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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.

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 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 (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs) 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.Significance StatementThe immunoglobulin superfamily (IgSF) is one of the largest and most functionally versatile domain families in animal genomes. Although their amino acid sequences can vary considerably, IgSF domains have been traditionally defined by conserved residues at several key positions in their fold. Here, we sequenced an invertebrate histocompatibility complex and discovered a family of IgSF genes with amino acid sequences that lack most of these residues yet are predicted to adopt folds virtually identical to canonical V-set and I-set IgSF domains. This work broadens the definition of the IgSF and shows that the V-set domain was present earlier in animal evolution than previously appreciated.

SUMMARYN6-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 cnidarianHydractinia 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 downAlkbh1, 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 inHydractinia, 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.

Pathogen-induced cancers account for 15% of human tumors and are a growing concern for endangered wildlife. Fibropapillomatosis is an expanding virally and environmentally co-induced sea turtle tumor epizootic. Chelonid herpesvirus 5 (ChHV5) is implicated as a causative virus, but its transmission method and specific role in oncogenesis and progression is unclear. We applied environmental (e)DNA-based viral monitoring to assess viral shedding as a direct means of transmission, and the relationship between tumor burden, surgical resection and ChHV5 shedding. To elucidate the abundance and transcriptional status of ChHV5 across early, established, regrowth and internal tumors we conducted genomics and transcriptomics. We determined that ChHV5 is shed into the water column, representing a likely transmission route, and revealed novel temporal shedding dynamics and tumor burden correlations. ChHV5 was more abundant in the water column than in marine leeches. We also revealed that ChHV5 is latent in fibropapillomatosis, including early stage, regrowth and internal tumors; higher viral transcription is not indicative of poor patient outcome, and high ChHV5 loads predominantly arise from latent virus. These results expand our knowledge of the cellular and shedding dynamics of ChHV5 and can provide insights into temporal transmission dynamics and viral oncogenesis not readily investigable in tumors of terrestrial species.

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/.

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. Stem cells expressing Piwi1 and Soxb1, which have a 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 neurological disorders in all animals, including humans.

Sex determination occurs across animal species, but most of our knowledge about the mechanisms of sex determination 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 this map to determine that this species has an XX/XY sex determination system. We delineate the pseudoautosomal and non-recombining regions of the Y chromosome and show that the latter encodes a number of genes with male gonad-specific expression. These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination.Significance StatementWhat determines whether an animal is male or female? The answer depends on the species. Some rely on signals from their environment, while others take cues from their genome. Most of what we understand about sex determination comes from traditional model organisms (such as mice, flies, and worms) or groups of well-studied vertebrates and insects. Studying sex determination in other animals, especially those from phyla that diverged early in animal evolution, would allow us to better understand how sex has evolved across the animal kingdom and could reveal pathways present in the eumetazoan ancestor >600 million years ago. In this study, we show that the cnidarian Hydractinia symbiolongicarpus has XY sex determination, establishing this species as a model system for sex determination in an understudied group of animals.

Conserved gene specifies germ cell Germ cells are the exclusive progenitors of gametes. In most studied animals, including humans, germ cells are produced only once during embryogenesis and are not replenished in adult life. DuBuc et al. studied germ cell induction in the clonal cnidarian Hydractinia symbiolongicarpus , an animal that forms germ cells continuously in adult life from stem cells that also generate somatic cells. A single transcription factor is capable of converting the animal's adult stem cells to germ cells. A similar gene also controls germ cell induction in mammalian embryos, but its action there is limited to a single event in early embryogenesis. Science , this issue p. 757

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression. Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFβ and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers. Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic., Yetsko, Farrell, Duffy, and colleagues conduct transcriptomic and immunohistological profiling of tumors from sea turtles with fibropapillomatosis. Internal tumors are distinct from more common external tumors, but share some oncogenic signaling pathways that may serve as treatment targets in future.

Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

Performing gene function analyses in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarianHydractinia symbiolongicarpusvia electroporation, yielding gene-targeted knockdowns that can be assessed throughout embryogenesis, larval settlement, and metamorphosis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilizedH.symbiolongicarpuseggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing significant knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2). We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. This is the first time that electroporation as a delivery system has been developed forHydractinia. Our detailed protocol for electroporation of shRNAs inH. symbiolongicarpusembryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

The maternal-zygotic transition (MZT) describes the developmental reprogramming of gene expression marked by the degradation of maternally supplied gene products and activation of the zygotic genome. While the timing and duration of the MZT vary among taxa, little is known about early stage transcriptional dynamics in the non-bilaterian phylum Ctenophora. We sought to better understand the extent of maternal mRNA loading and subsequent differential transcript abundance during the earliest stages of development by performing comprehensive RNA-sequencing-based analyses of mRNA abundance in single- and eight-cell-stage embryos in the lobate ctenophore Mnemiopsis leidyi. We found 1,908 contigs with significant differential abundance between single- and eight-cell stages, of which 1,208 contigs were more abundant at the single-cell stage and 700 contigs were more abundant at the eight-cell stage. Of the differentially abundant contigs, 267 were exclusively present in the eight-cell samples, providing strong evidence that both the MZT and zygotic genome activation (ZGA) have commenced by the eight-cell stage. Many highly abundant transcripts encode genes involved in molecular mechanisms critical to the MZT, such as maternal transcript degradation, serine/threonine kinase activity, and chromatin remodeling. Our results suggest that chromosomal restructuring, which is critical to ZGA and the initiation of transcriptional regulation necessary for normal development, begins by the third cleavage within 1.5 hours post-fertilization in Mnemiopsis leidyi. This article is protected by copyright. All rights reserved

Dinoflagellate-cnidarian associations form both the trophic and structural foundation of coral-reef ecosystems. Previous studies have highlighted the role of host innate immunity in regulation of these partnerships. This study reveals the presence of a transforming growth factor beta (TGF beta) in the coral Fungia scutaria that clusters with TGF beta sensu stricto (ss) from other animals. In functional studies of F. scutaria larvae, we show that (1) TGF beta ss mRNA is expressed during early stages of development prior to the onset of symbiosis; (2) apparent interference of the TGF beta pathway impairs the onset of symbiosis; and (3) this effect is associated with an increase of cytotoxic nitric oxide secretion, an immune response. This work highlights the importance of the TGF beta pathway in early life-history stages of corals by suggesting that its inhibition impacts the onset of symbiosis.

Sea turtle populations are directly and indirectly under threat from a range of anthropogenic processes. Perhaps the most visibly apparent of these is the disfiguring tumor disease epizootic (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at a number of affected sites globally. Environmental exposures seem key to inducing tumor development, possibly through weakening host immune systems to the point of enabling pathogen-induced tumor formation. However, we do not yet understand the precise molecular and mutational events driving fibropapillomatosis tumor formation and progression. Similarly, many open questions remain about the role of the herpesvirus (chelonid herpesvirus 5, ChHV5) associated with the disease as a potential co-trigger, and whether its occurrence within tumors is causative or opportunistic. Without improved understanding of the basic biology of this disease epizootic, treatment, containment and mitigation options are severely hampered.To address fundamental questions relating to the oncogenic signaling, mutational spectrum, viral load, viral transcriptional status (lytic or latent) and spread, we employed transcriptomic profiling, whole genome sequencing, immunohistochemistry and environmental (e)DNA-based monitoring of viral shedding. In particular we focused on the mutational landscape of tumors and assessing the transcriptional similarity of external (skin) and internal (visceral organs) tumors, and the oncogenic signaling events driving early stage tumor growth and post-surgical tumor regrowth. These analyses revealed that internal fibropapillomatosis tumors are molecularly distinct from the more common external tumors. However, our molecular analyses also revealed that there are a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common between internal and external tumors, such as the MAPK, Wnt, TGFβ and TNF oncogenic signaling pathways. We also determined that the tumor genomes can harbor copy number gains, indicating potentially viral-independent oncogenic processes. Genes within such mutated genomic regions have known roles in human skin cancer, including MAPK-associated genes. Turtles attempt to mount an immune response, but in some animals this appears to be insufficient to prevent tumor development and growth. ChHV5 was transcriptionally latent in all tumor stages sequenced, including early stage and recurrent tumors. We also revealed that the tumors themselves are the primary source of viral shedding into the marine environment and, if they are surgically removed, the level of ChHV5 in the water column drops.Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, therapeutic treatment, and appropriate quarantine responses for this wildlife epizootic. Furthermore, they provide insights into human pathogen-induced cancers, particularly mechanisms which are difficult to study in the human and terrestrial context, such as time-course quantification-based monitoring of viral shedding.

