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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.

Hydractinia symbiolongicarpus is a pioneering model organism for stem cell biology, being one of only a few animals with adult pluripotent stem cells (known as i-cells). However, the unavailability of a chromosome-level genome assembly has hindered a comprehensive understanding of global gene regulatory mechanisms underlying the function and evolution of i-cells. Here, we report the first chromosome-level genome assembly of H. symbiolongicarpus (HSymV2.0) using PacBio HiFi long-read sequencing and Hi-C scaffolding. The final assembly is 483 Mb in total length with 15 chromosomes representing 99.8% of the assembly. Repetitive sequences were found to account for 296 Mb (61%) of the total genome; we provide evidence for at least two periods of repeat expansion in the past. A total of 25,825 protein-coding genes were predicted in this assembly, which include 93.1% of the metazoan Benchmarking Universal Single-Copy Orthologs (BUSCO) gene set. 92.8% (23,971 genes) of the predicted proteins were functionally annotated. The H. symbiolongicarpus genome showed a high degree of macrosynteny conservation with the Hydra vulgaris genome. This chromosome-level genome assembly of H. symbiolongicarpus will be an invaluable resource for the research community that enhances broad biological studies on this unique model organism.

Abstract 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.

Abstract Background 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. Results 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. Conclusions These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination and the evolution of sex chromosomes.

Background: 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. Results: 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. Conclusions: These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination and the evolution of sex chromosomes. [ABSTRACT FROM AUTHOR]

Hydractinia symbiolongicarpus is a colonial hydrozoan that displays a division of labor through morphologically distinct and functionally specialized polyp types. As with all cnidarians, their venoms are housed in nematocysts, which are scattered across an individual. Here, we investigate the spatial distribution of a specific protein family, jellyfish toxins, in which multiple paralogs are differentially expressed across the functionally specialized polyps. Jellyfish toxins (JFTs) are known pore-forming toxins in the venoms of medically relevant species such as box jellyfish (class Cubozoa), but their role in other medusozoan venoms is less clear. Utilizing a publicly available single-cell dataset, we confirmed that four distinct H. symbiolongicarpus JFT paralogs are expressed in nematocyst-associated clusters, supporting these as true venom components in H. symbiolongicarpus. In situ hybridization and immunohistochemistry were used to localize the expression of these JFTs across the colony. These expression patterns, in conjunction with known nematocyst type distributions, suggest that two of these JFTs, HsymJFT1c-I and HsymJFT1c-II, are localized to specific types of nematocysts. We further interpret JFT expression patterns in the context of known regions of nematogenesis and differential rates of nematocyst turnover. Overall, we show that JFT expression patterns in H. symbiolongicarpus are consistent with the subfunctionalization of JFT paralogs across a partitioned venom system within the colony, such that each JFT is expressed within a specific set of functionally distinct polyp types and, in some cases, specific nematocyst types. [ABSTRACT FROM AUTHOR]

• We report the characterization of two cnidarian trefoil factor genes and in the cnidarian model Hydractinia symbiolongicarpus. • We present a new gene encoding a protein with a TFF domain flanked by two ShKT domains which is overexpressed after an immune challenge. • The TFF gene is expressed mainly in the endodermal canals of H. symbiolongicarpus as a high-molecular complex, most likely together with mucin. Proteins of the trefoil factor family (TFF) participate in mucosal repair and are formed by single or tandemly repeated trefoil domains. TFFs have been extensively studied in mammals and amphibians, but they have not been functionally characterized in other animals. Here we report the identification of two genes expressed in the hydroid Hydractinia symbiolongicarpus , predicted to encode trefoil domain-containing peptides, one with four trefoil domains in tandem and the other one with a trefoil domain flanked by two ShKT domains. Differential expression analyses by qPCR after an immune challenge and an induced mechanical damage, reveal that the former gene (hysyTFF) had no significant changes in expression after the inductions. However, the latter (hysyTFF-like) was overexpressed after three hours post immune challenge and was downregulated after the first hour post epithelial damage. Immunoblot analyses using specific IgY antibodies revealed that hysyTFF is secreted as a high molecular weight complex. Finally, whole mount immunofluorescence assays showed that hysyTFF was predominantly expressed in the endoderm of stolons and polyps, and sparsely in the ectoderm of both polyps and larvae. Thus, the tissue distribution and expression dynamics of trefoil factor genes in H. symbiolongicarpus suggest that hysyTFF is part of an ancient mechanism of epithelial restitution, and the newly reported hysy TFF-like might act as an immune effector gene, perhaps encoding an antibacterial peptide. [ABSTRACT FROM AUTHOR]

