 70 years, it has also been used in allorecognition research within the field of comparative immunology. More recently, other aspects of Hydractinia biology, including sensory biology, have been explored. Hydractinia colonies are composed of a limited number of repeating structural units: polyps and the stolon tissue that connects polyps. Polyps are divided into three major types used for feeding, reproduction, or defense. Clonal lines grown in the lab provide unlimited material from a single genetic unit. Colonies have separate sexes and spawn regularly with exposure to light. Recently, genomic and transcriptomic resources have been released for two species of Hydractinia: H. symbiolongicarpus and H. echinata. Tools for gene expression manipulation have been developed for this organism, including CRISPR/Cas9 knockout, shRNA knockdown, and overexpression via synthetic RNA. Fluorescent transgenic reporter lines have been created via random integration of circular DNA plasmids and CRISPR/Cas9-mediated gene knockin. We recently constructed an updated single-cell transcriptomic atlas of adult Hydractinia colonies to explore the cellular biology and cell-type expression profiles of the animal. We are investigating known and novel cell types and validating spatial expression patterns of cell-type specific markers to enable further understanding of the animal's cellular biology. This includes gaining a deeper understanding of the genetic control of cell differentiation of specific cell types from progenitor populations and uncovering the diversity of transcriptional subtypes that may be relevant to specific functions. Since Hydractinia is a model for whole-body regeneration, the identification and validation of new cell type and cell state markers will now allow for the elucidation of potential pathways involved in regenerating specific cell types, including testing alternative pathways for regeneration that include dedifferentiation and transdifferentiation. Hydractinia is poised to become a model for sensory biology research, as we can now fully explore their sensory cell types, including cnidocytes and neurons, and the expression and evolution of their gene complement with modern approaches and tools.

Adult pluripotent stem cells are found in diverse animals, including cnidarians, acoels, and planarians, and confer remarkable abilities such as whole-body regeneration. The mechanisms by which these pluripotent stem cells orchestrate the replacement of all lost cell types, however, remains poorly understood. Underlying heterogeneity within the stem cell populations of these animals is often obscured when focusing on certain tissue types or life history stages, which tend to have indistinguishable spatial expression patterns of stem cell marker genes. Here, we focus on the adult pluripotent stem cells (i-cells) of Hydractinia symbiolongicarpus , a colonial marine cnidarian with distinct polyp types and stolonal tissue. Recently, a single-cell expression atlas was generated for H. symbiolongicarpus which revealed two distinct clusters with i-cell signatures, potentially representing heterogeneity within this species’ stem cell population. Considering this finding, we investigated eight new putative stem cell marker genes from the atlas including five expressed in both i-cell clusters ( Pcna , Nop58 , Mcm4 , Ubr7 , and Uhrf1 ) and three expressed in one cluster or the other ( Pter, FoxQ2-like, and Zcwpw1 ). We characterized their expression patterns in various contexts – feeding and sexual polyps, juvenile feeding polyps, stolon, and during feeding polyp head regeneration – revealing context-dependent gene expression patterns and a transcriptionally dynamic i-cell population. We uncover previously unknown differences within the i-cell population of Hydractinia and demonstrate that its colonial nature serves as an excellent system for investigating and visualizing heterogeneity in pluripotent stem cells. ### Competing Interest Statement The authors have declared no competing interest.

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.

Hydractinia is a colonial marine hydroid that exhibits remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, H. symbiolongicarpus and H. echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from non-self.

Sex determination occurs across animal species, but most of our knowledge about its mechanisms comes from only a handful of bilaterian taxa. This limits our ability to infer the evolutionary history of sex determination within animals. In this study, we generated a linkage map of the genome of the colonial cnidarian Hydractinia symbiolongicarpus and used it to demonstrate that this species has an XX/XY sex determination system. We demonstrate that the X and Y chromosomes have pseudoautosomal and non-recombining regions. We then use the linkage map and a method based on the depth of sequencing coverage to identify genes encoded in the non-recombining region and show that many of them have male gonad-specific expression. In addition, we demonstrate that recombination rates are enhanced in the female genome and that the haploid chromosome number in Hydractinia is n = 15. These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination and the evolution of sex chromosomes.

