A G protein-coupled receptor mediates neuropeptide-induced oocyte maturation in the jellyfish Clytia.

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.

A developmental role for the chromatin-regulating CoREST complex in the cnidarian Nematostella vectensis.

BACKGROUND: Chromatin-modifying proteins are key players in the regulation of development and cell differentiation in animals. Most chromatin modifiers, however, predate the evolution of animal multicellularity, and how they gained new functions and became integrated into the regulatory networks underlying development is unclear. One way this may occur is the evolution of new scaffolding proteins that integrate multiple chromatin regulators into larger complexes that facilitate coordinated deposition or removal of different chromatin modifications. We test this hypothesis by analyzing the evolution of the CoREST-Lsd1-HDAC complex. RESULTS: Using phylogenetic analyses, we show that a bona fide CoREST homolog is found only in choanoflagellates and animals. We then use the sea anemone Nematostella vectensis as a model for early branching metazoans and identify a conserved CoREST complex by immunoprecipitation and mass spectrometry of an endogenously tagged Lsd1 allele. In addition to CoREST, Lsd1 and HDAC1/2 this complex contains homologs of HMG20A/B and PHF21A, two subunits that have previously only been identified in mammalian CoREST complexes. NvCoREST expression overlaps fully with that of NvLsd1 throughout development, with higher levels in differentiated neural cells. NvCoREST mutants, generated using CRISPR-Cas9, fail to develop beyond the primary polyp stage, thereby revealing essential roles during development and for the differentiation of cnidocytes that phenocopy NvLsd1 mutants. We also show that this requirement is cell autonomous using a cell-type-specific rescue approach. CONCLUSIONS: The identification of a Nematostella CoREST-Lsd1-HDAC1/2 complex, its similarity in composition with the vertebrate complex, and the near-identical expression patterns and mutant phenotypes of NvCoREST and NvLsd1 suggest that the complex was present before the last common cnidarian-bilaterian ancestor and thus represents an ancient component of the animal developmental toolkit.

Molecular characterisation of a cellular conveyor belt in Clytia medusae.

The tentacular system of Clytia hemisphaerica medusa (Cnidaria, Hydrozoa) has recently emerged as a promising experimental model to tackle the developmental mechanisms that regulate cell lineage progression in an early-diverging animal phylum. From a population of proximal stem cells, the successive steps of tentacle stinging cell (nematocyte) elaboration, are spatially ordered along a "cellular conveyor belt". Furthermore, the C. hemisphaerica tentacular system exhibits bilateral organisation, with two perpendicular polarity axes (proximo-distal and oral-aboral). We aimed to improve our knowledge of this cellular system by combining RNAseq-based differential gene expression analyses and expression studies of Wnt signalling genes. RNAseq comparisons of gene expression levels were performed (i) between the tentacular system and a control medusa deprived of all tentacles, nematogenic sites and gonads, and (ii) between three samples staggered along the cellular conveyor belt. The behaviour in these differential expression analyses of two reference gene sets (stem cell genes; nematocyte genes), as well as the relative representations of selected gene ontology categories, support the validity of the cellular conveyor belt model. Expression patterns obtained by in situ hybridisation for selected highly differentially expressed genes and for Wnt signalling genes are largely consistent with the results from RNAseq. Wnt signalling genes exhibit complex spatial deployment along both polarity axes of the tentacular system, with the Wnt/ß-catenin pathway probably acting along the oral-aboral axis rather than the proximo-distal axis. These findings reinforce the idea that, despite overall radial symmetry, cnidarians have a full potential for elaboration of bilateral structures based on finely orchestrated deployment of an ancient developmental gene toolkit.

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species.

In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has greatly advanced our understanding of biological processes, including developmental regulation. In situ protocols are today routinely followed in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acid sequence. This step is generally carried out at high temperatures and in a denaturing solution, called hybridization buffer, commonly containing 50% (v/v) formamide - a hazardous chemical. When applied to the soft-bodied hydrozoan medusa Clytia hemisphaerica, we found that this traditional hybridization approach was not fully satisfactory, causing extensive deterioration of morphology and tissue texture which compromised our observation and interpretation of results. We thus tested alternative solutions for in situ detection of gene expression and, inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8M urea solution in the hybridization buffer. Our new protocol not only yielded better morphologies and tissue consistency, but also notably improved the resolution of the signal, allowing more precise localization of gene expression and reducing aspecific staining associated with problematic areas. Given the improved results and reduced manipulation risks, we tested the urea protocol on other metazoans, two brachiopod species (Novocrania anomala and Terebratalia transversa) and the priapulid worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea during in situ hybridization offers a safer alternative, potentially of widespread use in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

Development of the aboral domain in Nematostella requires ß-catenin and the opposing activities of Six3/6 and Frizzled5/8.