Polycomb group proteins are essential regulators of developmental processes across animals. Despite their importance, studies on Polycomb are often restricted to classical model systems and, as such, little is known about the evolution of these important chromatin regulators. Here we focus on Polycomb Repressive Complex 1 (PRC1) and trace the evolution of core components of canonical and non-canonical PRC1 complexes in animals. Previous work suggested that a major expansion in the number of PRC1 complexes occurred in the vertebrate lineage. Here we show that the expansion of the PCGF protein family, an essential step for the establishment of the large diversity of PRC1 complexes found in vertebrates, predates the bilaterian-cnidarian ancestor. This means that the genetic repertoire necessary to form all major vertebrate PRC1 complexes emerged early in animal evolution, over 550 million years ago. We further show thatPCGF5, a gene conserved in cnidarians and vertebrates but lost in all other studied groups, is expressed in the nervous system in the sea anemoneNematostella vectensis, similar to its mammalian counterpart. Together this work provides an evolutionary framework to understand PRC1 complex diversity and evolution and establishesNematostellaas a promising model system in which this can be further explored.

Hydractinia, a genus of colonial marine cnidarians, has been used as a model organism for developmental biology and comparative immunology for over a century. It was this animal where stem cells and germ cells were first studied. However, protocols for efficient genetic engineering have only recently been established by a small but interactive community of researchers. The animal grows well in the lab, spawns daily, and its relatively short life cycle allows genetic studies. The availability of genomic tools and resources opens further opportunities for research using this animal. Its accessibility to experimental manipulation, growth- and cellular-plasticity, regenerative ability, and resistance to aging and cancer place Hydractinia as an emerging model for research in many biological and environmental disciplines.

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

SoxB transcription factors and histone deacetylases (HDACs) are each major players in the regulation of neurogenesis, but a functional link between them has not been previously demonstrated. Here, we show that SoxB2 and Hdac2 act together to regulate neurogenesis in the cnidarian Hydractinia echinata during tissue homeostasis and head regeneration. We find that misexpression of SoxB genes modifies the number of neural cells in all life stages and interferes with head regeneration. Hdac2 was coexpressed with SoxB2, and its downregulation phe-nocopied SoxB2 knockdown. We also show that SoxB2 and Hdac2 promote each other's transcript levels, but Hdac2 counteracts this amplification cycle by deacetylating and destabilizing SoxB2 protein. Finally, we present evidence for conservation of these interactions in human neural progenitors. We hypothesize that crosstalk between SoxB transcription factors and Hdac2 is an ancient feature of metazoan neurogenesis and functions to stabilize the correct levels of these multifunctional proteins.

The 2018 Cnidarian Model Systems Meeting (Cnidofest) was held September 6–9th at the University of Florida Whitney Laboratory for Marine Bioscience in St. Augustine, FL. Cnidofest 2018, which built upon the momentum of Hydroidfest 2016, brought together research communities working on a broad spectrum of cnidarian organisms from North America and around the world. Meeting talks covered diverse aspects of cnidarian biology, with sessions focused on genomics, development, neurobiology, immunology, symbiosis, ecology, and evolution. In addition to interesting biology, Cnidofest also emphasized the advancement of modern research techniques. Invited technology speakers showcased the power of microfluidics and single-cell transcriptomics and demonstrated their application in cnidarian models. In this report, we provide an overview of the exciting research that was presented at the meeting and discuss opportunities for future research.

