For marine species that reproduce by external fertilization, spawning is precisely coordinated within a local population to maximize the chances of producing offspring. Gamete release is often synchronized with respect to the diel light changes at dawn and dusk. In the hydrozoan jellyfish Clytia hemisphaerica, spawning occurs when oocyte maturation and gamete release are induced by maturation-inducing hormone (MIH) neuropeptides released from opsin-expressing cells in the gonad, directly upon light stimulus. Here, we characterize the distinct spawning cycle of a previously undescribed species Clytia sp. IZ-D, identified on the Pacific coast of Japan, which releases gametes in the evening. Clytia sp. IZ-D jellyfish spawn 14 hours after a light stimulus under a 24-hour light cycle and exhibit autonomous and synchronized spawning cycles with a 20-hour interval under constant light. We find that the female spawning cycle reflects the oocyte growth and their acquisition of competence for maturation, such that each day a new batch of growing oocytes becomes responsive to MIH at a time that correlates with the timing of actual spawning. We propose that the synchronized evening spawning in this species is controlled by an atypical circadian timing mechanism based on the progressive development of gamete competence to MIH and modulation of the opsin-controlled MIH signaling pathway. This mechanism may provide resilience to light cycle instability due to local climate variation and ensure reproductive isolation from other Clytia species by shifting the gamete release timing.

Transgenic markers and tools have revolutionised how we study cells and developing organisms. Some of the elements needed to construct those tools are universally applicable (e.g. fluorescent proteins), while others are species-specific (e.g. cis-regulatory elements driving transcription). Membrane-localising signals that target proteins to the plasma membrane have been identified in several model organisms. Unfortunately, the efficacy of these signals varies greatly across species. To address this problem, we generated a toolkit of 11 membrane-localising tags that can be screened rapidly in diverse organisms. The toolkit includes tags that target the plasma membrane through different mechanisms, including signal peptides, the attachment of lipids, and fusion with lipid-binding domains. Each tag has been fused to the red fluorescent protein mScarlet3 and placed downstream of a T7 promoter, allowing the in vitro production of mRNA that can be readily delivered in a wide range of embryos and cells of interest. Through a collaborative effort, we tested this toolkit in ten species of animals spanning diverse phyla, including chordates, echinoderms, arthropods, nematodes, annelids, flatworms and cnidarians. We identify robust membrane-localising tags in each of these animals, and in one of animals' closest relatives, the choanoflagellates. Three tags (KRas, GAP43 and Src64B) work in all of the species tested.

Body axis specification is a crucial event in animal embryogenesis and was an essential evolutionary innovation for founding the animal kingdom. This process involves two distinct components that coordinate to establish the spatial organisation of the embryo: initiation of cascades of regionalised gene expression and orientation of morphogenetic processes such as body elongation. Intense interest in the first component has revealed Wnt/β-catenin signalling as ancestrally responsible for initiating regional gene expression, but the evolutionary origin of oriented morphogenesis has received little attention. Here, by addressing the cell and morphological basis of body axis development in embryos of the cnidarian Clytia hemisphaerica, we have uncovered a simple and likely ancestral coordination mechanism between Wnt/β-catenin signalling and directed morphogenesis. We show that the ligand Wnt3, known to initiate oral gene expression via localised Wnt/β-catenin pathway activation, also has a key β-catenin-independent role in globally orienting planar cell polarity (PCP) to direct morphogenesis along the oral-aboral axis. This PCP orientation occurs in two distinct steps: local orientation by Wnt3 and global propagation by conserved core PCP protein interactions along the body axis. From these findings, we propose novel scenarios for PCP-driven symmetry-breaking underlying the emergence of the animal body plan.

