Evelyn Houliston

Lucas Leclère

Catriona Munro

Richard R Copley

Tsuyoshi Momose

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

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

Evelyn Houliston, Lucas Leclère, Catriona Munro, Richard R Copley,

Tsuyoshi Momose

To cite this version:

Evelyn Houliston, Lucas Leclère, Catriona Munro, Richard R Copley, Tsuyoshi Momose. Past, present and future of Clytia hemisphaerica as a laboratory jellyfish. Current Topics in Developmental Biology, 2022, Emerging Model Systems in Developmental Biology, 147, pp.121-151. 10.1016/bs.ctdb.2021.12.014. hal-03346217

1) Introduction and historical background

Our original, pragmatic, motivation to select Clytia hemisphaerica as a laboratory model was because of unique features suited for studying embryonic axis specification. Specifically, we were seeking an experimentally tractable species in which embryo polarity was established de novo following fertilization, rather than being directed by positional cues ("maternal determinants") in the egg. Such determinants, often as localized mRNAs, are responsible for axis specification in almost all traditional model species, but the key events of symmetry breaking are challenging to image and analyse because they occur over protracted periods of oogenesis within the mother's ovary [START_REF] Bashirullah | RNA localization in development[END_REF].

An important body of previous experimental embryology indicated at the time (mid 2000s) that animals from the clades Ctenophora or Cnidaria might offer uniquely accessible material for studying embryo polarity establishment. Dye tracing and egg manipulation experiments on many species from these taxa, including the cnidarian Clytia (a.k.a. Phialidium) hydrozoan jellyfish species, had indicated that the orientation of their single principle body axis was determined de novo in the fertilized egg, the position of the zygote nucleus dictating that of the future 'oral' pole [START_REF] Freeman | The role of cleavage in the localization of developmental potential in the ctenophore Mnemiopsis leidyi[END_REF][START_REF] Freeman | The establishment of the oral-aboral axis in the ctenophore embryo[END_REF](Freeman, , 1981a)). Thus embryo polarity establishment mechanisms in cnidarian and ctenophore species could potentially be analyzed in embryos developing in sea water on the bench.

Against expectations it turned out that the embryonic axis in Clytia is positioned by localized mRNAs that cause polarized Wnt pathway activation, revealing close and unexpected parallels with axis determination in traditional model species from Bilateria and allowing the historical experiments to be reinterpreted in a new light (see Section 4). By then, Clytia had proved its worth as an experimental model, and the initial investment in techniques and resources provided a platform for further studies on many topics. This included early realisation that the ovary of female Clytia has particular properties that make it an exceptional model for studying the regulation of oogenesis, oocyte maturation, and spawning, as detailed in Section 3. Furthermore, Clytia's phylogenetic distance from other laboratory models, including the increasingly popular cnidarian model Nematostella vectensis [START_REF] Layden | The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate development and regeneration[END_REF], proved increasingly to have significant added value in providing an evolutionary perspective on many processes, as illustrated across different sections of this chapter. In parallel the Clytia model has been exploited for more explicit 'EvoDevo' studies to map the genomic changes accompanying animal evolution, notably in the early years by Michael Manuel and colleagues in Paris, with possibilities augmented more recently by availability of the whole genome sequence as outlined in Section 6.

The choice of Clytia hemisphaerica among many candidate cnidarian species available in Villefranche was essentially based on ease of culture and suitability for embryo experimentation.

Clytia jellyfish reliably provide a daily supply of large (180 µm diameter), transparent, eggs devoid of extracellular envelopes or jelly, which are robust to manipulate and develop reliably. Crucially, previous studies on the site had shown that C. hemisphaerica could be cultured as 'immortal'

vegetatively propagating polyp colonies supplying medusae by constant budding (Figure 1; [START_REF] Carré | Origin of germ cells, sex determination, and sex inversion in medusae of the genus Clytia (Hydrozoa, leptomedusae): the influence of temperature[END_REF]. The adults are a convenient size to be maintained in modestly-dimensioned beaker or tank based aquarium systems, and survive on a simple diet of Artemia nauplii [START_REF] Lechable | An improved whole life cycle culture protocol for the hydrozoan genetic model Clytia hemisphaerica[END_REF].

Other unanticipated practical advantages became apparent as the model developed. The clonal origin of jellyfish by vegetative budding from stable polyp colonies was a huge boon, not only increasing reproducibility between experiments but meaning that when ESTs [START_REF] Chevalier | Polarised expression of FoxB and FoxQ2 genes during development of the hydrozoan Clytia hemisphaerica[END_REF][START_REF] Forêt | New tricks with old genes: the genetic bases of novel cnidarian traits[END_REF] and later full transcriptome and genome resources [START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF] were established, the sequences exactly matched those of the laboratory animals. This alleviated the polymorphism problems experienced for many marine animal models collected from wild populations, facilitating successful selection of sequence targets for gene knockdown approaches such as Morpholino antisense and later CRISPR/Cas9 (Section 2). The partial temperature dependence of colony sex determination [START_REF] Carré | Origin of germ cells, sex determination, and sex inversion in medusae of the genus Clytia (Hydrozoa, leptomedusae): the influence of temperature[END_REF]) allowed a set of highly genetically-related core colonies to be established via self-crossing [START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF]. These colonies can be conveniently distributed to colleagues around the world as cuttings on glass slides [START_REF] Lechable | An improved whole life cycle culture protocol for the hydrozoan genetic model Clytia hemisphaerica[END_REF]. Since the first 'molecular era' papers on Clytia [START_REF] Chevalier | Polarised expression of FoxB and FoxQ2 genes during development of the hydrozoan Clytia hemisphaerica[END_REF][START_REF] Jager | Expansion of the SOX gene family predated the emergence of the Bilateria[END_REF][START_REF] Momose | Two oppositely localised frizzled RNAs as axis determinants in a cnidarian embryo[END_REF][START_REF] Momose | A maternally localised Wnt ligand required for axial patterning in the cnidarian Clytia hemisphaerica[END_REF] which introduced ESTs, in situ hybridisation and gene function analysis via Morpholino/mRNA microinjection, technical possibilities have steadily widened and techniques refined. These advances, along with the progressive accumulation of in silico resources, are detailed in Section 2 and Table 1. In parallel, attention has extended from embryos and larvae to the adult medusa stage. This one-centimeter diameter transparent, actively swimming and sexually reproducing animal (Figure 1E) is already showing great promise as a model in the fields of regeneration and neural systems biology (see Section 5). Clytia thus illustrates how the impact of a new model organism can extend far beyond the original field of interest.

