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.

Nitric oxide controls shoot meristem activity via regulation of DNA methylation.

Despite the importance of Nitric Oxide (NO) as signaling molecule in both plant and animal development, the regulatory mechanisms downstream of NO remain largely unclear. Here, we show that NO is involved in Arabidopsis shoot stem cell control via modifying expression and activity of ARGONAUTE 4 (AGO4), a core component of the RNA-directed DNA Methylation (RdDM) pathway. Mutations in components of the RdDM pathway cause meristematic defects, and reduce responses of the stem cell system to NO signaling. Importantly, we find that the stem cell inducing WUSCHEL transcription factor directly interacts with AGO4 in a NO dependent manner, explaining how these two signaling systems may converge to modify DNA methylation patterns. Taken together, our results reveal that NO signaling plays an important role in controlling plant stem cell homeostasis via the regulation of de novo DNA methylation.

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.

Negative phototaxis in the photosymbiotic sea anemone Aiptasia as a potential strategy to protect symbionts from photodamage.

Photosymbiotic cnidarians generally seek bright environments so that their symbionts can be photosynthetically active. However, excess light may result in a breakdown of symbiosis due to the accumulation of photodamage in symbionts causing symbiont loss (bleaching). It is currently unknown if photosymbiotic cnidarians sense light only to regulate spawning time and to facilitate predation, or whether they also use their light-sensing capacities to protect their symbionts from photodamage. In this study, we examined how the sea anemone Aiptasia changes its behaviour when exposed to excess light. We reveal that Aiptasia polyps, when carrying symbionts, contract their bodies when exposed to high light intensities and subsequently migrate away in a direction perpendicular to the light source. Interestingly, this negative phototaxis was only evident under blue light and absent upon UV, green and red light exposure. Non-symbiotic Aiptasia did not exhibit this light response. Our study demonstrates that photosymbiotic Aiptasia polyps display negative phototactic behaviour in response to blue light, and that they also can perceive its direction, despite lacking specialized eye structures. We postulate that Aiptasia uses blue light, which penetrates seawater efficiently, as a general proxy for sunlight exposure to protect its symbionts from photodamage.

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.

Host nutrient sensing is mediated by mTOR signaling in cnidarian-dinoflagellate symbiosis.

To survive in the nutrient-poor waters of the tropics, reef-building corals rely on intracellular, photosynthetic dinoflagellate symbionts. Photosynthates produced by the symbiont are translocated to the host, and this enables corals to form the structural foundation of the most biodiverse of all marine ecosystems. Although the regulation of nutrient exchange between partners is critical for ecosystem stability and health, the mechanisms governing how nutrients are sensed, transferred, and integrated into host cell processes are largely unknown. Ubiquitous among eukaryotes, the mechanistic target of the rapamycin (mTOR) signaling pathway integrates intracellular and extracellular stimuli to influence cell growth and cell-cycle progression and to balance metabolic processes. A functional role of mTOR in the integration of host and symbiont was demonstrated in various nutritional symbioses, and a similar role of mTOR was proposed for coral-algal symbioses. Using the endosymbiosis model Aiptasia, we examined the role of mTOR signaling in both larvae and adult polyps across various stages of symbiosis. We found that symbiosis enhances cell proliferation, and using an Aiptasia-specific antibody, we localized mTOR to symbiosome membranes. We found that mTOR signaling is activated by symbiosis, while inhibition of mTOR signaling disrupts intracellular niche establishment and symbiosis altogether. Additionally, we observed that dysbiosis was a conserved response to mTOR inhibition in the larvae of a reef-building coral species. Our data confim that mTOR signaling plays a pivotal role in integrating symbiont-derived nutrients into host metabolism and symbiosis stability, ultimately allowing symbiotic cnidarians to thrive in challenging environments.

Hydrozoan sperm-specific SPKK motif-containing histone H2B variants stabilise chromatin with limited compaction.