For marine species that reproduce by external fertilisation, spawning is precisely coordinated within a local cohort to maximise the chances of producing offspring. Gamete release is often synchronised with respect to the daily light changes at sunrise and sunset. In the hydrozoan jellyfish Clytia hemisphaerica , morning spawning occurs when oocyte maturation and gamete release are induced by maturation-inducing hormone (MIH) neuropeptides released from opsin-expressing cells in the gonad directly upon light stimulus. Here, we characterise the distinct spawning cycle of a previously undescribed species Clytia sp. IZ-D identified on the Pacific coast of Japan, which releases gametes in the evening. Clytia sp. IZ-D jellyfish spawn 14 hours after light stimuli under a 24-hour light-dark cycle and exhibit autonomous and synchronised spawning cycles with a 20-hour interval under constant light. We found that the female spawning cycle reflects the growth and acquisition of maturation competence by oocytes, such that each day a new batch of growing oocytes becomes responsive to MIH at a time that correlates with the timing of actual spawning. We propose that the synchronised evening spawning in this species is controlled by an unusual circadian timing mechanism based on the progressive development of gamete competence to MIH and modulation of the opsin-controlled MIH signalling pathway. This mechanism may provide resilience to light cycle instability due to local climate variation and ensure reproductive isolation from other Clytia species by alternating the gamete release timing. ### Competing Interest Statement The authors have declared no competing interest. * MIH : Maturation-Inducing Hormone hps : hours post-spawning GVBD : Germinal Vesicle Breakdown PBFI/PBFII : 1st and 2nd Polar Body Formation MRC : MIH-Reactive Competence

Adult hydrozoan cnidarians undergo extensive tissue turnover, generating neural cell types including nematocytes (stinging cells) from interstitial stem cells (i-cells) expressing stemness proteins such as Piwi and Nanos. The contribution of i-cells during embryogenesis, however, has been unclear. Here we address the origin of neural cells during development of the Clytia hemisphaerica ‘planula’ larva. Marker gene in situ hybridisation revealed that Piwi/Nanos1-expressing cells within the presumptive endoderm of the early gastrula generate a substantial pool of nematoblasts, some of which migrate and differentiate into the planula ectoderm. Neurogenic transcription factor expression showed a markedly distinct pattern, predominantly in a basal layer of the aboral/lateral ectoderm during gastrula stages. Embryo bisection and lineage tracing experiments confirmed that neurosensory and secretory cell types derive from gastrula ectoderm, while nematocytes and at least some ganglionic cells derive from i-cells. Knockdown and inhibitor treatments revealed steps of both neurogenesis and nematogenesis regulated by Wnt-β-catenin. We conclude that two distinct neurogenesis pathways operate in parallel during Clytia embryogenesis, one involving aboral ectoderm delamination, and one generating mainly nematocytes from i-cell-like precursors. ### Competing Interest Statement The authors have declared no competing interest. European Union, https://ror.org/019w4f821, 766053

The hydrozoan species Clytia hemisphaerica was selected in the mid-2000s to address the cellular and molecular basis of body axis specification in a cnidarian, providing a reliable daily source of gametes and building on a rich foundation of experimental embryology. The many practical advantages of this species include genetic uniformity of laboratory jellyfish, derived clonally from easily-propagated polyp colonies. Phylogenetic distance from other laboratory models adds value in providing an evolutionary perspective on many biological questions. Here we outline the current state of the art regarding available experimental approaches and in silico resources, and illustrate the contributions of Clytia to understanding embryo patterning mechanisms, oogenesis and regeneration. Looking forward, the recent establishment of transgenesis methods is now allowing gene function and imaging studies at adult stages, making Clytia particularly attractive for whole organism biology studies across fields and extending its scientific impact far beyond the original question of interest.

Jellyfish are radially symmetric organisms without a brain that arose more than 500 million years ago. They achieve organismal behaviors through coordinated interactions between autonomously functioning body parts. Jellyfish neurons have been studied electrophysiologically, but not at the systems level. We introduce Clytia hemisphaerica as a transparent, genetically tractable jellyfish model for systems and evolutionary neuroscience. We generate stable F1 transgenic lines for cell-type-specific conditional ablation and whole-organism GCaMP imaging. Using these tools and computational analyses, we find that an apparently diffuse network of RFamide-expressing umbrellar neurons is functionally subdivided into a series of spatially localized subassemblies whose synchronous activation controls directional food transfer from the tentacles to the mouth. These data reveal an unanticipated degree of structured neural organization in this species. Clytia affords a platform for systems-level studies of neural function, behavior, and evolution within a clade of marine organisms with growing ecological and economic importance.