2) Practical State of the Art

Animal culture and life cycle

Clytia hemisphaerica culture methods have been refined progressively over the last 15 years, and a detailed update is available [START_REF] Lechable | An improved whole life cycle culture protocol for the hydrozoan genetic model Clytia hemisphaerica[END_REF]. A specific challenge compared to polyp-only cnidarian model species (notably Hydra, Nematostella and Hydractinia) is maintenance of the fragile and planktonic jellyfish (medusa) stage (Figure 1D,E), which requires constant gentle water flow. A convenient solution is to use adapted "Kreisel'' tanks for juvenile and adult stages. Clytia jellyfish reach sexual maturity 2~3 weeks after budding from specialized polyps called gonozooids (Figure 1C). They then release gametes for the rest of their lifespan, typically around 2 months total. Between 400 and 1000 eggs can be collected every day from a batch of 30-40 females in one tank, and successfully fertilized within one hour of spawning by mixing with sperm released in parallel from a few males. Embryogenesis (see Section 3) results in formation of a planula larva (Figure 1A), described in detail in Section 4. After metamorphosis of the planula into a primary feeding polyp (gastrozooid) on glass slides (Figure 1B), extension of the stolon network creates a polyp colony, which is nourished by prey captured by the gastrozooids. Individual gastrozooids and gonozooids periodically atrophy, but colony growth allows their indefinite renewal. The colony is thus an extremely convenient form to amplify, maintain and distribute genetically uniform strains including mutants without need for crossing.

Functional genomics

Functional approaches, notably gene loss-and gain-of-function, enable a range of molecular and genetics studies. For C. hemisphaerica as a model, the first successful approach was microinjection of Morpholino antisense oligonucleotide (MO) and synthetic mRNAs into eggs before fertilization. MOs designed to block mRNA splicing or translation generated reproducible loss-of polarity phenotypes for developmental regulators, starting with the Wnt receptors Fz1 and Fz3 [START_REF] Momose | Two oppositely localised frizzled RNAs as axis determinants in a cnidarian embryo[END_REF]. Other examples are illustrated in Sections 3-4 below. Injection of in vitro synthesized mRNA allows gene overexpression, verification of MO phenotype specificity, and expression of fluorescent protein markers to visualize protein localization [START_REF] Momose | A conserved function for Strabismus in establishing planar cell polarity in the ciliated ectoderm during cnidarian larval development[END_REF].

MO and mRNA egg microinjection can address biological events only between early blastula stage and metamorphosis, so other gene manipulation methods are required for studying processes in the polyp and jellyfish stages. Efforts to achieve reliable and effective targeted gene knockout started in the early 2010s, initially exploring the use of TALEN [START_REF] Zhang | Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription[END_REF] before switching to the CRISPR/Cas9 system [START_REF] Momose | High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion[END_REF]. We were able to establish a protocol that consistently generates gene knock-out Clytia strains (ie F0 polyp colonies) with very low mosaicism in a single generation, involving prediction of the dominant mutation that will be induced by each sgRNA [START_REF] Momose | High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion[END_REF]. This method exploits the observation that the Microhomology Mediated End-Joining (MMEJ) pathway is the principal pathway for DNA double strand break repair active in the early Clytia embryo. It has so far been used to knock-out endogenous GFPs (Figure 1D) as well as to identify key genes mediating oocyte maturation and spawning (see Section 3).

Development of high-efficiency transgenesis and gene knock-in approaches has been more difficult.

The MMEJ pathway likely favours rapid and error-prone DNA repair over homology-mediated insertion, explaining the relatively low incorporation of transgenes achieved in trials with CRISPR/Cas9 methods using various template DNA types. Observed accumulation of injected DNA around nuclei (T.M. and B. Weissbourd unpublished) suggests that effective DNA delivery to the genomic target is also an issue. A recent breakthrough is the successful establishment of a transposonmediated transgenesis protocol, through extensive collaboration between Caltech (B. Weissbourd and D. J. Anderson) and Villefranche teams. This achieves highly efficient random transgenesis using the Tol2 transposon vector system, co-injected with purified Tol2 protein [START_REF] Weissbourd | Functional modules within a distributed neural network control feeding in a model medusa[END_REF]. A first study successfully employed this method to create a stable F1 transgenic strain expressing the calcium indicator GCaMP in RFamide-expressing neurons in jellyfish [START_REF] Weissbourd | Functional modules within a distributed neural network control feeding in a model medusa[END_REF]. The use of polycistronic expression with 2A-peptide [START_REF] Liu | Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector[END_REF], further allowed metronidazoleinducible specific ablation of RFamide positive neurons by driving expression of nitroreductase with the ChePP5 promoter. So far, strong and uniform transgene expression has been obtained using promoters from a non-muscle actin gene and from the RFamide precursor gene ChePP5. Methods to predict and select promoter sequences are currently not fully established. Further development will be aided by more information concerning the gene regulatory landscape in Clytia.

Medusa cell type atlas and single cell sequencing

The medusa (jellyfish) stage is the sexual phase of the life cycle, and has much more complex anatomy than the polyp stage. Clytia jellyfish are tetraradial, with 4 gonads each connected via radial canals to the manubrium (mouth) in the center and the circular canal around the periphery (Figure 2A). The circular canal bears the tentacle bulbs, which continually replenish the tentacles. Adjacent to the circular canal, positioned between each tentacle bulb, are sensory balance organs called statocysts [START_REF] Singla | Statocysts of hydromedusae[END_REF]. The nervous system consists of various nerve nets and two nerve rings running around the margin of the bell, connected to the statocysts. The jellyfish has both smooth muscle (radially orientated) and striated muscle (circularly orientated). The nervous system and muscles enable complex behaviours such as swimming and feeding [START_REF] Houliston | Clytia hemisphaerica: a jellyfish cousin joins the laboratory[END_REF][START_REF] Leclère | Diversity of Cnidarian Muscles: Function, Anatomy, Development and Regeneration[END_REF][START_REF] Weissbourd | Functional modules within a distributed neural network control feeding in a model medusa[END_REF]. Until recently, the diversity of different cell types comprising the Clytia jellyfish was underexplored. A significant addition to available resources is a cell type atlas documenting the presence, distribution and transcriptomes of 36 distinct cell types [START_REF] Chari | Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types[END_REF]. This cell atlas was derived from analysis of single cell transcriptome data (scRNAseq) combined with in situ hybridisation to localize the expression of marker genes.

3) Gametogenesis

The Clytia gonad is one of the simplest organs in the animal kingdom. Its basic structure comprises the two canonical cnidarian body layers, with the basal surfaces of the epidermal and gastrodermal epithelial cells facing each other across a common, loosely-structured extracellular matrix (ECM).

Within this ECM layer, gametogenic cells proliferate and differentiate. In female medusae, daily batches of small oocytes grow from around 50µm diameter to the full size of around 180 µm ready for the spawning cue (Amiel and Houliston, 2009;[START_REF] Jessus | Managing the Oocyte Meiotic Arrest-Lessons from Frogs and Jellyfish[END_REF]Figure 2A). The four gonads hang as pouches beneath the medusa bell, with the gonad gastrodermis derived from localized thickening of the radial canals and made up of specialized digestive-absorptive and secretory cell types (Figure 2A, D) [START_REF] Chari | Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types[END_REF].

Two principal features render Clytia gonads particularly suited to gametogenesis research compared to the most studied species in this field (notably Xenopus, mouse and starfish; see [START_REF] Jessus | Managing the Oocyte Meiotic Arrest-Lessons from Frogs and Jellyfish[END_REF].

