Hydractinia symbiolongicarpus and Hydractinia echinata are sister species of colonial Hydrozoan Cnidarians. Hydractinia symbiolongicarpus occurs along the eastern coast of North America, from Maine to South Carolina. Hydractinia echinata found along North European Atlantic coasts. In the field, they are found exclusively on Gastropod Shells occupied by Hermit Crabs (e.g., Pagurus longicarpus). Colonies consist of polyps specialized for feeding, reproduction, or defense, which grow from a sheet of tissue called the stolonal mat. Unlike many of its Hydrozoan relatives, Hydractinia does not produce a free-living medusa stage (Jellyfish). Instead, gametes mature in a rudimentary medusoid that remains attached to sexual polyps. All polyps within a colony are clonally derived and therefore genetically identical. The mat consists of two epidermal cell layers, which sandwich a network of gastrodermal canals connecting polyps to each other and forming a gastrovascular system. Colonies grow by expanding the edge of the mat or by elongating individual stolons, extensions of gastrovascular canals encased in a thin, chitinous integument called the periderm. Colonies are dioecious and spawn about 90 min after first light. Eggs sink to the bottom after fertilization and develop into a planula larva within 2–3 days. Mature larvae latch onto a passing Hermit Crab shell by firing nematocysts located in their posterior ends. Once on the shell, the larvae metamorphose into a primary polyp in response to a Bacterial cue. The juvenile colony then grows as described, frequently covering the entire shell.
In a paper published in the journal EvoDevo on 26 March 2020, Uri Frank of the Centre for Chromosome Biology at the University of Ireland Galway, Matthew Nicotra of the Departments of Surgery and Immunology at the University of Pittsburgh, and Christine Schnitzler of the Whitney Laboratory for Marine Bioscience and Department of Biology at the University of Florida, discuss the cultivation of Hydractinia spp. in the laboratory, and its usefulness as a model organism.
Hydractinia morphology, life history, and culture. (a) Colony growing on a microscope slide. Major morphological structures are labeled. This colony was explanted from a larger colony. The yellow-brown rectangle at the center is a layer of chitin that is slowly deposited below the mat as the colony grows and indicates the outline of the original explant. Scale bar is 1 mm. (b) Life cycle of Hydractinia. (c) Typical setup of a 39 litre glass aquarium for culturing Hydractinia. Frank et al. (2020).
Cnidarians are an interesting and highly diverse group of Animals. This phylum diverged from the lineage leading to Bilaterian Animals (the group that includes Flies, Worms, and Vertebrates) at least 600 million years ago, providing sufficient time for substantial diversification within the Cnidarian lineage. Most extant Cnidarians share a body wall consisting of an epithelial bilayer, a gastric cavity, and a unique cell type, the stinging cell or cnidocyte (also known as nematocyte) from which the phylum name derives. Cnidarians are phylogenetically positioned as the sister group to Bilaterians; therefore, studying biological phenomena in Cnidarians can provide insight into their origin and how they have changed over evolutionary time between and within phyla. The past two decades have brought substantial progress in Cnidarian molecular biology and genetics, enabling functional genetic studies at least in some Cnidarian representatives. Overall, Cnidarians’ relative morphological simplicity, sequenced genomes, amenability to genetic manipulation, and phylogenetic position promise a fruitful future in research on these Animals that will inform areas spanning all the way from evolutionary biology to biomedical sciences.
Cladogram showing evolutionary relationships between Hydractinia and other model organisms. Frank et al. (2020).
Current research on Hydractinia focuses on a number of topics, including embryonic development, neurogenesis, stem cells, germ cells, and regeneration, allorecognition, metabolism, immunity, and natural product chemistry. Allorecognition refers to the ability to discriminate ‘self’ from ‘non-self’ within the same species, a phenomenon observed in most colonial Cnidarians, but not in Hydra or Nematostella, the two most commonly used Cnidarian model systems for molecular work. At present, Hydractinia is the only Cnidarian in which genes controlling allorecognition have been identified and functionally characterised.
Other areas of interest are stem cells and regeneration. These topics have been well studied in Hydra and are emerging topics for Nematostella researchers too. Interestingly, data published to date suggest that both stem cell behavior and the mode of regeneration differ substantially between Cnidarian species. For example, Hydrozoan neuronal cells derive from migratory i-cells, whereas in Anthozoans, neural progenitor cells are epithelial. As to regeneration modes, Hydra can reform the main head structures following decapitation in the absence of cell proliferation whereas in Hydractinia and Nematostella cell proliferation is essential for regeneration. These findings highlight the importance of studying more than one animal in order to prevent false conceptual generalizations and underestimation of the complexity underlying biological phenomena.
Hydractinia does not show any evidence for age-related deterioration, s highly resistant to ionizing irradiation, and develops tumors only very rarely following genetic manipulation but not spontaneously. These features are consistent with high genomic stability in this Animal, a feature that remains to be investigated.
Manipulating gene expression has so far only been established in four Cnidarians: Hydra, Nematostella, Hydractinia, and Clytia. This can be done either by permanent modification of the Animal’s genome or by transient interference with specific gene products. Both approaches have their pros and cons and their usage depends on the type of experiment being conducted and availability of appropriate protocols for a given species and life stage.