N6-methyldeoxyadenosine (6mA) is a chemical alteration of DNA, observed across all realms of life. Although the functions of 6mA are well understood in bacteria and protists, its roles in animal genomes have been controversial. We show that 6mA randomly accumulates in early embryos of the cnidarian Hydractinia symbiolongicarpus, with a peak at the 16-cell stage followed by clearance to background levels two cell cycles later, at the 64-cell stage-the embryonic stage at which zygotic genome activation occurs in this animal. Knocking down Alkbh1, a putative initiator of animal 6mA clearance, resulted in higher levels of 6mA at the 64-cell stage and a delay in the initiation of zygotic transcription. Our data are consistent with 6mA originating from recycled nucleotides of degraded m6A-marked maternal RNA postfertilization. Therefore, while 6mA does not function as an epigenetic mark in Hydractinia, its random incorporation into the early embryonic genome inhibits transcription. In turn, Alkbh1 functions as a genomic 6mA "cleaner," facilitating timely zygotic genome activation. Given the random nature of genomic 6mA accumulation and its ability to interfere with gene expression, defects in 6mA clearance may represent a hitherto unknown cause of various pathologies.

The second annual Cnidarian Model Systems Meeting, aka "Cnidofest", took place in Davis, California from 7 to 10th of September, 2022. The meeting brought together scientists using cnidarians to study molecular and cellular biology, development and regeneration, evo-devo, neurobiology, symbiosis, physiology, and comparative genomics. The diversity of topics and species represented in presentations highlighted the importance and versatility of cnidarians in addressing a wide variety of biological questions. In keeping with the spirit of the first meeting (and its predecessor, Hydroidfest), almost 75% of oral presentations were given by early career researchers (i.e., graduate students and postdocs). In this review, we present research highlights from the meeting.

N6-methyldeoxyadenosine (6mA) is a chemical alteration of DNA, observed across all realms of life. The functions of 6mA are well understood in bacteria but its roles in animal genomes have been controversial. We show that 6mA randomly accumulates in early embryos of the cnidarian Hydractinia symbiolongicarpus , with a peak at the 16-cell stage followed by clearance to background levels two cell cycles later, at the 64-cell stage – the embryonic stage at which zygotic genome activation occurs in this animal. Knocking down Alkbh1 , a putative initiator of animal 6mA clearance, resulted in higher levels of 6mA at the 64-cell stage and a delay in the commencement of zygotic transcription. Our data are consistent with 6mA originating from recycled nucleotides of degraded m6A-marked maternal RNA post-fertilization. Therefore, while 6mA does not function as an epigenetic mark in Hydractinia , its random incorporation into the early embryonic genome inhibits transcription. Alkbh1 functions as a genomic 6mA ‘cleaner’, facilitating timely zygotic genome activation. Given the random nature of genomic 6mA accumulation and its ability to interfere with gene expression, defects in 6mA clearance may represent a hitherto unknown cause of various pathologies.

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/ .

Most colonial marine invertebrates are capable of allorecognition, the ability to distinguish between themselves and conspecifics. One long-standing question is whether invertebrate allorecognition genes are homologous to vertebrate histocompatibility genes. In the cnidarian Hydractinia symbiolongicarpus, allorecognition is controlled by at least two genes, Allorecognition 1 ( Alr1 ) and Allorecognition 2 ( Alr2 ), which encode highly polymorphic cell-surface proteins that serve as markers of self. Here, we show that Alr1 and Alr2 are part of a family of 41 Alr genes, all of which reside in a single genomic interval called the Allorecognition Complex (ARC). Using sensitive homology searches and highly accurate structural predictions, we demonstrate that the Alr proteins are members of the immunoglobulin superfamily (IgSF) with V-set and I-set Ig domains unlike any previously identified in animals. Specifically, their primary amino acid sequences lack many of the motifs considered diagnostic for V-set and I-set domains, yet they adopt secondary and tertiary structures nearly identical to canonical Ig domains. Thus, the V-set domain, which played a central role in the evolution of vertebrate adaptive immunity, was present in the last common ancestor of cnidarians and bilaterians. Unexpectedly, several Alr proteins also have immunoreceptor tyrosine-based activation motifs and immunoreceptor tyrosine-based inhibitory motifs in their cytoplasmic tails, suggesting they could participate in pathways homologous to those that regulate immunity in humans and flies. This work expands our definition of the IgSF with the addition of a family of unusual members, several of which play a role in invertebrate histocompatibility.