Firstly, they are highly transparent, allowing the entire progression of events to be observed in whole animals, live or fixed. Secondly, they function largely autonomously, such that gonads separated by simple dissection from adult mature jellyfish support repeated cycles of gamete production and release for several days until nutritional resources (including recycled somatic tissue) are exhausted [START_REF] Freeman | The role of oocyte maturation in the ontogeny of the fertilization site in the hydrozoan Hydractinia echinata[END_REF][START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF]. So far, molecular studies of oogenesis have focussed mainly on females. This reflects the historical interest in maternal determinants for axis specification, as outlined in sections 1 and 3. Identification of the maternal determinant mRNAs Wnt3, Fz1 and Fz3 allowed the cellular mechanisms responsible for their localisation to be linked to different oogenesis events: Fz1 mRNA localizes to the animal hemisphere (future oral) cytoplasm by a microtubule-based mechanism during the major daily oocyte growth phase (Figure 2B), while Wnt3 and Fz3mRNA segregate respectively to oral and aboral domains of the oocyte cortex during the two hour process of lightinduced oocyte maturation (Figure 2C; Amiel & Houliston, 2009).

Oocyte maturation

Oocyte meiotic maturation transforms a fully grown, tetraploid, diplotene oocyte into a haploid gamete ready to be released from the gonad for fertilization. Completion of the first meiotic division terminates in emission of a first polar body containing one set of replicated homologous chromosomes, the second meiotic division separates sister chromatids and expels one set in the second polar body. In Clytia, meiotic maturation ends in an unfertilized egg containing a haploid female pronucleus (Figure 2C). These events can be followed in isolated gonads exposed to light after darkness, or in isolated oocytes treated with oocyte maturation hormone (MIH) or with cell-permeable cAMP analogues (Amiel et al., 2009;[START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF]. As extensively discussed recently in a recent review [START_REF] Jessus | Managing the Oocyte Meiotic Arrest-Lessons from Frogs and Jellyfish[END_REF] they are regulated by core kinase systems highly conserved across the animal kingdom: the Cdk1/Cyclin B complex (also known as MPF) active during each M phase, MAP-kinase which progressively activates as maturation proceeds, and cAMP-dependent kinase (PKA) activation during the initial triggering mechanism [START_REF] Freeman | The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans[END_REF][START_REF] Takeda | Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae[END_REF]. A study focussed on Clytia orthologues of the animal oocyte cytoplasmic kinase Mos, which initiates MAP-kinase activation by phosphorylating the MAP-kinsase kinase MEK in animal oocytes, illustrates how Clytia can reveal both core and novel features of this regulation (Amiel et al., 2009).

Mos activity is regulated by the timing of its translation during meiotic maturation, as extensively analysed in the Xenopus and mouse vertebrate models [START_REF] Gebauer | Synthesis and function of Mos: the control switch of vertebrate oocyte meiosis[END_REF]. In Clytia, hydrozoan Mos gene family expansion has generated several paralogs, including two expressed in oocytes. Morpholino-mediated inhibition of their translation revealed a conserved role for Clytia CheMos1 in the oocyte-specific processes of polar body formation and post-meiotic arrest, but suggest that CheMos2 has evolved a distinct function in preparing oocytes for maturation. Since the kinase activities of the two proteins appear equivalent, this difference appears to be mediated by evolution of UTR sequence mediating translation timing (Amiel et al., 2009). It will be of interest to explore in Clytia the role and evolution of other oocytes regulatory kinases such as PKA and Greatwall, to help understand how oocyte maturation regulation has adapted to different speciesspecific requirements to synchronize gamete release and thus optimize reproductive success.

Triggering oocyte maturation and spawning

Studies exploiting CRISPR/Cas9-mediated gene knockdown have dissected the initiation mechanism for oocyte meiotic maturation in Clytia. This process takes about two hours and ends with the release of unfertilized eggs via localized rupturing of the gonad epidermis. This spawning process in females, and also the parallel process of sperm activation and release in males, is initiated by a light cue after darkness [START_REF] Roosen-Runge | On the Biology of Sexual Reproduction of Hydromedusae, Genus Phialidium Leuckhart[END_REF][START_REF] Freeman | The role of oocyte maturation in the ontogeny of the fertilization site in the hydrozoan Hydractinia echinata[END_REF][START_REF] Miller | Sperm chemotaxis in the hydromedusae. I. Species-specificity and sperm behavior[END_REF][START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF]. Specialized cells with neural-like morphology in the gonad ectoderm co-express one of 10 Clytia opsin proteins, Opsin9, and precursors of the oocyte maturation-inducing neuropeptide hormone MIH [START_REF] Quiroga Artigas | A gonad-expressed opsin mediates light-induced spawning in the jellyfish[END_REF][START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF]. Upon light stimulation, Opsin9 mediates the release of MIH both inside and outside the gonad. Subsequent binding of MIH to a specific G-protein coupled receptor in the oocyte membrane initiates activation of meiotic maturation via GalphaS, cytoplasmic cAMP rise and PKA activation (Figure 2C) [START_REF] Quiroga Artigas | A G protein-coupled receptor mediates neuropeptideinduced oocyte maturation in the jellyfish Clytia[END_REF][START_REF] Takeda | Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae[END_REF].Thus medusae from F0 female polyp colonies carrying mutations in the Opsin9 or MIH receptor (MIHR) genes accumulate fully grown oocytes but fail to spawn. In both cases the absence of light-induced oocyte maturation can be overridden by treatment with cell-permeable cAMP analogues.

Origins of the germ cells

In both male and female medusae, the germ cells originate from a population of cells positioned within the gastrodermis close to the canal. These are characterized by small cytoplasm: nuclear ratio, expression of a "Germ line multipotency" gene set [START_REF] Juliano | A conserved germline multipotency program[END_REF] including Piwi and Nanos genes [START_REF] Leclère | Maternally localized germ plasm mRNAs and germ cell/stem cell formation in the cnidarian Clytia[END_REF], and perinuclear clumps of "nuage" material (Figure 2E). Knowledge from other hydrozoans, notably Hydra, suggests that this cell population likely includes both i-cells ("interstitial cells"), the classic hydrozoan multipotent stem cells [START_REF] Hobmayer | Stemness in Hydra -a current perspective[END_REF], and their derivatives that have embarked towards a germ cell fate. Whether a separate germ-line stem cell population derived from i-cells undergoes self renewal in the adult, as suspected in Hydra (Nishimiya-Fujisawa & Kobayashi, 2012), remains to be addressed.