BackgroundHydractinia symbiolongicarpus, a colonial cnidarian, is a tractable model system for many cnidarian-specific and general biological questions. Until recently, tests of gene function in Hydractinia have relied on laborious forward genetic approaches, randomly integrated transgenes, or transient knockdown of mRNAs.ResultsHere, we report the use of CRISPR/Cas9 genome editing to generate targeted genomic insertions in H. symbiolonigcarpus. We used CRISPR/Cas9 to promote homologous recombination of two fluorescent reporters, eGFP and tdTomato, into the Eukaryotic elongation factor 1 alpha (Eef1a) locus. We demonstrate that the transgenes are expressed ubiquitously and are stable over two generations of breeding. We further demonstrate that CRISPR/Cas9 genome editing can be used to mark endogenous proteins with FLAG or StrepII-FLAG affinity tags to enable in vivo and ex vivo protein studies.ConclusionsThis is the first account of CRISPR/Cas9 mediated knockins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9 inserted transgene in a cnidarian. The ability to precisely insert exogenous DNA into the Hydractinia genome will enable sophisticated genetic studies and further development of functional genomics tools in this understudied cnidarian model.

Three studies presenting genomes and transcriptomes from different life stages of nearly all major lineages of medusozoans present radically different gene expression profiles between life cycle stages and many medusa-specific genes.

The Kruppel-like factor and specificity protein (KLF/SP) genes play key roles in critical biological processes including stem cell maintenance, cell proliferation, embryonic development, tissue differentiation, and metabolism and their dysregulation has been implicated in a number of human diseases and cancers. Although many KLF/SP genes have been characterized in a handful of bilaterian lineages, little is known about the KLF/SP gene family in nonbilaterians and virtually nothing is known outside the metazoans. Here, we analyze and discuss the origins and evolutionary history of the KLF/SP transcription factor family and associated transactivation/repression domains. We have identified and characterized the complete KLF/SP gene complement from the genomes of 48 species spanning the Eukarya. We have also examined the phylogenetic distribution of transactivation/repression domains associated with this gene family. We report that the origin of the KLF/SP gene family predates the divergence of the Metazoa. Furthermore, the expansion of the KLF/SP gene family is paralleled by diversification of transactivation domains via both acquisitions of pre-existing ancient domains as well as by the appearance of novel domains exclusive to this gene family and is strongly associated with the expansion of cell type complexity.

Wildlife populations are under intense anthropogenic pressures, with the geographic range of many species shrinking, dramatic reductions in population numbers and undisturbed habitats, and biodiversity loss. It is postulated that we are in the midst of a sixth (Anthropocene) mass extinction event, the first to be induced by human activity. Further, threatening vulnerable species is the increased rate of emerging diseases, another consequence of anthropogenic activities. Innovative approaches are required to help maintain healthy populations until the chronic underlying causes of these issues can be addressed. Fibropapillomatosis in sea turtles is one such wildlife disease. Here, we applied precision-medicine-based approaches to profile fibropapillomatosis tumors to better understand their biology, identify novel therapeutics, and gain insights into viral and environmental triggers for fibropapillomatosis. We show that fibropapillomatosis tumors share genetic vulnerabilities with human cancer types, revealing that they are amenable to treatment with human anti-cancer therapeutics.

The function of Notch signaling was previously studied in two cnidarians, Hydra and Nematostella, representing the lineages Hydrozoa and Anthozoa, respectively. Using pharmacological inhibition in Hydra and a combination of pharmacological and genetic approaches in Nematostella, it was shown in both animals that Notch is required for tentacle morphogenesis and for late stages of stinging cell maturation. Surprisingly, a role for Notch in neural development, which is well documented in bilaterians, was evident in embryonic Nematostella but not in adult Hydra. Adult neurogenesis in the latter seemed to be unaffected by DAPT, a drug that inhibits Notch signaling. To address this apparent discrepancy, we studied the role of Notch in Hydractinia echinata, an additional hydrozoan, in all life stages. Using CRISPR-Cas9 mediated mutagenesis, transgenesis, and pharmacological interference we show that Notch is dispensable for Hydractinia normal neurogenesis in all life stages but is required for the maturation of stinging cells and for tentacle morphogenesis. Our results are consistent with a conserved role for Notch in morphogenesis and nematogenesis across Cnidaria, and a lineage-specific loss of Notch dependence in neurogenesis in hydrozoans.