Jellyfish are free-swimming, radially symmetric organisms with complex behaviors that arise from coordinated interactions between distinct, autonomously functioning body parts. This behavioral complexity evolved without a corresponding cephalization of the nervous system. The systems-level neural mechanisms through which such decentralized control is achieved remain unclear. Here, we address this question using the jellyfish, Clytia, and present it as a new neuroscience model. We describe a coordinated, asymmetric behavior in which food is passed from the umbrellar margin to the central mouth via directed margin folding. Using newly developed transgenic jellyfish lines to ablate or image specific neuronal subpopulations, we find, unexpectedly, that margin folding reflects the local activation of neural subnetworks that tile the umbrella. Modeling suggests that this structured ensemble activity emerges from sparse, local connectivity rules. These findings reveal how an organismal behavior can emerge from local interactions between functional modules in the absence of a central brain.

The reproductive hormones that trigger oocyte meiotic maturation and release from the ovary vary greatly between animal species. Identification of receptors for these maturation-inducing hormones (MIHs) and understanding how they initiate the largely conserved maturation process remain important challenges. In hydrozoan cnidarians including the jellyfish Clytia hemisphaerica, MIH comprises neuropeptides released from somatic cells of the gonad. We identified the receptor (MIHR) for these MIH neuropeptides in Clytia using cell culture-based "deorphanization" of candidate oocyte-expressed G protein-coupled receptors (GPCRs). MIHR mutant jellyfish generated using CRISPR-Cas9 editing had severe defects in gamete development or in spawning both in males and females. Female gonads, or oocytes isolated from MIHR mutants, failed to respond to synthetic MIH. Treatment with the cAMP analogue Br-cAMP to mimic cAMP rise at maturation onset rescued meiotic maturation and spawning. Injection of inhibitory antibodies to the alpha subunit of the Gs heterodimeric protein (GαS) into wild-type oocytes phenocopied the MIHR mutants. These results provide the molecular links between MIH stimulation and meiotic maturation initiation in hydrozoan oocytes. Molecular phylogeny grouped Clytia MIHR with a subset of bilaterian neuropeptide receptors, including neuropeptide Y, gonadotropin inhibitory hormone (GnIH), pyroglutamylated RFamide, and luqin, all upstream regulators of sexual reproduction. This identification and functional characterization of a cnidarian peptide GPCR advances our understanding of oocyte maturation initiation and sheds light on the evolution of neuropeptide-hormone systems.

The jellyfish species Clytia hemisphaerica (Cnidaria, Hydrozoa) has emerged as a new experimental model animal in the last decade. Favorable characteristics include a fully transparent body suitable for microscopy, daily gamete production and a relatively short life cycle. Furthermore, whole genome sequence assembly and efficient gene editing techniques using CRISPR/Cas9 have opened new possibilities for genetic studies. The quasi-immortal vegetatively-growing polyp colony stage provides a practical means to maintain mutant strains. In the context of developing Clytia as a genetic model, we report here an improved whole life cycle culture method including an aquarium tank system designed for culture of the tiny jellyfish form. We have compared different feeding regimes using Artemia larvae as food and demonstrate that the stage-dependent feeding control is the key for rapid and reliable medusa and polyp rearing. Metamorphosis of the planula larvae into a polyp colony can be induced efficiently using a new synthetic peptide. The optimized procedures detailed here make it practical to generate genetically modified Clytia strains and to maintain their whole life cycle in the laboratory.

Jellyfish (medusae) are a distinctive life-cycle stage of medusozoan cnidarians. They are major marine predators, with integrated neurosensory, muscular and organ systems. The genetic foundations of this complex form are largely unknown. We report the draft genome of the hydrozoan jellyfish Clytia hemisphaerica and use multiple transcriptomes to determine gene use across life-cycle stages. Medusa, planula larva and polyp are each characterised by distinct transcriptome signatures reflecting abrupt life cycle transitions, and all deploy a mixture of phylogenetically old and new genes. Medusa specific transcription factors, including many with bilaterian orthologs, associate with diverse neurosensory structures. Compared to Clytia , the polyp-only hydrozoan Hydra has lost many of the medusa-expressed transcription factors, despite similar overall rates of gene content and sequence evolution. Absence of expression and gene loss among Clytia orthologs of genes patterning the anthozoan aboral pole, secondary axis and endomesoderm support simplification of planulae and polyps in Hydrozoa, including loss of bilateral symmetry. Consequently, although the polyp and planula are generally considered the ancestral cnidarian forms, in Clytia the medusa maximally deploys ancestral cnidarian–bilaterian transcription factor gene complexity.