Many animals achieve sperm chromatin compaction and stabilisation by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines during spermatogenesis. Hydrozoan cnidarians and echinoid sea urchins lack protamines and have evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N termini rich in SPK(K/R) motifs. Echinoid sperm packaging is regulated by spH2Bs. Their sperm is negatively buoyant and fertilises on the sea floor. Hydroid cnidarians undertake broadcast spawning but their sperm properties are poorly characterised. We show that Hydractinia echinata and H. symbiolongicarpus sperm chromatin possesses higher stability than somatic chromatin, with reduced accessibility to transposase Tn5 integration and to endonucleases in vitro. In contrast, nuclear dimensions are only moderately reduced in mature Hydractinia sperm. Ectopic expression of spH2B in the background of H2B.1 knockdown results in downregulation of global transcription and cell cycle arrest in embryos, without altering their nuclear density. Taken together, SPKK-containing spH2B variants act to stabilise chromatin and silence transcription in Hydractinia sperm with only limited chromatin compaction. We suggest that spH2Bs could contribute to sperm buoyancy as a reproductive adaptation.

Mitochondrial Genomes in Perkinsus Decode Conserved Frameshifts in All Genes.

Mitochondrial genomes of apicomplexans, dinoflagellates, and chrompodellids that collectively make up the Myzozoa, encode only three proteins (Cytochrome b [COB], Cytochrome c oxidase subunit 1 [COX1], Cytochrome c oxidase subunit 3 [COX3]), contain fragmented ribosomal RNAs, and display extensive recombination, RNA trans-splicing, and RNA-editing. The early-diverging Perkinsozoa is the final major myzozoan lineage whose mitochondrial genomes remained poorly characterized. Previous reports of Perkinsus genes indicated independent acquisition of non-canonical features, namely the occurrence of multiple frameshifts. To determine both ancestral myzozoan and novel perkinsozoan mitochondrial genome features, we sequenced and assembled mitochondrial genomes of four Perkinsus species. These data show a simple ancestral genome with the common reduced coding capacity but disposition for rearrangement. We identified 75 frameshifts across the four species that occur as distinct types and that are highly conserved in gene location. A decoding mechanism apparently employs unused codons at the frameshift sites that advance translation either +1 or +2 frames to the next used codon. The locations of frameshifts are seemingly positioned to regulate protein folding of the nascent protein as it emerges from the ribosome. The cox3 gene is distinct in containing only one frameshift and showing strong selection against residues that are otherwise frequently encoded at the frameshift positions in cox1 and cob. All genes lack cysteine codons implying a reduction to 19 amino acids in these genomes. Furthermore, mitochondrion-encoded rRNA fragment complements are incomplete in Perkinsus spp. but some are found in the nuclear DNA suggesting import into the organelle. Perkinsus demonstrates further remarkable trajectories of organelle genome evolution including pervasive integration of frameshift translation into genome expression.

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.

Development of the Myzozoan Aquatic Parasite Perkinsus marinus as A Versatile Experimental Genetic Model Organism.

The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation was previously achieved for Perkinsus spp. but with few tools for transgene expression and limited selection efficacy. We sought to expand the power of experimental genetic tools for Perkinsus using P. marinus as a model. We constructed a modular plasmid assembly system for expression of multiple genes simultaneously. We developed efficient selection systems for three drugs, puromycin, bleomycin and blasticidin, that are effective in as little as three weeks. We developed eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination but with transgene fragmentation including deletion of some elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide great new capacity to genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.

Dinoflagellate symbionts escape vomocytosis by host cell immune suppression.