Hydroidfest 2016 took place on September 23–25 at the UC Davis Bodega Marine Laboratory in Bodega Bay, CA. The meeting brought together cnidarian researchers, with an emphasis on those studying hydrozoans, from North America and other parts of the world. The scientific topics discussed were diverse, including sessions focused on development, regeneration, aging, immunology, symbiosis, and neurobiology. Thanks to the application of modern biological technologies, hydrozoans and other cnidarians are now fertile ground for research in numerous disciplines. Moreover, their amenability to comparative approaches is a powerful asset that was repeatedly showcased during the meeting. Here, we give a brief account of the work that was presented and the opportunities that emerged from the ensuing discussions.

Since its founding, the Monterey Bay Aquarium Research Institute (MBARI) has pioneered unique capabilities for accessing the deep ocean and its inhabitants through focused peer relationships between scientists and engineers. This focus has enabled breakthroughs in our understanding of life in the sea, leading to fundamental advances in describing the biology and the ecology of open-ocean and deep-sea animals. David Packard’s founding principle was the application of technological advances to studying the deep ocean, in part because he recognized the critical importance of this habitat in a global context. Among other fields, MBARI’s science has benefited from applying novel methodologies in molecular biology and genetics, imaging systems, and in situ observations. These technologies have allowed MBARI’s bioluminescence and biodiversity laboratory and worldwide collaborators to address centuries-old questions related to the biodiversity, behavior, and bio-optical properties of organisms living in the water column, from the surface into the deep sea. Many of the most interesting of these phenomena are in the midwater domain—the vast region of ocean between the sunlit surface waters and the deep seafloor.

Background Cnidarians are a group of early branching animals including corals, jellyfish and hydroids that are renowned for their high regenerative ability, growth plasticity and longevity. Because cnidarian genomes are conventional in terms of protein-coding genes, their remarkable features are likely a consequence of epigenetic regulation. To facilitate epigenetics research in cnidarians, we analysed the histone complement of the cnidarian model organism Hydractinia echinata using phylogenomics, proteomics, transcriptomics and mRNA in situ hybridisations. Results We find that the Hydractinia genome encodes 19 histones and analyse their spatial expression patterns, genomic loci and replication-dependency. Alongside core and other replication-independent histone variants, we find several histone replication-dependent variants, including a rare replication-dependent H3.3, a female germ cell-specific H2A.X and an unusual set of five H2B variants, four of which are male germ cell-specific. We further confirm the absence of protamines in Hydractinia. Conclusions Since no protamines are found in hydroids, we suggest that the novel H2B variants are pivotal for sperm DNA packaging in this class of Cnidaria. This study adds to the limited number of full histone gene complements available in animals and sets a comprehensive framework for future studies on the role of histones and their post-translational modifications in cnidarian epigenetics. Finally, it provides insight into the evolution of spermatogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0085-1) contains supplementary material, which is available to authorized users.

Background Fluorescent proteins are optically active proteins found across many clades in metazoans. A fluorescent protein was recently identified in a ctenophore, but this has been suggested to derive from a cnidarian, raising again the question of origins of this group of proteins. Results Through analysis of transcriptome data from 30 ctenophores, we identified a member of an orthologous group of proteins similar to fluorescent proteins in each of them, as well as in the genome of Mnemiopsis leidyi. These orthologs lack canonical residues involved in chromophore formation, suggesting another function. Conclusions The phylogenetic position of the ctenophore protein family among fluorescent proteins suggests that this gene was present in the common ancestor of all ctenophores and that the fluorescent protein previously found in a ctenophore actually derives from a siphonophore. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0738-5) contains supplementary material, which is available to authorized users.