The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvß-catenin regulates both oral and aboral development. In the oral hemisphere, Nvß-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvß-catenin knockdown. In addition, Nvß-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvß-catenin in the patterning of the entire oral-aboral axis of Nematostella.

The bilaterian head patterning gene six3/6 controls aboral domain development in a cnidarian.

The origin of the bilaterian head is a fundamental question for the evolution of animal body plans. The head of bilaterians develops at the anterior end of their primary body axis and is the site where the brain is located. Cnidarians, the sister group to bilaterians, lack brain-like structures and it is not clear whether the oral, the aboral, or none of the ends of the cnidarian primary body axis corresponds to the anterior domain of bilaterians. In order to understand the evolutionary origin of head development, we analysed the function of conserved genetic regulators of bilaterian anterior development in the sea anemone Nematostella vectensis. We show that orthologs of the bilaterian anterior developmental genes six3/6, foxQ2, and irx have dynamic expression patterns in the aboral region of Nematostella. Functional analyses reveal that NvSix3/6 acts upstream of NvFoxQ2a as a key regulator of the development of a broad aboral territory in Nematostella. NvSix3/6 initiates an autoregulatory feedback loop involving positive and negative regulators of FGF signalling, which subsequently results in the downregulation of NvSix3/6 and NvFoxQ2a in a small domain at the aboral pole, from which the apical organ develops. We show that signalling by NvFGFa1 is specifically required for the development of the apical organ, whereas NvSix3/6 has an earlier and broader function in the specification of the aboral territory. Our functional and gene expression data suggest that the head-forming region of bilaterians is derived from the aboral domain of the cnidarian-bilaterian ancestor.

The Xenopus doublesex-related gene Dmrt5 is required for olfactory placode neurogenesis.

The Dmrt (doublesex and mab-3 related transcription factor) genes encode a large family of evolutionarily conserved transcription factors whose function in sex specific differentiation has been well studied in all animal lineages. In vertebrates, their function is not restricted to the developing gonads. For example, Xenopus Dmrt4 is essential for neurogenesis in the olfactory system. Here we have isolated and characterized Xenopus Dmrt5 and found that it is coexpressed with Dmrt4 in the developing olfactory placodes. As Dmrt4, Dmrt5 is positively regulated in the ectoderm by neural inducers and negatively by proneural factors. Both Dmrt5 and Dmrt4 genes are also activated by the combined action of the transcription factor Otx2, broadly transcribed in the head ectoderm and of Notch signaling, activated in the anterior neural ridge. As for Dmrt4, knockdown of Dmrt5 impairs neurogenesis in the embryonic olfactory system and in neuralized animal caps. Conversely, its overexpression promotes neuronal differentiation in animal caps, a property that requires the conserved C-terminal DMA and DMB domains. We also found that the sea anenome Dmrt4/5 related gene NvDmrtb also induces neurogenesis in Xenopus animal caps and that conversely, its knockdown in Nematostella reduces elav-1 positive neurons. Together, our data identify Dmrt5 as a novel important regulator of neurogenesis whose function overlaps with that of Dmrt4 during Xenopus olfactory system development. They also suggest that Dmrt may have had a role in neurogenesis in the last common ancestor of cnidarians and bilaterians.

Expanding roles for the evolutionarily conserved Dmrt sex transcriptional regulators during embryogenesis.

Dmrt genes encode a large family of transcription factors characterized by the presence of a DM domain, an unusual zinc finger DNA binding domain. While Dmrt genes are well known for their important role in sexual development in arthropodes, nematodes and vertebrates, several new findings indicate emerging functions of this gene family in other developmental processes. Here, we provide an overview of the evolution, structure and mechanisms of action of Dmrt genes. We summarize recent findings on their function in sexual regulation and discuss more extensively the role played by these proteins in somitogenesis and neural development.

Maternally localized germ plasm mRNAs and germ cell/stem cell formation in the cnidarian Clytia.

The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm ("preformation" or "maternal inheritance") or by inductive signaling (classically termed "epigenesis" or "zygotic induction"). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent "interstitial" cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic.

Establishing Bilateral Symmetry in Hydrozoan Planula Larvae, a Review of Siphonophore Early Development.

Siphonophores are colonial hydrozoans, characterized by complex colony organization and unparalleled zooid functional specialization. Recent genomic studies have offered an evolutionary perspective on how this morphological complexity arose, but a molecular characterization of symmetry breaking in siphonophore embryonic development is still largely missing. Here, bringing together historical data on early development with new immunohistochemical data, we review the diversity of developmental trajectories that lead to the formation of bilaterally symmetric planula larvae in siphonophores. Embryonic development, up to the planula stage, is remarkably similar across siphonophore phylogeny. Then, with the appearance of the lateral endodermal thickening (= ventral endoderm), larval development diverges between taxa, differing in the location and patterning of the primary buds, chronology of budding, establishment of growth zones, and retention of larval zooids. Our work also uncovers a number of open questions in siphonophore development, including homology of different zooids, mechanisms underlying formation and maintenance of spatially restricted growth zone(s), and molecular factors establishing a secondary dorsal-ventral axis in planulae. By discussing siphonophore development and body axes within the broader cnidarian context, we then set the framework for future work on siphonophores, which is finally achievable with the advent of culturing methods.