The genetics of allorecognition has been studied extensively in inbred lines of Hydractinia symbiolongicarpus, in which genetic control is attributed mainly to the highly polymorphic loci allorecognition 1 (Alr1) and allorecognition 2 (Alr2), located within the Allorecognition Complex (ARC). While allelic variation at Alr1 and Alr2 can predict the phenotypes in inbred lines, these two loci do not entirely predict the allorecognition phenotypes in wild-type colonies and their progeny, suggesting the presence of additional uncharacterized genes that are involved in the regulation of allorecognition in this species. Comparative genomics analyses were used to identify coding sequence differences from assembled chromosomal intervals of the ARC and from genomic scaffold sequences between two incompatible H. symbiolongicarpus siblings from a backcross population. New immunoglobulin superfamily (Igsf) genes are reported for the ARC, where five of these genes are closely related to the Alr1 and Alr2 genes, suggesting the presence of multiple Alr-like genes within this complex. Complementary DNA sequence evidence revealed that the allelic polymorphism of eight Igsf genes is associated with allorecognition phenotypes in a backcross population of H. symbiolongicarpus, yet that association was not found between parental colonies and their offspring. Alternative splicing was found as a mechanism that contributes to the variability of these genes by changing putative activating receptors to inhibitory receptors or generating secreted isoforms of allorecognition proteins. Our findings demonstrate that allorecognition in H. symbiolongicarpus is a multigenic phenomenon controlled by genetic variation in at least eight genes in the ARC complex. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Hydractinia symbiolongicarpus is an emerging model organism in which cutting-edge genomic tools and resources are being developed for use in a growing number of research fields. One limitation of this model system is the lack of long-term storage for genetic resources. In this study, approaches for Hydractinia sperm cryopreservation were established for the first time. Open hardware and 3-D printing were used to facilitate animal husbandry, sperm handling, and cryopreservation. Hydractinia sperm at a concentration of 2 × 107 cells/mL stored at 4 °C for as long as 6 d were able to achieve 50% fertilization rate. A fertilization rate of 41–69% was observed using sperm equilibrated with 5, 10, or 15% (v/v) cryoprotectant (dimethyl sulfoxide or methanol) for 20 min, cooled at a rate of 5, 10, or 20 °C/min from 4 °C to −80 °C, at a cell concentration of 1 × 108 sperm/mL, in 0.25 mL French straws. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, these generalizable approaches can be used as a model to develop germplasm repositories for other cnidarian species. Hydractinia symbiolongicarpus is an emerging model organism in which cutting-edge genomic tools and resources are being developed for use in a growing number of research fields. One limitation of this model system is the lack of long-term storage for genetic resources. The goal of this study was to establish a generalizable cryopreservation approach for Hydractinia that would support future repository development for other cnidarian species. Specific objectives were to: (1) characterize basic parameters related to sperm quality; (2) develop a generalizable approach for sperm collection; (3) assess the feasibility of in vitro fertilization (IVF) with sperm after refrigerated storage; (4) assess the feasibility of IVF with sperm cryopreserved with various sperm concentrations; (5) evaluate feasibility of cryopreservation with various freezing conditions, and (6) explore the feasibility of cryopreservation by use of a 3-D printed open-hardware (CryoKit) device. Animal husbandry and sperm collection were facilitated by use of 3-D printed open hardware. Hydractinia sperm at a concentration of 2 × 107 cells/mL stored at 4 °C for 6 d were able to achieve 50% fertilization rate. It appeared that relatively higher sperm concentration (>5 × 107 cells/mL) for cryopreservation could promote fertilization. A fertilization rate of 41–69% was observed using sperm equilibrated with 5, 10, or 15% (v/v) cryoprotectant (dimethyl sulfoxide or methanol) for 20 min, cooled at a rate of 5, 10, or 20 °C/min from 4 °C to −80 °C, at a cell concentration of 108/mL, in 0.25 mL French straws. Samples cryopreserved with the CryoKit produced a fertilization rate of 72–82%. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, these generalizable approaches can be used as a model to develop germplasm repositories for other cnidarian species. [ABSTRACT FROM AUTHOR]