Across the animal kingdom, environmental light cues are widely involved in regulating gamete release, but the molecular and cellular bases of the photoresponsive mechanisms are poorly understood. In hydrozoan jellyfish, spawning is triggered by dark-light or light-dark transitions acting on the gonad, and is mediated by oocyte maturation-inducing neuropeptide hormones (MIHs) released from the ectoderm. We determined in Clytia hemisphaerica that blue-cyan light triggers spawning in isolated gonads. A candidate opsin (Opsin9) was found co-expressed with MIH within specialised ectodermal cells. Opsin9 knockout jellyfish generated by CRISPR/Cas9 failed to undergo oocyte maturation and spawning, a phenotype reversible by synthetic MIH. Gamete maturation and release in Clytia is thus regulated by gonadal photosensory-neurosecretory cells that secrete MIH in response to light via Opsin9. Similar cells in ancestral eumetazoans may have allowed tissue-level photo-regulation of diverse behaviours, a feature elaborated in cnidarians in parallel with expansion of the opsin gene family.

Targeted mutagenesis using CRISPR/Cas9 technology has been shown to be a powerful approach to examine gene function in diverse metazoan species. One common drawback is that mixed genotypes, and thus variable phenotypes, arise in the F0 generation because incorrect DNA repair produces different mutations amongst cells of the developing embryo. We report here an effective method for gene knockout (KO) in the hydrozoan Clytia hemisphaerica, by injection into the egg of Cas9/sgRNA ribonucleoprotein complex (RNP). Expected phenotypes were observed in the F0 generation when targeting endogenous GFP genes, which abolished fluorescence in embryos, or CheRfx123 (that codes for a conserved master transcriptional regulator for ciliogenesis) which caused sperm motility defects. When high concentrations of Cas9 RNP were used, the mutations in target genes at F0 polyp or jellyfish stages were not random but consisted predominantly of one or two specific deletions between pairs of short microhomologies flanking the cleavage site. Such microhomology-mediated (MM) deletion is most likely caused by microhomology-mediated end-joining (MMEJ), which may be favoured in early stage embryos. This finding makes it very easy to isolate uniform, largely non-mosaic mutants with predictable genotypes in the F0 generation in Clytia, allowing rapid and reliable phenotype assessment.

The fresh water polyp Hydra provides textbook experimental demonstration of positional information gradients and regeneration processes. Developmental biologists are thus familiar with Hydra, but may not appreciate that it is a relatively simple member of the Hydrozoa, a group of mostly marine cnidarians with complex and diverse life cycles, exhibiting extensive phenotypic plasticity and regenerative capabilities. Hydrozoan species offer extensive opportunities to address many developmental mechanisms relevant across the animal kingdom. Here we review recent work from non-Hydra hydrozoans - hydromedusae, hydroids and siphonophores - shedding light on mechanisms of oogenesis, embryonic patterning, allorecognition, stem cell regulation and regeneration. We also highlight potential research directions in which hydrozoan diversity can illuminate the evolution of developmental processes at micro-and macro-evolutionary time scales.

More and more genomes are sequenced and a great range of biological questions can be examined at the genomic level in a growing number of organisms. Testing the function of genome features, from gene networks, genome organization, conserved non-coding sequences to microRNAs, and, more generally, experimentally addressing the genotype-phenotype relationship is now possible owing to the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 revolution of genome editing. In the present review, we give a brief overview of the CRISPR/Cas9 toolbox and different strategies for genome editing currently available. We list the first examples of applications to marine organisms and also draw from studies in more common laboratory models to suggest both guidelines for design of genome editing experiments as well as discuss challenges specific to marine organisms. In addition, we discuss future perspectives, including applications of CRISPR/Cas9 to base editing and targeted reprogramming of gene transcription.

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at “oral” and “aboral” poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.