Animals must manage interactions with beneficial as well as detrimental microbes. Immunity therefore includes strategies for both resistance to and tolerance of microbial invaders. Transforming growth factor beta (TGFβ) cytokines have many functions in animals including a tolerance-promoting (tolerogenic) role in immunity in vertebrates. TGFβ pathways are present in basal metazoans such as cnidarians but their potential role in immunity has never been explored. This study takes a two-part approach to examining an immune function for TGFβ in cnidarians. First bioinformatic analyses of the model anemone Aiptasia pallida were used to identify TGFβ pathway components and explore the hypothesis that an immune function for TGFβs existed prior to the evolution of vertebrates. A TGFβ ligand from A. pallida was identified as one that groups closely with vertebrate TGFβs that have an immune function. Second, cellular analyses of A. pallida were used to examine a role for a TGFβ pathway in the regulation of cnidarian–dinoflagellate mutualisms. These interactions are stable under ambient conditions but collapse under elevated temperature, a phenomenon called cnidarian bleaching. Addition of exogenous human TGFβ suppressed an immune response measured as LPS-induced nitric oxide (NO) production by the host. Addition of anti-TGFβ to block a putative TGFβ pathway resulted in immune stimulation and a failure of the symbionts to successfully colonize the host. Finally, addition of exogenous TGFβ suppressed immune stimulation in heat-stressed animals and partially abolished a bleaching response. These findings suggest that the dinoflagellate symbionts somehow promote host tolerance through activation of tolerogenic host immune pathways, a strategy employed by some intracellular protozoan parasites during their invasion of vertebrates. Insight into the ancient, conserved nature of host–microbe interactions gained from this cnidarian–dinoflagellate model is valuable to understanding the evolution of immunity and its role in the regulation of both beneficial and detrimental associations.

The cellular mechanisms controlling the successful establishment of a stable mutualism between cnidarians and their dinoflagellate partners are largely unknown. The planula larva of the solitary Hawaiian scleractinian coral Fungia scutaria and its dinoflagellate symbiont Symbiodinium sp. type C1f represents an ideal model for studying the onset of cnidarian-dinoflagellate endosymbiosis due to the predictable availability of gametes, the ability to raise non-symbiotic larvae and establish the symbiosis experimentally, and the ability to precisely quantify infection success. The goal of this study was to identify genes differentially expressed in F. scutaria larvae during the initiation of endosymbiosis with Symbiodinium sp. C1f. Newly symbiotic larvae were compared to non-symbiotic larvae using a custom cDNA microarray. The 5184-feature array was constructed with cDNA libraries from newly symbiotic and non-symbiotic F. scutaria larvae, including 3072 features (60%) that were enriched for either state by subtractive hybridization. Our analyses revealed very few changes in the F. scutaria transcriptome as a result of infection with Symbiodinium sp. C1f, similar to other studies focused on the early stages of this symbiotic interaction. We suggest that these results may be due, in part, to an inability to detect the transcriptional signal from the small percentage of infected cells compared to uninfected cells. We discuss several other potential explanations for this result, including suggesting that certain types of Symbiodinium sp. may have evolved mechanisms to suppress or circumvent cnidarian host responses to infection.

Elevated temperature and solar radiation, including ultraviolet radiation, are now recognized as the primary environmental stresses that lead to mass cnidarian bleaching. This study takes a functional genomics approach to identifying genes that change expression soon after exposure to these stressors in the temperate sea anemone Anthopleura elegantissima that harbors Symbiodinium, the same genus of symbionts found in reef-building corals. Symbiotic anemones were subjected to elevated temperature or UV over a 24 h period. cDNA from these animals was hybridized to a 10,000-feature cDNA microarray of A. elegantissima. Overall 2.7% of the 10,000 features were found to be differentially expressed as a function of temperature or UV stress. Of the 86 features sequenced, 45% displayed significant homology to sequences in GenBank. There are 27 features that were differentially expressed in both stress conditions. Gene ontology analysis placed the differentially expressed genes in a wide range of categories including cytoskeleton organization and biogenesis, protein biosynthesis, cell proliferation, apoptosis and transport. This suggests that the early stress response to elevated temperature and UV involves essentially all aspects of host cellular regulation and machinery and that downstream cnidarian bleaching is a complex cellular response in host tissues.