Alveolata comprises diverse taxa of single-celled eukaryotes, many of which are renowned for their ability to live inside animal cells. Notable examples are apicomplexan parasites and dinoflagellate symbionts, the latter of which power coral reef ecosystems. Although functionally distinct, they evolved from a common, free-living ancestor and must evade their host's immune response for persistence. Both the initial cellular events that gave rise to this intracellular lifestyle and the role of host immune modulation in coral-dinoflagellate endosymbiosis are poorly understood. Here, we use a comparative approach in the cnidarian endosymbiosis model Aiptasia, which re-establishes endosymbiosis with free-living dinoflagellates every generation. We find that uptake of microalgae is largely indiscriminate, but non-symbiotic microalgae are expelled by vomocytosis, while symbionts induce host cell innate immune suppression and form a lysosomal-associated membrane protein 1-positive niche. We demonstrate that exogenous immune stimulation results in symbiont expulsion and, conversely, inhibition of canonical Toll-like receptor signalling enhances infection of host animals. Our findings indicate that symbiosis establishment is dictated by local innate immune suppression, to circumvent expulsion and promote niche formation. This work provides insight into the evolution of the cellular immune response and key steps involved in mediating endosymbiotic interactions.

Photoreceptor Diversification Accompanies the Evolution of Anthozoa.

Anthozoan corals are an ecologically important group of cnidarians, which power the productivity of reef ecosystems. They are sessile, inhabit shallow, tropical oceans and are highly dependent on sun- and moonlight to regulate sexual reproduction, phototaxis, and photosymbiosis. However, their exposure to high levels of sunlight also imposes an increased risk of UV-induced DNA damage. How have these challenging photic environments influenced photoreceptor evolution and function in these animals? To address this question, we initially screened the cnidarian photoreceptor repertoire for Anthozoa-specific signatures by a broad-scale evolutionary analysis. We compared transcriptomic data of more than 36 cnidarian species and revealed a more diverse photoreceptor repertoire in the anthozoan subphylum than in the subphylum Medusozoa. We classified the three principle opsin classes into distinct subtypes and showed that Anthozoa retained all three classes, which diversified into at least six subtypes. In contrast, in Medusozoa, only one class with a single subtype persists. Similarly, in Anthozoa, we documented three photolyase classes and two cryptochrome (CRY) classes, whereas CRYs are entirely absent in Medusozoa. Interestingly, we also identified one anthozoan CRY class, which exhibited unique tandem duplications of the core functional domains. We next explored the functionality of anthozoan photoreceptors in the model species Exaiptasia diaphana (Aiptasia), which recapitulates key photo-behaviors of corals. We show that the diverse opsin genes are differentially expressed in important life stages common to reef-building corals and Aiptasia and that CRY expression is light regulated. We thereby provide important clues linking coral evolution with photoreceptor diversification.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

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

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Transcription factor AP2 controls cnidarian germ cell induction.

Clonal animals do not sequester a germ line during embryogenesis. Instead, they have adult stem cells that contribute to somatic tissues or gametes. How germ fate is induced in these animals, and whether this process is related to bilaterian embryonic germline induction, is unknown. We show that transcription factor AP2 (Tfap2), a regulator of mammalian germ lines, acts to commit adult stem cells, known as i-cells, to the germ cell fate in the clonal cnidarian Hydractinia symbiolongicarpus Tfap2 mutants lacked germ cells and gonads. Transplanted wild-type cells rescued gonad development but not germ cell induction in Tfap2 mutants. Forced expression of Tfap2 in i-cells converted them to germ cells. Therefore, Tfap2 is a regulator of germ cell commitment across germ line-sequestering and germ line-nonsequestering animals.

The Biochemistry and Evolution of the Dinoflagellate Nucleus.

Dinoflagellates are known to possess a highly aberrant nucleus-the so-called dinokaryon-that exhibits a multitude of exceptional biological features. These include: (1) Permanently condensed chromosomes; (2) DNA in a cholesteric liquid crystalline state, (3) extremely large DNA content (up to 200 pg); and, perhaps most strikingly, (4) a deficit of histones-the canonical building blocks of all eukaryotic chromatin. Dinoflagellates belong to the Alveolata clade (dinoflagellates, apicomplexans, and ciliates) and, therefore, the biological oddities observed in dinoflagellate nuclei are derived character states. Understanding the sequence of changes that led to the dinokaryon has been difficult in the past with poor resolution of dinoflagellate phylogeny. Moreover, lack of knowledge of their molecular composition has constrained our understanding of the molecular properties of these derived nuclei. However, recent advances in the resolution of the phylogeny of dinoflagellates, particularly of the early branching taxa; the realization that divergent histone genes are present; and the discovery of dinoflagellate-specific nuclear proteins that were acquired early in dinoflagellate evolution have all thrown new light nature and evolution of the dinokaryon.