Entry into meiosis

Clytia gonads, through their transparency and accessibility within the living animal, have excellent potential for studying the very early events of meiosis including the transition from mitotic to meiotic cycles, synapsis, and recombination. In female jellyfish oocytes are produced continuously, and earlystage meiotic oocytes are present in the gonads of female medusae throughout its lifespan. Meiosis progresses through leptotene, zygotene and pachytene before arresting in diplotene during oocyte growth [START_REF] Jessus | Managing the Oocyte Meiotic Arrest-Lessons from Frogs and Jellyfish[END_REF]. The early meiotic stages are most accessible for study inside young jellyfish within the first 1 to 2 weeks following their release from the gonozooid. During this period they dominate the population compared to growing and fully grown oocytes. On the first day of jellyfish release, a handful of germ cells are found in a patch along the radial canal at the future site of the gonad. After several days, the number of cells has increased and oocytes at zygotene and pachytene stages can be recognized. In one week old female jellyfish, the gonad is full of early oocytes at all stages of prophase I (Figure 2D). After 2 weeks, diplotene stage oocytes have already started to grow (Figure 2B,E), while oocytes of earlier stages are still present in the periphery of the gonad (Figure 2E) [START_REF] Amiel | Clytia hemisphaerica: A Cnidarian Model for Studying Oogenesis[END_REF]Amiel & Houliston, 2009;[START_REF] Jessus | Managing the Oocyte Meiotic Arrest-Lessons from Frogs and Jellyfish[END_REF]. In a collaboration between the Villefranche lab and Jean-René Huynh at Collège de France, we have recently initiated studies of entry into meiosis and the early meiotic events in prophase I.

CRISPR/Cas9 is being used to target key genes, including the highly conserved meiotic gene Spo11, which is responsible for initiating meiotic double stranded breaks.

Recent technical advances open up exciting possibilities for future studies of gametogenesis in Clytia based on live imaging, particularly of the cellular mechanisms of early meiotic entry where cells are inaccessible for injection. As well as studying meiosis in females, many issues concerning spermatogenesis could also be addressed using the transparent and highly structured male gonads [START_REF] Carré | Biologie et étude ultrastructurale des spermatozoïdes du cnidaire Clytia hemispherica (Leptomedusae): mise en évidence d'un processus acrosomal[END_REF][START_REF] Roosen-Runge | On biology and structure of the testis of Phialidium Leuckhart (Leptomedusae)[END_REF][START_REF] Szollosi | The Structure and function of centrioles and their satellites in the jellyfish Phialidium gregarium[END_REF].

4) Embryogenesis

The daily spawning from laboratory cultures of hundreds of transparent eggs provides ideal material for studies of embryogenesis, allowing the comparison of mechanisms within Cnidaria as well as more widely across animals. The cnidarian larval form, the planula, is typically elongated along the principle "oral-aboral" body axis, and made up of the two basic cnidarian tissue layers: epidermis and gastrodermis. Unlike as in some other cnidarian larvae, the oral end of the Clytia planula is not marked by a blastopore or later by development of a mouth opening, but it transforms into the feeding "head" of the polyp following the drastic morphological transformations of metamorphosis [START_REF] Freeman | Is the remodeling of the polyp prepattern in a hydrozoan planula a function of larval age or size and is it of "adaptive[END_REF]. Coordinated ciliary beating propels the larva with the aboral pole in front. It becomes competent to metamorphose after about three days of development, and at that time will settle on a suitable substrate and metamorphose into a primary polyp (Figure 1B). The different cell types of the planula have not yet been fully characterized. Nematocytes (cnidocytes) are scattered through the lateral epidermis and concentrated at the oral pole, while specialized secretory cells involved in substrate adhesion are concentrated at the aboral pole (Bodo and Bouillon, 1969). Nematocytes are the stinging cells that characterize Cnidaria, whose pressurized stinging capsules include products of genes acquired by horizontal transfer from bacteria (Denker et al., 2008a). Various neural cell types classed as sensory and ganglionic are distributed within the epidermis and in the underlying mesoglea [START_REF] Thomas | The Embryonic Origin of Neurosensory Cells and the Role of Nerve Cells in Metamorphosis inPhialidium gregarium(Cnidaria, Hydrozoa)[END_REF], with an aboral plexus likely involved in coordinating sensory inputs. Neural cells in the larva, including the nematocytes [START_REF] Rentzsch | The cellular and molecular basis of cnidarian neurogenesis[END_REF], are likely generated principally from hydrozoan-specific multipotent stem cells called i-cells occupying the endodermal region, although neurosensory and secretory cells appear to derive from the aboral ectoderm during gastrulation [START_REF] Kraus | Cell shape changes during larval body plan development in Clytia hemisphaerica[END_REF][START_REF] Thomas | The Embryonic Origin of Neurosensory Cells and the Role of Nerve Cells in Metamorphosis inPhialidium gregarium(Cnidaria, Hydrozoa)[END_REF].

Axis establishment

As introduced in Section 1, historically important studies (Freeman, 1981a,b) demonstrated that polarity of the fertilized egg presages the larval oral-aboral body axis, and that endoderm derives from the egg animal/future oral pole. In the early 2000s, experimental manipulation of embryos from the hydrozoan Podocoryne [START_REF] Momose | Animal pole determinants define oral-aboral axis polarity and endodermal cell-fate in hydrozoan jellyfish Podocoryne carnea[END_REF] and demonstration of localized Wnt/β-catenin pathway activation in relation to endoderm formation during Nematostella embryogenesis [START_REF] Wikramanayake | An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation[END_REF] together suggested that cnidarian eggs likely contained axis determinants at the animal (future oral) pole that initiate Wnt/β-catenin signalling. Such determinants could be identified for the first time in Clytia, in the form of localized mRNAs for the Frizzled family receptors, CheFz1 and CheFz3 and the Wnt ligand CheWnt3 (Figure 2BC, Figure 3) [START_REF] Momose | A maternally localised Wnt ligand required for axial patterning in the cnidarian Clytia hemisphaerica[END_REF][START_REF] Momose | Two oppositely localised frizzled RNAs as axis determinants in a cnidarian embryo[END_REF]. Experimental reorientation of the oral-aboral axis achieved by low-speed centrifugation of fertilized eggs (Freeman, 1981a), can be explained by the codisplacement of Fz1 mRNA with the zygote nucleus in these experiments (Amiel & Houliston, 2009).

Morphogenesis directed by global polarity

Based on the extensive ability of embryo fragments, and even dissociated cells, to reestablish polarity, Gary Freeman proposed that the embryonic axis was determined by a global, ie non-localized, polarity property (Freeman, 1981b). We can now equate this property with Planar Cell Polarity (PCP), the common alignment of cells within epithelia or other coherent tissues mediated by specific interactions between neighboring cells [START_REF] Butler | Planar cell polarity in development and disease[END_REF]. Clytia possesses a full set of proteins for the conserved 'Wnt-PCP" pathway, including Strabismus, Flamingo and Prickle orthologs [START_REF] Momose | A conserved function for Strabismus in establishing planar cell polarity in the ciliated ectoderm during cnidarian larval development[END_REF]. Morpholino-mediated knockdown of Clytia Strabismus (CheStbm) revealed a requirement for PCP both for cell alignment in the ciliated ectoderm and for planula elongation (Figure 3C). The role in elongation likely involves intercalation between the ectodermal epithelial cells perpendicular to the axis of elongation [START_REF] Byrum | An analysis of hydrozoan gastrulation by unipolar ingression[END_REF], but also between ingressing presumptive endoderm cells (Figure 3D) [START_REF] Kraus | Cell shape changes during larval body plan development in Clytia hemisphaerica[END_REF][START_REF] Van Der Sande | A cell-based boundary model of gastrulation by unipolar ingression in the hydrozoan cnidarian Clytia hemisphaerica[END_REF].