Background The recent expansion of whole-genome sequence data available from diverse animal lineages provides an opportunity to investigate the evolutionary origins of specific classes of human disease genes. Previous studies have observed that human disease genes are of particularly ancient origin. While this suggests that many animal species have the potential to serve as feasible models for research on genes responsible for human disease, it is unclear whether this pattern has meaningful implications and whether it prevails for every class of human disease. Results We used a comparative genomics approach encompassing a broad phylogenetic range of animals with sequenced genomes to determine the evolutionary patterns exhibited by human genes associated with different classes of disease. Our results support previous claims that most human disease genes are of ancient origin but, more importantly, we also demonstrate that several specific disease classes have a significantly large proportion of genes that emerged relatively recently within the metazoans and/or vertebrates. An independent assessment of the synonymous to non-synonymous substitution rates of human disease genes found in mammals reveals that disease classes that arose more recently also display unexpected rates of purifying selection between their mammalian and human counterparts. Conclusions Our results reveal the heterogeneity underlying the evolutionary origins of (and selective pressures on) different classes of human disease genes. For example, some disease gene classes appear to be of uncommonly recent (i.e., vertebrate-specific) origin and, as a whole, have been evolving at a faster rate within mammals than the majority of disease classes having more ancient origins. The novel patterns that we have identified may provide new insight into cases where studies using traditional animal models were unable to produce results that translated to humans. Conversely, we note that the larger set of disease classes do have ancient origins, suggesting that many non-traditional animal models have the potential to be useful for studying many human disease genes. Taken together, these findings emphasize why model organism selection should be done on a disease-by-disease basis, with evolutionary profiles in mind. Electronic supplementary material The online version of this article (doi:10.1186/s12862-014-0212-1) contains supplementary material, which is available to authorized users.

Mnemiopsis leidyi is a ctenophore native to the coastal waters of the western Atlantic Ocean. A number of studies on Mnemiopsis have led to a better understanding of many key biological processes, and these studies have contributed to the emergence of Mnemiopsis as an important model for evolutionary and developmental studies. Recently, we sequenced, assembled, annotated, and performed a preliminary analysis on the 150-megabase genome of the ctenophore, Mnemiopsis. This sequencing effort has produced the first set of whole-genome sequencing data on any ctenophore species and is amongst the first wave of projects to sequence an animal genome de novo solely using next-generation sequencing technologies. The Mnemiopsis Genome Project Portal ( http://research.nhgri.nih.gov/mnemiopsis/ ) is intended both as a resource for obtaining genomic information on Mnemiopsis through an intuitive and easy-to-use interface and as a model for developing customized Web portals that enable access to genomic data. The scope of data available through this Portal goes well beyond the sequence data available through GenBank, providing key biological information not available elsewhere, such as pathway and protein domain analyses; it also features a customized genome browser for data visualization. We expect that the availability of these data will allow investigators to advance their own research projects aimed at understanding phylogenetic diversity and the evolution of proteins that play a fundamental role in metazoan development. The overall approach taken in the development of this Web site can serve as a viable model for disseminating data from whole-genome sequencing projects, framed in a way that best-serves the specific needs of the scientific community.