Cnidarians (jellyfish, hydroids, sea anemones, and corals) possess a unique method for venom production, maintenance, and deployment through a decentralized system composed of different types of venom-filled stinging structures called nematocysts. In many species, nematocyst types are distributed heterogeneously across functionally distinct tissues. This has led to a prediction that different nematocyst types contain specific venom components. The colonial hydrozoan, Hydractinia symbiolongicarpus, is an ideal system to study the functional distribution of nematocyst types and their venoms, given that they display a division of labor through functionally distinct polyps within the colony. Here, we characterized the composition and distribution of nematocysts (cnidome) in the different polyp types and show that the feeding polyp (gastrozooid) has a distinct cnidome compared to the reproductive (gonozooid) and predatory polyp (dactylozooid). We generated a nematocyst-specific reporter line to track nematocyst development (nematogenesis) in H. symbiolongicarpus, and were able to confirm that nematogenesis primarily occurs in the mid-region of the gastrozooid and throughout stolons (tubes of epithelia that connect the polyps in the colony). This reporter line enabled us to isolate a nematocyst-specific lineage of cells for de novo transcriptome assembly, annotate venom-like genes (VLGs) and determine differential expression (DE) across polyp types. We show that a majority of VLGs are upregulated in gastrozooids, consistent with it being the primary site of active nematogenesis. However, despite gastrozooids producing more nematocysts, we found a number of VLGs significantly upregulated in dactylozooids, suggesting that these VLGs may be important for prey-capture. Our transgenic Hydractinia reporter line provides an opportunity to explore the complex interplay between venom composition, nematocyst diversity, and ecological partitioning in a colonial hydrozoan that displays a division of labor.

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.

In situ hybridization allows the detection of nucleic acid sequences in fixed cells and tissues. The gelatinous nature of cnidarians and Hydractinia demands extensive and exhausting protocols to detect RNA transcripts with traditional methods (e.g., colorimetric in situ hybridization). Signal amplification by exchange reaction (SABER) fluorescence in situ hybridization (FISH) enables simplifying and multiplex imaging of RNA targets in a rapid and cost-effective manner. In one enzymatic reaction, SABER-FISH uses a strand-displacing polymerase and catalytic DNA hairpin to generate FISH probes with adjustable signal amplification, allowing highly sensitive detection of nucleic acids and reducing the number of required probes. Here I describe the methodology to detect transcripts within the cells of Hydractinia by SABER-FISH in whole-mount samples., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

An ancient evolutionary innovation of a novel cell type, the stinging cell (cnidocyte), appeared >600 million years ago in the phylum Cnidaria (sea anemones, corals, hydroids, and jellyfish). A complex bursting nano-injector of venom, the cnidocyst, is embedded in cnidocytes and enables cnidarians to paralyze their prey and predators, contributing to this phylum's evolutionary success. In this work, we show that post-transcriptional regulation by a pan-cnidarian microRNA, miR-2022, is essential for biogenesis of these cells in the sea anemone Nematostella vectensis. By manipulation of miR-2022 levels in a transgenic reporter line of cnidocytes, followed by transcriptomics, single-cell data analysis, prey paralysis assays, and cell sorting of transgenic cnidocytes, we reveal that miR-2022 enables cnidocyte biogenesis in Nematostella , while exhibiting a conserved expression domain with its targets in cnidocytes of other cnidarian species. Thus, here we revealed a functional basis to the conservation of one of nature's most ancient microRNAs. [Display omitted] • miR-2022 is crucial for formation of the cnidocyte, an ancient complex cell type in Cnidaria • Manipulating miR-2022 affects cnidocyte biogenesis and prey/predator paralysis • Conservation of miR-2022 suggests its functional significance across diverse cnidarian species Fridrich et al. unravel a role for miR-2022 in the formation of cnidocytes, ancient cnidarian-specific cells. By manipulating miR-2022 levels in Nematostella , they demonstrate it is essential for cnidocyte biogenesis and prey paralysis. The conservation of miR-2022 across all major cnidarian classes suggests its contribution to this phylum's evolutionary success. [ABSTRACT FROM AUTHOR]

