Randomly incorporated genomic N6-methyldeoxyadenosine delays zygotic transcription initiation in a cnidarian.

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

The genome of the colonial hydroid Hydractinia reveals that their stem cells use a toolkit of evolutionarily shared genes with all animals.

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

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

The epithelial and interstitial stem cells of the freshwater polyp Hydra are the best-characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation in Hydra is needed. To this end, we generated extensive new resources for characterizing transcriptional regulation in Hydra, including new genome assemblies for Hydra oligactis and the AEP strain of Hydra vulgaris, an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large kilobase-scale chromatin interaction domains in the Hydra genome that contain transcriptionally coregulated genes. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types: isorhiza-type nematocytes and somatic gonad ectoderm. Finally, we identified novel candidate regulators of cell type-specific transcription, several of which have likely been conserved at least since the divergence of Hydra and the jellyfish Clytia hemisphaerica more than 400 million years ago.

A family of unusual immunoglobulin superfamily genes in an invertebrate histocompatibility complex.

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.

XY sex determination in a cnidarian.

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.

The genetic basis for PRC1 complex diversity emerged early in animal evolution.

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. We show that the expansion of the Polycomb Group RING Finger (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 that PCGF5, a gene conserved in cnidarians and vertebrates but lost in all other studied groups, is expressed in the nervous system in the sea anemone Nematostella vectensis, similar to its mammalian counterpart. Together this work provides a framework for understanding the evolution of PRC1 complex diversity and it establishes Nematostella as a promising model system in which the functional ramifications of this diversification can be further explored.

Functional studies on the role of Notch signaling in Hydractinia development.

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.

CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus.

BACKGROUND: Hydractinia 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. RESULTS: Here, 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. CONCLUSIONS: This 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.

The maternal-zygotic transition and zygotic activation of the Mnemiopsis leidyi genome occurs within the first three cleavage cycles.

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 hr post-fertilization in M. leidyi.

Non-excitable fluorescent protein orthologs found in ctenophores.

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.

Evolutionary profiling reveals the heterogeneous origins of classes of human disease genes: implications for modeling disease genetics in animals.

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.

A customized Web portal for the genome of the ctenophore Mnemiopsis leidyi.

BACKGROUND: 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. DESCRIPTION: 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. CONCLUSIONS: 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.

Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes.

BACKGROUND: Calcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis. RESULTS: The Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light emission, raising the hypothesis that light production and light reception may be functionally connected in ctenophore photocytes. We also present genomic evidence of a complete ciliary phototransduction cascade in Mnemiopsis. CONCLUSIONS: This study elucidates the genomic organization, evolutionary history, and developmental expression of photoprotein and opsin genes in the ctenophore Mnemiopsis leidyi, introduces a novel dual role for ctenophore photocytes in both bioluminescence and phototransduction, and raises the possibility that light production and light reception are linked in this early-branching non-bilaterian animal.

Cnidofest 2022: hot topics in cnidarian research.

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.

The genome of the colonial hydroid Hydractinia reveals their stem cells utilize a toolkit of evolutionarily shared genes with all animals.

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

MicroRNAs and essential components of the microRNA processing machinery are not encoded in the genome of the ctenophore Mnemiopsis leidyi.

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.

GNL3 is an evolutionarily conserved stem cell gene influencing cell proliferation, animal growth and regeneration in the hydrozoan Hydractinia.

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

A cellular and molecular analysis of SoxB-driven neurogenesis in a cnidarian.

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.

The colonial cnidarian Hydractinia.

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.

Publisher Correction: Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.