Background: The Sox genes, a family of transcription factors characterized by the presence of a high mobility group (HMG) box domain, are among the central groups of developmental regulators in the animal kingdom. They are indispensable in progenitor cell fate determination, and various Sox family members are involved in managing the critical balance between stem cells and differentiating cells. There are 20 mammalian Sox genes that are divided into five major groups (B, C, D, E, and F). True Sox genes have been identified in all animal lineages but not outside Metazoa, indicating that this gene family arose at the origin of the animals. Whole-genome sequencing of the lobate ctenophore Mnemiopsis leidyi allowed us to examine the full complement and expression of the Sox gene family in this early-branching animal lineage. Results: Our phylogenetic analyses of the Sox gene family were generally in agreement with previous studies and placed five of the six Mnemiopsis Sox genes into one of the major Sox groups: SoxB (MleSox1), SoxC (MleSox2), SoxE (MleSox3, MleSox4), and SoxF (MleSox5), with one unclassified gene (MleSox6). We investigated the expression of five out of six Mnemiopsis Sox genes during early development. Expression patterns determined through in situ hybridization generally revealed spatially restricted Sox expression patterns in somatic cells within zones of cell proliferation, as determined by EdU staining. These zones were located in the apical sense organ, upper tentacle bulbs, and developing comb rows in Mnemiopsis, and coincide with similar zones identified in the cydippid ctenophore Pleurobrachia. Conclusions: Our results are consistent with the established role of multiple Sox genes in the maintenance of stem cell pools. Both similarities and differences in juvenile cydippid stage expression patterns between Mnemiopsis Sox genes and their orthologs from Pleurobrachia highlight the importance of using multiple species to characterize the evolution of development within a given phylum. In light of recent phylogenetic evidence that Ctenophora is the earliest-branching animal lineage, our results are consistent with the hypothesis that the ancient primary function of Sox family genes was to regulate the maintenance of stem cells and function in cell fate determination. publishedVersion

The sequential association of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 and a seven-transmembrane segment coreceptor such as CCR5 or CXCR4 initiates entry of the virus into its target cell. The N terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in the CD4-dependent association of gp120 with CCR5 and in viral entry. Here we demonstrate that a tyrosine-sulfated peptide based on the N terminus of CCR5, but not its unsulfated analogue, inhibits infection of macrophages and peripheral blood mononuclear cells by CCR5-dependent, but not CXCR4-dependent, HIV-1 isolates. The sulfated peptide also inhibited the association of CCR5-expressing cells with gp120-soluble CD4 complexes and, less efficiently, with MIP-1alpha. Moreover, this peptide inhibited the precipitation of gp120 by 48d and 23e antibodies, which recognize CD4-inducible gp120 epitopes, but not by several other antibodies that recognize proximal epitopes. The ability of the sulfated peptide to block 48d association with gp120 was dependent in part on seven tropism-determining residues in the third variable (V3) and fourth conserved (C4) domains of gp120. These data underscore the important role of the N-terminal sulfate moieties of CCR5 in the entry of R5 HIV-1 isolates and localize a critical contact between gp120 and CCR5.

An understanding of ctenophore biology is critical for reconstructing events that occurred early in animal evolution. Toward this goal, we have sequenced, assembled, and annotated the genome of the ctenophore Mnemiopsis leidyi. Our phylogenomic analyses of both amino acid positions and gene content suggest that ctenophores rather than sponges are the sister lineage to all other animals. Mnemiopsis lacks many of the genes found in bilaterian mesodermal cell types, suggesting that these cell types evolved independently. The set of neural genes in Mnemiopsis is similar to that of sponges, indicating that sponges may have lost a nervous system. These results present a newly supported view of early animal evolution that accounts for major losses and/or gains of sophisticated cell types, including nerve and muscle cells.

Background MicroRNAs play a vital role in the regulation of gene expression and have been identified in every animal with a sequenced genome examined thus far, except for the placozoan Trichoplax. The genomic repertoires of metazoan microRNAs have become increasingly endorsed as phylogenetic characters and drivers of biological complexity. Results In this study, we report the first investigation of microRNAs in a species from the phylum Ctenophora. We use short RNA sequencing and the assembled genome of the lobate ctenophore Mnemiopsis leidyi to show that this species appears to lack any recognizable microRNAs, as well as the nuclear proteins Drosha and Pasha, which are critical to canonical microRNA biogenesis. This finding represents the first reported case of a metazoan lacking a Drosha protein. Conclusions Recent phylogenomic analyses suggest that Mnemiopsis may be the earliest branching metazoan lineage. If this is true, then the origins of canonical microRNA biogenesis and microRNA-mediated gene regulation may postdate the last common metazoan ancestor. Alternatively, canonical microRNA functionality may have been lost independently in the lineages leading to both Mnemiopsis and the placozoan Trichoplax, suggesting that microRNA functionality was not critical until much later in metazoan evolution.