The most common approach in Hydractinia is microinjection of nucleic acids and/or proteins into the zygote. Hydractinia spawning is light-induced without the need for any further induction. Eggs are not embedded in jelly and can be directly microinjected upon fertilisation. Electroporation techniques are currently being developed in Frank et al.’s labs with promising results. Circular plasmids readily integrate into the Hydractinia genome. The site of integration is unknown, but the process is highly efficient; in excess of 80% of injected embryos become transgenic in the hands of experienced researchers. This approach has been used to create fluorescent reporter lines for many developmental genes and cell type-specific markers. A more targeted way to genetically manipulate the Animals is provided by CRISPR–Cas9 technology (a technology that enables geneticists to edit parts of the genome, which was adapted from a naturally occurring genome editing system in Bacteria). In Hydractinia, this is performed by microinjecting site-specific short guide RNAs (sgRNA) together with recombinant Cas9 to generate loss-of-function mutations. Adding to the injecting cocktail a plasmid including a fragment of DNA, flanked by two homology arms, can be used for targeted knock-in of fragments. As with all plasmids, this DNA could also integrate randomly into the genome. Designing the injected DNA such that it must rely on the promotor of the target gene limits the likelihood that it would be expressed if integrating non-specifically.
Live imaging of transgenic Hydractinia gastrozooids. (a) A polyp expressing eGFP under an RFamide precursor promoter, labeling a subset of neurons. The animal was created via random integration of a circular DNA plasmid. (b) A polyp expressing eGFP under the endogenous Eef1a promoter. The animal was created using CRISPR/Cas9 to target integration of the eGFP coding sequence into the Hydractinia Eef1a locus. Frank et al. (2020).
Gene expression manipulation without genetic alteration can be achieved by injecting short hairpin RNA (shRNA) or morpholino oligonucleotides to lower expression of genes, or synthetic RNA to overexpress them. Finally, incubating polyps in seawater containing double stranded RNA (dsRNA) transiently lowers the expression of the corresponding gene, albeit with low efficiency.
Hydractinia is also unique among model Cnidarians for being the only species in which a forward genetic approach has been used to identify the genetic basis of a phenotype. The reasons for this are almost entirely logistical. First, Hydractinia colonies can produce hundreds of embryos per day, making it possible to quickly generate large populations of bred Animals. Second, the Animals grow as encrustations on a surface that can be labeled, making it possible to co-culture large populations of genetically distinct animals in a small number of tanks. To date, forward genetic approaches have been used to identify genes responsible for allorecognition and sex determination. Given the availability of a sequenced genome and the cost efficiency of high-throughput genotyping, it seems feasible to consider mutagenesis screens as well.
An additional experimental approach in Hydractinia is grafting of tissues. This can be done for, e.g., introducing transgenic cells into a colony. Grafting of tissues from genetically distinct individuals requires at least partial matching of allorecognition alleles to prevent allogeneic rejection.
Single-cell RNA sequencing methods are also under development in our labs with the first single-cell sequencing libraries giving encouraging results. Frank et al's current goal is to develop a robust cellular atlas to define major cell types and subtypes in Hydractinia and to identify marker genes for all cell types as was recently done in Hydra and Nematostella. With a robust genome and cellular atlas in place, Hydractinia will be poised to answer biological questions in a more comprehensive way. Flow cytometry and fluorescence activated cell sorting protocols are available, and together with many transgenic reporter strains it allows for generating cell type-specific transcriptomes following fluorescence activated cell sorting-sorting of defined cell populations.
As with any model organism, Hydractinia has limitations. Perhaps most obvious one is that it lacks a medusa stage, so researchers interested in this feature must look elsewhere, notably to the Hydroid Clytia and the Scyphozoan Aurelia. The existing Hydractinia research community also remains small compared to that for Hydra and Nematostella, so the availability of shared reagents and techniques is somewhat more limited. This concern is increasingly mitigated by additional labs beginning to study Hydractinia, and an upsurge in crosstalk between researchers.
The Hydractinia research community is relatively small but growing as Hydractinia is gaining recognition as a tractable Cnidarian research model. A recent National Science Foundation Enabling Discovery through GEnomic Tools grant has been awarded to Frank et al., ensuring that the genetic toolkit and community of Hydractinia researchers will continue to blossom and grow. Current resources include high-quality genomes and transcriptomes from both Hydractinia symbiolongicarpus and Hydractinia echinata. Draft Illumina genome and transcriptome assemblies are publicly available through the Hydractinia Genome Project Portal, and long-read PacBio genome assemblies for both species are forthcoming. With an estimated genome size of 774 Mb for Hydractinia echinata and 514 Mb for Hydractinia symbiolongicarpus, the Hydractinia genomes are larger than the genome of Nematostella (329 Mb) but smaller than that of Hydra (1086 Mb). Annotated reference genomes and transcriptomes can be used for mapping standard RNA sequencing data. Laboratory selected, fast-growing strains are available to anyone. Frank et al. are developing a community portal to be completed in the coming months, which will link to written and video-based protocols and to a community forum, and provide an online form to request Animals. Newcomers to the field are encouraged to attend the two biennial research conferences, the American Cnidofest and the European Tutzing meeting.
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