Functional and morphological planar cell polarity (PCP) oriented along the oral-aboral body axis is clearly evident in the ectoderm of torpedo-shaped planula larvae of hydrozoan cnidarians such as Clytia hemisphaerica. Ectodermal epithelial cells bear a single motile cilium the beating of which is coordinated between cells, causing directional swimming towards the blunt, aboral pole. We have characterised PCP during Clytia larval development and addressed its molecular basis. PCP is first detectable in ectodermal cells during gastrulation as coordinated basal body positioning, the ciliary root becoming consistently positioned on the oral side of the apical surface of the cell. At later stages, more pronounced structural polarity develops around the base of each cilium in relation to the cilia beating direction, including a characteristic asymmetric cortical actin organisation. Morpholino antisense oligonucleotide and mRNA injection studies showed that PCP development requires the Clytia orthologues of the core Fz-PCP pathway components Strabismus (CheStbm), Frizzled (CheFz1) and Dishevelled (CheDsh). Morpholinos targeting any of these components prevented ectodermal PCP, disrupted ciliogenesis and inhibited embryo elongation during gastrulation, which involves cell intercalation. We show that YFP-tagged CheStbm adopts a polarised intracellular distribution, localising preferentially to the aboral boundary of each cell, as has been demonstrated in Drosophila and some vertebrate PCP studies. Our findings in a cnidarian strongly suggest that the Fz-PCP pathway is a highly conserved and evolutionary ancient metazoan feature that is probably widely responsible for oriented swimming and/or feeding in relation to body axis in the many ciliated larval types found throughout the animal kingdom.

Taxonomically restricted genes or lineage-specific genes contribute to morphological diversification in metazoans and provide unique functions for particular taxa in adapting to specific environments. To understand how such genes arise and participate in morphological evolution, we have investigated a gene called nematogalectin in Hydra, which has a structural role in the formation of nematocysts, stinging organelles that are unique to the phylum Cnidaria. Nematogalectin is a 28-kDa protein with an N-terminal GlyXY domain (glycine followed by two hydrophobic amino acids), which can form a collagen triple helix, followed by a galactose-binding lectin domain. Alternative splicing of the nematogalectin transcript allows the gene to encode two proteins, nematogalectin A and nematogalectin B. We demonstrate that expression of nematogalectin A and B is mutually exclusive in different nematocyst types: Desmonemes express nematogalectin B, whereas stenoteles and isorhizas express nematogalectin B early in differentiation, followed by nematogalectin A. Like Hydra, the marine hydrozoan Clytia also has two nematogalectin transcripts, which are expressed in different nematocyte types. By comparison, anthozoans have only one nematogalectin gene. Gene phylogeny indicates that tandem duplication of nematogalectin B exons gave rise to nematogalectin A before the divergence of Anthozoa and Medusozoa and that nematogalectin A was subsequently lost in Anthozoa. The emergence of nematogalectin A may have played a role in the morphological diversification of nematocysts in the medusozoan lineage.

Clytia hemisphaerica, a member of the early-branching animal phylum Cnidaria, is emerging rapidly as an experimental model for studies in developmental biology and evolution. Unlike the two existing genome-sequenced cnidarian models Nematostella and Hydra, Clytia has a free-swimming jellyfish form, which like "higher" animals (the Bilateria) has a complex organization including striated musculature, specialized nervous system and structured sensory and reproductive organs. Clytia has proved well suited to laboratory culture and to gene function analysis during early development. Initial studies have shed light on the origins of embryonic polarity and of the nematocyte as a specialized neurosensory cell, and on the regulation of oocyte maturation. With a full genome sequence soon to become available, and a clear potential for genetic approaches, Clytia is well placed to provide invaluable information on core mechanisms in cell and developmental biology, and on the evolution of key features of animal body plans.

Recent thought on genome evolution has focused on the creation of new genes and changes in regulatory mechanisms while ignoring the role of selective gene loss in shaping genomes. Using data from two cnidarians, the jellyfish Clytia and the coral Acropora, we examined the relative significance of new 'taxonomically restricted' genes and selectively retained ancestral genes in enabling the evolution of novel traits. Consistent with its more complex life-cycle, the proportion of novel genes identified in Clytia was higher than that in the 'polyp only' cnidarians Nematostella and Hydra, but each of these cnidarians has retained a proportion of ancestral genes not present in the other two. The ubiquity and nearstochastic nature of gene loss can explain the discord between patterns of gene distribution and taxonomy.