As first described at the end of the nineteenth century, gastrulation in Clytia involves ingression of individual cells from the future oral pole of a single cell-layered blastula [START_REF] Byrum | An analysis of hydrozoan gastrulation by unipolar ingression[END_REF][START_REF] Kraus | Cell shape changes during larval body plan development in Clytia hemisphaerica[END_REF][START_REF] Metchnikoff | Embryologische Studien an Medusen : Ein Beitrag zur Genealogie der Primitiv-organe[END_REF]. As they ingress, the presumptive endoderm cells crawl along the blastocoel wall and against each other, simultaneously narrowing and elongating the embryo along the oralaboral axis (Y. [START_REF] Kraus | Cell shape changes during larval body plan development in Clytia hemisphaerica[END_REF]. Computational simulations of gastrulation require lateral interactions between ingressing cells to be included, simulating PCP, to account for full embryo elongation [START_REF] Van Der Sande | A cell-based boundary model of gastrulation by unipolar ingression in the hydrozoan cnidarian Clytia hemisphaerica[END_REF]. Thus PCP acts in the Clytia embryo to generate the main morphological features of the larva: the axially elongated form and oriented cilia beating. It can be considered as a non-localized axial determinant acting in parallel with Wnt/β-catenin signalling to direct gene expression programmes across the embryo [START_REF] Lapébie | Differential responses to Wnt and PCP disruption predict expression and developmental function of conserved and novel genes in a cnidarian[END_REF].

Modes of embryogenesis between different species of Cnidaria vary widely, with the two body layers forming by ingression in Clytia, but by cell sheet invagination uncoupled from embryo elongation in Nematostella, and partitioning during the cleavage divisions in Hydractinia [START_REF] Kraus | The gastrulation in Cnidaria: A key to understanding phylogeny or the chaos of secondary modifications?[END_REF][START_REF] Technau | Gastrulation and germ layer formation in the sea anemone Nematostella vectensis and other cnidarians[END_REF]. Cnidarian embryos are thus a very valuable source of comparative material for addressing how morphogenetic processes function and evolve. In this context it was revealing to find that simulations of gastrulation in Clytia could be derived from simulations set up for Nematostella by changing parameters representing adhesion between cells, allowing apical cell contraction to occur in all cells rather than being orally restricted, and adding lateral interactions ('PCP') between ingressing cells (van der Sande et al., 2020).

5) Medusa formation and Regeneration

Hydrozoan jellyfish have provided a rich source of material for biologists since the late nineteenth century. Early workers documented for example their development, nervous systems, musculature and regeneration. Molecular studies to explore this rich heritage have only just begun [START_REF] Peron | The marine jellyfish model Clytia hemisphaerica[END_REF]Leclère et al., 2016). Here we will provide an overview of recent findings in each of these areas.

Medusa development

Clytia jellyfish are produced clonally by lateral budding of specialized polyps, the gonozooids (Figure 1C). This process appears highly similar to that described in other hydrozoan species (e.g. Boelsterli, 1977). Jellyfish budding starts with bulging of the gonozooid body wall. A specialized cell layer called the entocodon, formed from a group of cells that delaminate within the early bud, generates smooth and striated muscle layers of the subumbrella and manubrium [START_REF] Kraus | Adoption of conserved developmental genes in development and origin of the medusa body plan[END_REF]. The remaining epidermis of the bud gives rise to the exumbrella, the tentacle epidermis and the outer layer of the velum of the medusa, while the bud gastrodermis provides the gastrovascular system of the medusa. The precise ontogeny of medusa formation as well as the molecular processes underlying budding, growth and aging of Clytia jellyfish are still very largely unexplored.

Nervous system

The nervous system of the Clytia jellyfish is still poorly characterized. As in other hydrozoan jellyfish, two nerve rings running around the bell margin play a central role in integrating sensory inputs to coordinate behavioural responses, while subpopulations of neural cells also occupy epidermal and gastrodermal layers of the manubrium and gonads. Analysis of scRNAseq data [START_REF] Chari | Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types[END_REF] revealed at least 12 mature neural cell types expressing different neuropeptides, whose precise morphologies, locations and roles mainly remain to be determined. A recent pioneering functional study shows that RFamide-expressing neurons in the subumbrella mediate the oriented flexing of the bell that brings prey captured by the tentacle to the mouth [START_REF] Weissbourd | Functional modules within a distributed neural network control feeding in a model medusa[END_REF].

GCaMP imaging revealed these RFamide-positive neurons to be organized in distinct and largely independent modules under the control of the nerve ring.

Nerve cells and nematocytes likely derive principally from i-cells in the Clytia medusa, as shown in the polyps hydrozoans Hydra [START_REF] Hobmayer | Stemness in Hydra -a current perspective[END_REF] and Hydractinia [START_REF] Gahan | The interstitial stem cells in Hydractinia and their role in regeneration[END_REF]). In the Clytia medusa, i-cell pools are located not only in the gonads (see Section 3) but also at the base of the manubrium and, conspicuously, in the ectoderm at the base of each tentacle bulb (Condamine et al., 2019;[START_REF] Denker | Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria)[END_REF][START_REF] Leclère | Maternally localized germ plasm mRNAs and germ cell/stem cell formation in the cnidarian Clytia[END_REF]. Given the continuous growth of the tentacles and the constant renewal of nematocytes, the Clytia jellyfish tentacle bulb is an attractive system to investigate neurosensory cell differentiation. It can be viewed as a cellular conveyor belt, with stem cells close to the bell margin and differentiation into nematocytes progressing along the longitudinal axis of the bulb and into the tentacle [START_REF] Denker | Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria)[END_REF][START_REF] Chari | Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types[END_REF] (Figure 4A).

Transcriptome comparisons between the base, middle and distal parts of the bulb uncovered sets of genes differentially expressed along the bulb [START_REF] Condamine | Molecular characterisation of a cellular conveyor belt in Clytia medusae[END_REF], confirming previous in situ hybridization studies of particular regulatory gene families [START_REF] Denker | Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria)[END_REF][START_REF] Hwang | Nematogalectin, a nematocyst protein with GlyXY and galectin domains, demonstrates nematocyte-specific alternative splicing in Hydra[END_REF][START_REF] Jager | Multiple Sox genes are expressed in stem cells or in differentiating neuro-sensory cells in the hydrozoan Clytia hemisphaerica[END_REF][START_REF] Leclère | Maternally localized germ plasm mRNAs and germ cell/stem cell formation in the cnidarian Clytia[END_REF][START_REF] Steinmetz | Independent evolution of striated muscles in cnidarians and bilaterians[END_REF]. Regulation of this differentiation likely involves both the Hippo [START_REF] Coste | Comparative study of Hippo pathway genes in cellular conveyor belts of a ctenophore and a cnidarian[END_REF] and Wnt pathways [START_REF] Condamine | Molecular characterisation of a cellular conveyor belt in Clytia medusae[END_REF]. Wnt related genes are expressed differentially both along the proximo distal axis of the bulb and in relation to its perpendicular oral-aboral axis, likely reflecting complex spatial regulation of neural differentiation within the bulb.