Sperm Nuclear Basic Proteins of Marine Invertebrates.

In this chapter, a short evolutionary history and comparative analysis of sperm nuclear basic proteins (SNBPs) in marine invertebrates are presented based on some of the most recent publications in the field and building upon previously published reviews on the topic. Putative functions of SNBPs in sperm chromatin beyond DNA packaging will also be discussed with a primary focus on outstanding research questions.In somatic cells of all metazoans, DNA is packaged into tightly folded and dynamically accessible chromatin by canonical histones H2A, H2B, H3 and H4. Sperm chromatin of many animals, on the other hand, is organised by small yet structurally highly heterogeneous proteins called SNBPs, which can package sperm DNA on their own or in combination with each other. In extreme cases, sperm chromatin is condensed into a volume 6-10 times smaller than that of a somatic nucleus. SNBPs are classified into three major groups: H1 histone-type proteins (H-type SNBPs), protamines (P-type SNBPs) and protamine-like proteins (PL-type SNBPs). P-type SNBPs are mostly found in vertebrates, while PL-type SNBPs are ubiquitous in many invertebrate phyla. PL-type and P-type SNBPs evolved from histone H-type SNBP precursors through vertical evolution. Porifera, Ctenophora and Crustacea, Echinoidea (phylum Echinodermata) and Hydrozoa (phylum Hydrozoa) lack SNBPs. Echinoidea and Hydrozoa, however, evolved novel nucleosomal histone variants with specific roles during spermatogenesis. Seemingly, chromatin condensation plays a critical role in the silencing and tight packing of the genome within the sperm nucleus of most animals. However, the question of what necessitates the compaction of some sperm DNA beyond classical nucleosomal packaging while other sperm function using 'normal' histones remains unanswered to date.

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.

Major transitions in dinoflagellate evolution unveiled by phylotranscriptomics.

Dinoflagellates are key species in marine environments, but they remain poorly understood in part because of their large, complex genomes, unique molecular biology, and unresolved in-group relationships. We created a taxonomically representative dataset of dinoflagellate transcriptomes and used this to infer a strongly supported phylogeny to map major morphological and molecular transitions in dinoflagellate evolution. Our results show an early-branching position of Noctiluca, monophyly of thecate (plate-bearing) dinoflagellates, and paraphyly of athecate ones. This represents unambiguous phylogenetic evidence for a single origin of the group's cellulosic theca, which we show coincided with a radiation of cellulases implicated in cell division. By integrating dinoflagellate molecular, fossil, and biogeochemical evidence, we propose a revised model for the evolution of thecal tabulations and suggest that the late acquisition of dinosterol in the group is inconsistent with dinoflagellates being the source of this biomarker in pre-Mesozoic strata. Three distantly related, fundamentally nonphotosynthetic dinoflagellates, Noctiluca, Oxyrrhis, and Dinophysis, contain cryptic plastidial metabolisms and lack alternative cytosolic pathways, suggesting that all free-living dinoflagellates are metabolically dependent on plastids. This finding led us to propose general mechanisms of dependency on plastid organelles in eukaryotes that have lost photosynthesis; it also suggests that the evolutionary origin of bioluminescence in nonphotosynthetic dinoflagellates may be linked to plastidic tetrapyrrole biosynthesis. Finally, we use our phylogenetic framework to show that dinoflagellate nuclei have recruited DNA-binding proteins in three distinct evolutionary waves, which included two independent acquisitions of bacterial histone-like proteins.

The cnidarian Hydractinia echinata employs canonical and highly adapted histones to pack its DNA.

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.