Coevolution of the Tlx homeobox gene with medusa development (Cnidaria: Medusozoa).

Cnidarians display a wide diversity of life cycles. Among the main cnidarian clades, only Medusozoa possesses a swimming life cycle stage called the medusa, alternating with a benthic polyp stage. The medusa stage was repeatedly lost during medusozoan evolution, notably in the most diverse medusozoan class, Hydrozoa. Here, we show that the presence of the homeobox gene Tlx in Cnidaria is correlated with the presence of the medusa stage, the gene having been lost in clades that ancestrally lack a medusa (anthozoans, endocnidozoans) and in medusozoans that secondarily lost the medusa stage. Our characterization of Tlx expression indicate an upregulation of Tlx during medusa development in three distantly related medusozoans, and spatially restricted expression patterns in developing medusae in two distantly related species, the hydrozoan Podocoryna carnea and the scyphozoan Pelagia noctiluca. These results suggest that Tlx plays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage in the evolution of Hydrozoa.

Somatic stem cells express Piwi and Vasa genes in an adult ctenophore: ancient association of "germline genes" with stemness.

Stem cells are essential for animal development and adult tissue homeostasis, and the quest for an ancestral gene fingerprint of stemness is a major challenge for evolutionary developmental biology. Recent studies have indicated that a series of genes, including the transposon silencer Piwi and the translational activator Vasa, specifically involved in germline determination and maintenance in classical bilaterian models (e.g., vertebrates, fly, nematode), are more generally expressed in adult multipotent stem cells in other animals like flatworms and hydras. Since the progeny of these multipotent stem cells includes both somatic and germinal derivatives, it remains unclear whether Vasa, Piwi, and associated genes like Bruno and PL10 were ancestrally linked to stemness, or to germinal potential. We have investigated the expression of Vasa, two Piwi paralogues, Bruno and PL10 in Pleurobrachia pileus, a member of the early-diverging phylum Ctenophora, the probable sister group of cnidarians. These genes were all expressed in the male and female germlines, and with the exception of one of the Piwi paralogues, they showed similar expression patterns within somatic territories (tentacle root, comb rows, aboral sensory complex). Cytological observations and EdU DNA-labelling and long-term retention experiments revealed concentrations of stem cells closely matching these gene expression areas. These stem cell pools are spatially restricted, and each specialised in the production of particular types of somatic cells. These data unveil important aspects of cell renewal within the ctenophore body and suggest that Piwi, Vasa, Bruno, and PL10 belong to a gene network ancestrally acting in two distinct contexts: (i) the germline and (ii) stem cells, whatever the nature of their progeny.

Past, present and future of Clytia hemisphaerica as a laboratory jellyfish.

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.

Muscle systems and motility of early animals highlighted by cnidarians from the basal Cambrian.

Although fossil evidence suggests that various animal groups were able to move actively through their environment in the early stages of their evolution, virtually no direct information is available on the nature of their muscle systems. The origin of jellyfish swimming, for example, is of great interest to biologists. Exceptionally preserved muscles are described here in benthic peridermal olivooid medusozoans from the basal Cambrian of China (Kuanchuanpu Formation, ca. 535 Ma) that have direct equivalent in modern medusozoans. They consist of circular fibers distributed over the bell surface (subumbrella) and most probably have a myoepithelial origin. This is the oldest record of a muscle system in cnidarians and more generally in animals. This basic system was probably co-opted by early Cambrian jellyfish to develop capacities for jet-propelled swimming within the water column. Additional lines of fossil evidence obtained from ecdysozoans (worms and panarthropods) show that the muscle systems of early animals underwent a rapid diversification through the early Cambrian and increased their capacity to colonize a wide range of habitats both within the water column and sediment at a critical time of their evolutionary radiation.

The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes.

BACKGROUND: The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. RESULTS: Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. CONCLUSIONS: We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

Whole-animal multiplexed single-cell RNA-seq reveals transcriptional shifts across Clytia medusa cell types.

We present an organism-wide, transcriptomic cell atlas of the hydrozoan medusa Clytia hemisphaerica and describe how its component cell types respond to perturbation. Using multiplexed single-cell RNA sequencing, in which individual animals were indexed and pooled from control and perturbation conditions into a single sequencing run, we avoid artifacts from batch effects and are able to discern shifts in cell state in response to organismal perturbations. This work serves as a foundation for future studies of development, function, and regeneration in a genetically tractable jellyfish species. Moreover, we introduce a powerful workflow for high-resolution, whole-animal, multiplexed single-cell genomics that is readily adaptable to other traditional or nontraditional model organisms.