Muscle systems and regeneration

The Clytia jellyfish umbrella consists of exumbrella and subumbrella cell layers, separated by a thick ECM structure called the mesoglea (the 'jelly'). The exumbrella comprises flattened, non-contractile polygonal cells. The sub-umbrella is made up of three layers of cells: (i) the endodermal plate of polygonal non contractile epithelial cells, covered by two contractile epithelial layers; (ii) a muscle layer displaying smooth radial muscle fibers oriented radially and responsible for the crumpling of the bell, and between these (iii) a layer of circular striated muscle layer responsible for the pulsing bell contractions used for swimming. Clytia striated muscles strongly resemble those of bilaterian animals in structure, but molecular phylogeny and gene expression studies of the proteins comprising the typical periodic actomyosin arrays suggest that this similarity arose through convergent evolution [START_REF] Steinmetz | Independent evolution of striated muscles in cnidarians and bilaterians[END_REF]. The radial smooth muscles have been recently studied for their role in food transfer from the bell margin to the mouth, stimulated by the action of associated networks of RFamide positive neurons [START_REF] Weissbourd | Functional modules within a distributed neural network control feeding in a model medusa[END_REF]. Intriguingly, reorganisation of these radial muscle fibres upon injury driven by actomyosin-driven wound closure may also have a patterning role in the jellyfish, determining the site where a new manubrium will form [START_REF] Sinigaglia | Pattern regulation in a regenerating jellyfish[END_REF]. These observations on the jellyfish muscle systems open an exciting era of exploration of the molecular basis of their development and function, comparisons of the composition and structure of the contractile fibers with canonical bilaterian muscles, and understanding of their role in regeneration and body patterning [START_REF] Sinigaglia | Pattern regulation in a regenerating jellyfish[END_REF].

Cnidarians are well known for their extensive regenerative capacities, which have been well characterized in polyp-only species such as Hydra, Nematostella and Hydractinia, but less so in medusae. Building on a body of historical work (Neppi et al. 1979;[START_REF] Schmid | Regeneration in Medusa buds and Medusae of Hydrozoa[END_REF][START_REF] Schmid | Factors effecting manubrium-regeneration in hydromedusae (Coelenterata)[END_REF][START_REF] Schmid | The reconstitutional performances of the Leptomedusa Campanularia jonstoni[END_REF], recent cellular and molecular analyses have shed a new light on regeneration processes in Clytia. Studies of wound healing exploited the optical clarity of the exumbrella monolayered epithelium for high resolution imaging and revealed the extreme rapidity and simplicity of this process, which occurs much faster than for cultured cells or bilaterian embryos [START_REF] Kamran | In vivo imaging of epithelial wound healing in the cnidarian Clytia hemisphaerica demonstrates early evolution of purse string and cell crawling closure mechanisms[END_REF][START_REF] Malamy | An orientation-independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model[END_REF]. Both purse-string-like supra-cellular actomyosin contractions and lamellipodia-dependent cell crawling contribute to wound healing [START_REF] Kamran | In vivo imaging of epithelial wound healing in the cnidarian Clytia hemisphaerica demonstrates early evolution of purse string and cell crawling closure mechanisms[END_REF][START_REF] Malamy | An orientation-independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model[END_REF]. The balance between these mechanisms depends on the depth of the wound and the extent of damage to the basement membrane / ECM. Remarkably, the Clytia medusa can also cope with much more extreme damage. Even small fragments are able to rapidly restore shape, functionality and even missing organs [START_REF] Sinigaglia | Pattern regulation in a regenerating jellyfish[END_REF]. This occurs in three steps: initial wound healing is followed by a purse-string mediated remodeling process to re-establish the circular shape of the medusa. Finally, reformation of missing organs occurs, prioritizing the manubrium to allow feeding to resume (Figure 4B). Manubrium reformation requires both cell proliferation and cell migration from other parts of the medusa, notably the gonads. Grafting experiments revealed that the position of the new manubrium is not directed by a morphogen based system of coordinates within the subumbrella (Volker Schmid et al., 1976;[START_REF] Sinigaglia | Pattern regulation in a regenerating jellyfish[END_REF]. It seems rather that patterns of reorganization of the radial smooth muscles of the sub-umbrella during remodelling define the site of the new manubrium. These mechanical contraction cues lead to activation of Wnt signaling at the remodeling site, which promotes manubrium regeneration or not, depending on the organization of the muscle fibers in the regenerating fragment (Figure 4B). A similar patterning function for muscle is also emerging from studies of regeneration in Hydra [START_REF] Livshits | Structural Inheritance of the Actin Cytoskeletal Organization Determines the Body Axis in Regenerating Hydra[END_REF][START_REF] Maroudas-Sacks | Topological defects in the nematic order of actin fibers as organization centers of Hydra morphogenesis[END_REF]Maroudas-Sacks et al., 2020). Understanding of how this influence of muscle integrates with the well established role of Wnt in Hydra head regeneration awaits future studies [START_REF] Cazet | Generic injuries are sufficient to induce ectopic Wnt organizers in[END_REF][START_REF] Wang | Wnt signaling determines body axis polarity in regenerating Hydra tissue fragments[END_REF]. Another important player during manubrium regeneration in Clytia is the radial canal system, which both transports construction material to the blastema site and acts locally to direct manubrium morphogenesis.

Much remains to be understood about the extreme and fascinating regeneration capacity of Clytia medusae. Open questions to be addressed notably include i) the role of cell migration in regeneration,

ii) the integration of mechanical cues and signaling pathways during regeneration, iii) the link between signaling, ECM, wound healing and mechanical forces, and iv) the nature of the local cues that direct regeneration and morphogenesis.

6) Genomics of the jellyfish

Cnidarians are the closest living relatives of the bilaterians, the major group of bilaterally symmetric animals that includes most familiar species including humans. Cnidaria itself comprises two great clades, Anthozoa, consisting of corals and anemones (including the laboratory model Nematostella vectensis), and Medusozoa, most simply understood as those that have a jellyfish-like stage in their life-cycle. The divergence between Anthozoa and Medusozoa is ancient [START_REF] Dos Reis | Uncertainty in the Timing of Origin of Animals and the Limits of Precision in Molecular Timescales[END_REF], and both are large and diverse clades. The four medusozoan classes are Hydrozoa (including Clytia and Hydra) Staurozoa (stalked jellyfish), Scyphozoa (true jellyfish) and Cubozoa (box jellyfish). The last few years have seen the publication of high quality genomes of several Medusozoa, Nemopilema nomurai [START_REF] Kim | The genome of the giant Nomura's jellyfish sheds light on the early evolution of active predation[END_REF]; Rhopilema esculentum [START_REF] Li | Chromosome-level reference genome of the jellyfish Rhopilema esculentum[END_REF][START_REF] Nong | Jellyfish genomes reveal distinct homeobox gene clusters and conservation of small RNA processing[END_REF]; Sanderia malayensis [START_REF] Nong | Jellyfish genomes reveal distinct homeobox gene clusters and conservation of small RNA processing[END_REF]; Chrysaora quinquecirrha (W. [START_REF] Xia | High-Quality Genome Assembly of Chrysaora quinquecirrha Provides Insights Into the Adaptive Evolution of Jellyfish[END_REF][START_REF] Xia | High-continuity genome assembly of the jellyfish[END_REF]; Aurelia aurita [START_REF] Gold | The genome of the jellyfish Aurelia and the evolution of animal complexity[END_REF][START_REF] Khalturin | Medusozoan genomes inform the evolution of the jellyfish body plan[END_REF]; Morbakka virulenta [START_REF] Khalturin | Medusozoan genomes inform the evolution of the jellyfish body plan[END_REF]. Of all these, except the box jellyfish Morbakka, are Scyphozoa. Another box jellyfish, Alatina alata and a staurozoan Calvadosia cruxmelitensis are available as drafts [START_REF] Ohdera | Box, stalked, and upside-down? Draft genomes from diverse jellyfish (Cnidaria, Acraspeda) lineages: Alatina alata (Cubozoa), Calvadosia cruxmelitensis (Staurozoa), and Cassiopea xamachana (Scyphozoa)[END_REF].