Nucleostemin (NS) is a vertebrate gene preferentially expressed in stem and cancer cells, which acts to regulate cell cycle progression, genome stability and ribosome biogenesis. NS and its paralogous gene, GNL3-like (GNL3L), arose in the vertebrate clade after a duplication event from their orthologous gene, G protein Nucleolar 3 (GNL3). Research on invertebrate GNL3, however, has been limited. To gain a greater understanding of the evolution and functions of the GNL3 gene, we have performed studies in the hydrozoan cnidarian Hydractinia symbiolongicarpus, a colonial hydroid that continuously generates pluripotent stem cells throughout its life cycle and presents impressive regenerative abilities. We show that Hydractinia GNL3 is expressed in stem and germline cells. The knockdown of GNL3 reduces the number of mitotic and S-phase cells in Hydractinia larvae of different ages. Genome editing of Hydractinia GNL3 via CRISPR/Cas9 resulted in colonies with reduced growth rates, polyps with impaired regeneration capabilities, gonadal morphological defects, and low sperm motility. Collectively, our study shows that GNL3 is an evolutionarily conserved stem cell and germline gene involved in cell proliferation, animal growth, regeneration and sexual reproduction in Hydractinia, and sheds new light into the evolution of GNL3 and of stem cell systems.

Invertebrate histocompatibility—also known as allorecognition—has long interested marine ecologists, population geneticists, evolutionary biologists, and immunologists, but its genetic basis remains enigmatic in most species. Here, we report the nearly complete sequence of a histocompatibility complex from the colonial cnidarian, Hydractinia symbiolongicarpus. This sequence reveals that the two known Hydractinia allorecognition genes, Allorecognition 1 (Alr1) and Allorecognition 2 (Alr2) are part of a large family of immunoglobulin superfamily (IgSF) genes, several of which are candidates for new allodeterminants. These genes encode transmembrane proteins with domain architectures similar to cell-adhesion molecules and immune receptors. Several also contain cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Our data, which include highly accurate protein structure predictions, reveal that these proteins have V-set and I-set domains with unusual sequence signatures. This suggests the last common ancestor of cnidarians and bilaterians had distinct V-set and I-set Ig domains.

Neurogenesis is the generation of neurons from stem cells, a process that is regulated by SoxB transcription factors (TFs) in many animals. Although the roles of these TFs are well understood in bilaterians, how their neural function evolved is unclear. Here, we use Hydractinia symbiolongicarpus , a member of the early-branching phylum Cnidaria, to provide insight into this question. Using a combination of mRNA in situ hybridization, transgenesis, gene knockdown, transcriptomics, and in vivo imaging, we provide a comprehensive molecular and cellular analysis of neurogenesis during embryogenesis, homeostasis, and regeneration in this animal. We show that SoxB genes act sequentially at least in some cases. Stem cells expressing Piwi1 and Soxb1 , which have broad developmental potential, become neural progenitors that express Soxb2 before differentiating into mature neural cells. Knockdown of SoxB genes resulted in complex defects in embryonic neurogenesis. Hydractinia neural cells differentiate while migrating from the aboral to the oral end of the animal, but it is unclear whether migration per se or exposure to different microenvironments is the main driver of their fate determination. Our data constitute a rich resource for studies aiming at addressing this question, which is at the heart of understanding the origin and development of animal nervous systems.