Molecular phylogenetics of Thecata (Hydrozoa, Cnidaria) reveals long-term maintenance of life history traits despite high frequency of recent character changes.

Two fundamental life cycle types are recognized among hydrozoan cnidarians, the benthic (generally colonial) polyp stage either producing pelagic sexual medusae or directly releasing gametes elaborated from an attached gonophore. The existence of intermediate forms, with polyps producing simple medusoids, has been classically considered compelling evidence in favor of phyletic gradualism. In order to gain insights about the evolution of hydrozoan life history traits, we inferred phylogenetic relationships of 142 species of Thecata (= Leptothecata, Leptomedusae), the most species-rich hydrozoan group, using 3 different ribosomal RNA markers (16S, 18S, and 28S). In conflict with morphology-derived classifications, most thecate species fell in 2 well-supported clades named here Statocysta and Macrocolonia. We inferred many independent medusa losses among Statocysta. Several instances of secondary regain of medusoids (but not of full medusa) from medusa-less ancestors were supported among Macrocolonia. Furthermore, life cycle character changes were significantly correlated with changes affecting colony shape. For both traits, changes did not reflect graded and progressive loss or gain of complexity. They were concentrated in recent branches, with intermediate character states being relatively short lived at a large evolutionary scale. This punctuational pattern supports the existence of 2 alternative stable evolutionary strategies: simple stolonal colonies with medusae (the ancestral strategy, seen in most Statocysta species) versus large complex colonies with fixed gonophores (the derived strategy, seen in most Macrocolonia species). Hypotheses of species selection are proposed to explain the apparent long-term stability of these life history traits despite a high frequency of character change. Notably, maintenance of the medusa across geological time in Statocysta might be due to higher extinction rates for species that have lost this dispersive stage.

Dynamic Evolution of the Cthrc1 Genes, a Newly Defined Collagen-Like Family.

Collagen triple helix repeat containing protein 1 (Cthrc1) is a secreted glycoprotein reported to regulate collagen deposition and to be linked to the Transforming growth factor ß/Bone morphogenetic protein and the Wnt/planar cell polarity pathways. It was first identified as being induced upon injury to rat arteries and was found to be highly expressed in multiple human cancer types. Here, we explore the phylogenetic and evolutionary trends of this metazoan gene family, previously studied only in vertebrates. We identify Cthrc1 orthologs in two distant cnidarian species, the sea anemone Nematostella vectensis and the hydrozoan Clytia hemisphaerica, both of which harbor multiple copies of this gene. We find that Cthrc1 clade-specific diversification occurred multiple times in cnidarians as well as in most metazoan clades where we detected this gene. Many other groups, such as arthropods and nematodes, have entirely lost this gene family. Most vertebrates display a single highly conserved gene, and we show that the sequence evolutionary rate of Cthrc1 drastically decreased within the gnathostome lineage. Interestingly, this reduction coincided with the origin of its conserved upstream neighboring gene, Frizzled 6 (FZD6), which in mice has been shown to functionally interact with Cthrc1. Structural modeling methods further reveal that the yet uncharacterized C-terminal domain of Cthrc1 is similar in structure to the globular C1q superfamily domain, also found in the C-termini of collagens VIII and X. Thus, our studies show that the Cthrc1 genes are a collagen-like family with a variable short collagen triple helix domain and a highly conserved C-terminal domain structure resembling the C1q family.

Pattern regulation in a regenerating jellyfish.

Jellyfish, with their tetraradial symmetry, offer a novel paradigm for addressing patterning mechanisms during regeneration. Here we show that an interplay between mechanical forces, cell migration and proliferation allows jellyfish fragments to regain shape and functionality rapidly, notably by efficient restoration of the central feeding organ (manubrium). Fragmentation first triggers actomyosin-powered remodeling that restores body umbrella shape, causing radial smooth muscle fibers to converge around 'hubs' which serve as positional landmarks. Stabilization of these hubs, and associated expression of Wnt6, depends on the configuration of the adjoining muscle fiber 'spokes'. Stabilized hubs presage the site of the manubrium blastema, whose growth is Wnt/ß-catenin dependent and fueled by both cell proliferation and long-range cell recruitment. Manubrium morphogenesis is modulated by its connections with the gastrovascular canal system. We conclude that body patterning in regenerating jellyfish emerges mainly from local interactions, triggered and directed by the remodeling process.

An improved whole life cycle culture protocol for the hydrozoan genetic model Clytia hemisphaerica.

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.This article has an associated First Person interview with the two first authors of the paper.