Cell fate stability is essential to maintaining "law and order" in complex animals. However, high stability comes at the cost of reduced plasticity and, by extension, poor regenerative ability. This evolutionary trade-off has resulted in most modern animals being rather simple and regenerative or complex and non-regenerative. The mechanisms mediating cellular plasticity and allowing for regeneration remain unknown. We show that signals emitted by senescent cells can destabilize the differentiated state of neighboring somatic cells, reprogramming them into stem cells that are capable of driving whole-body regeneration in the cnidarian Hydractinia symbiolongicarpus. Pharmacological or genetic inhibition of senescence prevents reprogramming and regeneration. Conversely, induction of transient ectopic senescence in a regenerative context results in supernumerary stem cells and faster regeneration. We propose that senescence signaling is an ancient mechanism mediating cellular plasticity. Understanding the senescence environment that promotes cellular reprogramming could provide an avenue to enhance regeneration. [Display omitted] • Amputation injury induces senescence in a small number of head cells in Hydractinia • Senescent cells persist in the tissue for several hours before being expelled • Signals emitted by senescent cells induce reprogramming of neighboring cells • Reprogrammed cells proliferate and drive whole-body regeneration Cellular senescence is a form of permanent cell-cycle arrest in animals that is associated with aging and inflammation. Salinas-Saavedra et al. find that, in the absence of resident stem cells, senescent cells can instruct neighboring somatic cells to reprogram into stem cells that drive whole-body regeneration in the cnidarian Hydractinia symbiolongicarpus. [ABSTRACT FROM AUTHOR]

The Upper Eocene rocks (Qasr El-Sagha Formation) at northwestern Qarun Lake, in Fayum depression, revealed numerous abraded and bio-eroded Kerunia cornuta specimens, with calcified hydractinian encrustation. The symbiotic relationship between the encrusted genus Hydractinia and the host gastropod shell is discussed through thin section examination. A report of in situ hermit crab in the host gastropod shell in Kerunia cornuta is documented here for the first time from the Eocene rocks of Egypt. The studied bioerosion structures in Kerunia are attributed to three ichnospecies: Gastrochaenolites lapidicus Kelly and Bromley1984; Gastrochaenolites torpedo Kelly and Bromley1984 and Gastrochaenolites isp. These ichnospecies are related to Trypanites ichnofacies in an offshore environment under storm wave base. The taphonomic processes that affected the Kerunia cornuta specimens are also discussed in this work. The present study indicates that the paleoenvironment prevailed during the deposition of the Kerunia cornuta specimens reflects shallow agitated and bioturbated environments. • Kerunia cournuta collected from of Upper Eocene of Egypt. • Kerunia is encrusted with Hydractinia and affected by bioerosion. • Hermit crab is recorded in Keunia for the first time in Egypt. [ABSTRACT FROM AUTHOR]

Hydractinia symbiolongicarpus is a pioneering model organism for stem cell biology, being one of only a few animals with adult pluripotent stem cells (known as i-cells). However, the unavailability of a chromosome-level genome assembly has hindered a comprehensive understanding of global gene regulatory mechanisms underlying the function and evolution of i-cells. Here, we report the first chromosome-level genome assembly of H. symbiolongicarpus (HSymV2.0) using PacBio HiFi long-read sequencing and Hi-C scaffolding. The final assembly is 483 Mb in total length with 15 chromosomes representing 99.8% of the assembly. Repetitive sequences were found to account for 296 Mb (61%) of the total genome; we provide evidence for at least two periods of repeat expansion in the past. A total of 25,825 protein-coding genes were predicted in this assembly, which include 93.1% of the metazoan Benchmarking Universal Single-Copy Orthologs (BUSCO) gene set. 92.8% (23,971 genes) of the predicted proteins were functionally annotated. The H. symbiolongicarpus genome showed a high degree of macrosynteny conservation with the Hydra vulgaris genome. This chromosome-level genome assembly of H. symbiolongicarpus will be an invaluable resource for the research community that enhances broad biological studies on this unique model organism., Competing Interests: Conflicts of interest statement The authors declare that they have no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

The ability to regenerate lost body parts is irregularly distributed among animals, with substantial differences in regenerative potential between and within metazoan phyla. It is widely believed that regenerative animal clades inherited some aspects of their capacity to regenerate from their common ancestors but have also evolved new mechanisms that are not shared with other regenerative animals. Therefore, to gain a broad understanding of animal regenerative mechanisms and evolution, a broad sampling approach is necessary. Unfortunately, only few regenerative animals have been established as laboratory models with protocols for functional gene studies. Here, we describe the methods to establish transgenic individuals of the marine cnidarian Hydractinia. We also provide methods for transient gene expression manipulation without modifying the genome of the animals., (© 2022. The Author(s).)

Many proteins expressed on the cellular surface provide signaling and cell adhesion properties required for vital cellular functions. These binding interactions can occur between different but complementary proteins such as a ligand and receptor, or between the same protein acting as both ligand and receptor. The cell aggregation assay is a straightforward technique to identify homophilic interactions from such proteins. Here we describe the procedure for testing proteins via cell aggregation assays in HEK293T cells., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)