After Hydra [START_REF] Chapman | The dynamic genome of Hydra[END_REF][START_REF] Hamada | A Reference Genome from the Symbiotic Hydrozoan, Hydra viridissima[END_REF], Clytia was the second hydrozoan species for which the genome has been sequenced, and the first with planula and medusa stages [START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF]. The 445 Mb assembled genome of Clytia is typical for hydrozoans, with moderate GC and repeat contents (35% and 39% respectively). As in other hydrozoans, messenger RNAs in Clytia are frequently subject to transplicing, involving addition of short RNA leader sequences to their 5' ends [START_REF] Derelle | Convergent origins and rapid evolution of spliced leader trans-splicing in metazoa: insights from the ctenophora and hydrozoa[END_REF]. Work on improving the contiguity of the Clytia hemisphaerica genome continues; an improved assembly with N50 > 1Mb is now available at Ensembl (link in Table 1). A central result of the published Clytia genome analysis was that gene order in Clytia and Hydra is disrupted relative to the last common ancestor of cnidarians and bilaterians. Comparisons with the new scyphozoan genomes suggest that this disruption is indeed a hydrozoan phenomenon, with better gene order conservation between Anthozoa and Scyphozoa than between Anthozoa and Clytia or Hydra. These results will need to be confirmed as more contiguous hydrozoan genomes become available [START_REF] Zimmermann | Sea anemone genomes reveal ancestral metazoan chromosomal macrosynteny[END_REF]. In this context, it is interesting that Clytia appears to have the same number of chromosomes (15) as the ancestral Cnidarian [START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF][START_REF] Zimmermann | Sea anemone genomes reveal ancestral metazoan chromosomal macrosynteny[END_REF]. Gene order disruption of Hydrozoa does not, therefore, appear to have been an inevitable consequence of genomic restructuring necessary to form another life-cycle stage.

The accessibility of the planula, polyp and medusa in the laboratory environment has enabled detailed study of gene use in the different stages of Clytia. The likelihood that the jellyfish is an evolutionary novelty (i.e. 'invented' in the medusozoan lineage), raised the possibility that it might be enriched in lineage specific genes. All studies that have looked at this question, however, have concluded that this is not the case [START_REF] Gold | The genome of the jellyfish Aurelia and the evolution of animal complexity[END_REF][START_REF] Khalturin | Medusozoan genomes inform the evolution of the jellyfish body plan[END_REF][START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF]. Instead, genes showing differential expression between life cycle stages are more likely to be taxonomically restricted (i.e. more recently evolved), but they are not preferentially associated with the medusa stage [START_REF] Khalturin | Medusozoan genomes inform the evolution of the jellyfish body plan[END_REF][START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF]. In contrast, a careful analysis of transcription factors showing medusa specific expression showed that several had been lost in Hydra, despite being ancestral to Medusozoa and present in the closest medusoid-producing relatives of Hydra for which data were available [START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF]. These cases of gene loss are thus associated with loss of a life-cycle stage.

Analysis of life-cycle stage gene use also suggested that the Clytia planula and polyp stages have been secondarily simplified relative to the Anthozoa, as genes with apparent conserved roles in patterning the apical organ and directive axis formation were lost or are not expressed. In this context it is notable that Scyphozoa similarly lacks many of these genes (Gbx, Hox2 etc.). These analyses also suggested that, of all the stages, the medusa expresses the most complex set of transcriptional regulators. Many of these appear to associate with specific cell types, notably within the nervous system [START_REF] Chiori | Are Hox genes ancestrally involved in axial patterning? evidence from the hydrozoan Clytia hemisphaerica (Cnidaria)[END_REF][START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF]. A current research focus is understanding this phenomenon within the context of the medusa cell-types and their evolution [START_REF] Chari | Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types[END_REF].

More widely, the accumulation of genomic and sequence resources for Clytia (see Table 1) is providing a wealth of information valuable for the understanding of the evolution of animals and their genomes. Improvements to genome annotation and epigenomic studies in Clytia and other jellyfish species will fuel further insights into life cycle evolution and development in medusozoans, and the evolution of cell types in these different stages.

8) Perspectives

This The time is also ripe now for Clytia to be used also for studies at the level of the whole organism. One notable application in the context of climate change concern is to dissect biological responses to changing environmental parameters such as temperature and UV irradiation and food availability.

Transparent planktonic organisms are especially vulnerable to UV damage, especially when they rise to the ocean surface to spawn. One hypothesized adaptation for UV protection is its absorption by a set of endogenous GFPs, highly expressed in the epidermis of the gonad and tentacle bulbs as well as in the planula ectoderm and egg mitochondria [START_REF] Fourrage | An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows cotargeting to mitochondria and efficient bioluminescence energy transfer[END_REF]. This idea can now be tested using stable CRISPR/Cas9 Knockout strains lacking different GFP subtypes (Fig. 1D). Population genomics approaches to correlate the GFP repertoires of natural populations with their environment would also be informative. Another possible adaptation to UV exposure currently being explored is the differential deployment of alternative DNA repair pathways between life-cycle stages and cell types. This idea stems from analyses of repair responses to Cas9 mediated DNA breaks, which revealed microhomology mediated end-joining (MMEJ) pathway to be dominant over better known double strand break repair pathways such as non-homologous end-joining (NHEJ) or homologydependent repair pathway (HR) in the cells from the larva that found the polyp colony [START_REF] Momose | High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion[END_REF]. Another aspect of Clytia biology clearly impacted by environmental factors is life-cycle transitions. Metamorphosis of hydrozoan planulae is promoted by bacterial biofilms by largely unknown mechanisms [START_REF] Guo | Two distinct bacterial biofilm components trigger metamorphosis in the colonial hydrozoan Hydractinia echinata[END_REF]. The temperature at which the polyp colonies are established then influences the sex of the medusa which bud from it (D. [START_REF] Carré | Origin of germ cells, sex determination, and sex inversion in medusae of the genus Clytia (Hydrozoa, leptomedusae): the influence of temperature[END_REF]. The molecular and cellular basis of this intriguing observation is now open to be addressed.

Clytia also could be a useful model for investigating the physiological relationships between food processing and regulation of starvation and gametogenesis. Study of the impact of food availability on the medusa has recently been leveraged by a proof of concept Whole Animal Multiplexed Single-Cell RNA-Seq ("WHAM-seq") study in which cells from control and starved Clytia jellyfish were simultaneously processed [START_REF] Chari | Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types[END_REF]. This provided a rich body of information concerning the cells and genes affected by starvation, and revealed certain gastrodermal cell types to be particularly responsive. These likely include the "mobilising gastrodermal digestive cells" ("MGD cells"; [START_REF] Sinigaglia | Pattern regulation in a regenerating jellyfish[END_REF] that relocate through the gastrovascular canal system during starving and regeneration, hypothesized to mediate redistribution of nutrient resources between the different organs of the medusa. The functional specialisations of the many distinct secretory and absorptive cell types and physiological states of the jellyfish digestive system are now open for investigation.

To conclude, Clytia is now fully established as a laboratory animal, offering many opportunities to a community of researchers progressively expanding across many fields, as well as ideal material for educators. As well as in silico resources (Table 1) animal resources can be shared as "cuttings" of polyp colonies established on glass slides [START_REF] Lechable | An improved whole life cycle culture protocol for the hydrozoan genetic model Clytia hemisphaerica[END_REF]. Clytia wildtype strains are currently available through the Marine Biological Resource Centre (CRBM, request to be submitted from https://www.embrc-france.fr/) We hope that community expansion will continue, allowing further accelerated development of approaches and resources, and fuelling scientific discovery across domains for years to come.

Figure 1 -

 1 Figure 1 -Clytia hemisphaerica life cycle. On the left is a schematic of the full cycle, as maintained in the laboratory. Images on the right illustrate different stages. (A) 2-day planula larva. Its simple organization comprises two tissue layers: epidermis (ectoderm) and gastrodermis (endoderm) and an elongated "oral-aboral" axis (oral at the top). (B) Images from a time-lapse recording of planula metamorphosis recorded over 2 days following settlement on a substrate. The form drastically changes to generate the primary feeding polyp (gastrozooid). (C) Extension of a stolon from the base of the primary polyp and subsequent emergence of many polyps, both gastrozooids and gonozooids (budding polyps), leads to formation of a colony. Ga -Gastrozooid, Go -Gonozooid. (D) Bright field (left) and fluorescence (right) images of wildtype (bottom) and GFP1/GFP2 knockout (top) jellyfish shortly following budding. Multiple loci of GFP genes were simultaneously mutated by CRISPR/Cas9. (E) Adult jellyfish. Scale bars 50 µm for A, 200 µm for B, 500 µm for D, and 1 mm for C and E. Lifecycle diagram adapted from Munro (2021a). A-D adapted fromMomose (2021a,b,c,d); E adapted from[START_REF] Lechable | An improved whole life cycle culture protocol for the hydrozoan genetic model Clytia hemisphaerica[END_REF].

Figure 2 :

 2 Figure 2: Jellyfish anatomy and gametogenesis A. Schematic of a female jellyfish, with the organisation of the female gonad highlighted. Adapted from Munro, C. (2021a,b). B. Schematic of oocyte growth in a prophase I arrested oocyte. In fully grown jellyfish, cohorts of stage I oocytes (approx 50µm diameter) grow daily to stage III (approx 180µm diameter) prior to spawning. Fz1 mRNA becomes concentrated in the animal cytoplasm during this growth phase. C. Schematic of oocyte maturation. Light triggers the release of oocyte maturation hormone (MIH) from cells in the gonad epidermis, triggering oocyte maturation and spawning. Wnt3 and Fz3 mRNAs are localized around the periphery of the stage III oocyte. During meiotic maturation, Fz3 becomes localized to the vegetal cortex and then Wnt3 to the animal cortex. D. False coloured electron micrograph from the ovary of a 1 week-old jellyfish. Ectoderm is highlighted in blue, gastroderm in red and early oocytes in green (arrows indicate the perinuclear "nuage" characteristic of the germ cells). E. Confocal microscopy image of a two week ovary by Anna Ferraioli. Nuclei are stained with hoechst (cyan), and actin is stained with phalloidin (magenta). GC/ Early PI = Germ cell, Early Prophase I; Z/P = Zygotene/Pachytene; D = Diplotene; GrO = Growing oocyte.

  Morpholino and mRNA injection experiments showed that Fz1, Wnt3 and Fz3 proteins produced from the localized mRNAs act together to stabilize β-catenin in the oral domain and thus set up distinct programmes of gene expression along the axis (Fig 3A, B). Zygotic gene expression, which starts from mid/late blastula stage, is globally controlled by Wnt3. Two distinct transcriptional programmes activated downstream of Wnt3 can be distinguished in the oral ectoderm versus ingressing presumptive endoderm on the basis of sensitivity to PCP-mediated cellular interactions[START_REF] Lapébie | Differential responses to Wnt and PCP disruption predict expression and developmental function of conserved and novel genes in a cnidarian[END_REF].

Figure 3 :

 3 Figure 3: Establishment of oral-aboral polarity in the embryo A) Schematic of an unfertilized egg showing the localization of Wnt/β-catenin pathway component mRNAs. B) The proteins synthesized from these mRNAs locally activate the pathway and initiate transcriptional programmes that define oral and aboral regional characters, including presumptive endoderm fate at the oral pole. C) In parallel, conserved planar cell polarity (PCP) pathway proteins coordinate tissue polarity coordination in the ciliated ectoderm. (D) Individual ingression of presumptive endoderm cells from the oral pole, and PCP-mediated intercalation of cells in both layers underlie the morphogenetic changes during gastrulation that largely contribute to the elongated form of the planula.

Figure 4 :

 4 Figure 4: Nematogenesis and regeneration in Clytia medusae.(A) Nematocyte differentiation ordered along the proximo-distal axis of the Clytia medusa tentacle bulb[START_REF] Condamine | Molecular characterisation of a cellular conveyor belt in Clytia medusae[END_REF][START_REF] Denker | Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria)[END_REF]. (B) Successive stages of wound healing, remodeling and regeneration in Clytia medusa following bisection (orange dotted line;[START_REF] Sinigaglia | Pattern regulation in a regenerating jellyfish[END_REF]. Only the small fragment missing the manubrium (top) will reform one. The muscle fibers (green) merge into a hub (orange arrows), which becomes the regeneration site of a new manubrium 4 days after dissection. In larger fragments bearing a manubrium (bottom), re-circularization also causes the formation of a muscle hub, but this one later disaggregates, and no new manubrium forms.

  review has illustrated how practical advantages combined with phylogenetic position have enabled Clytia hemisphaerica to contribute a number of insights in the fields of cell and developmental biology, and animal genome evolution. Existing in silico resources and well established methods like egg MO/mRNA injection and CRISPR/Cas9-mediated gene knockout already allow efficient functional testing of genes expressed during larval development or specifically in the adult, respectively. The recent emergence of an efficient transposon-mediated transgenesis method opens significant further perspectives to the community, notably widening the scope of gene function exploration in adult (polyp and medusa) stages via the use of inducible promoters. A detailed protocol including workflows to select and maintain strains is being prepared (B. Weissbourd and T.Momose). Transgenesis will also allow researchers to tease out relationships between cell lineages via in vivo tracking, ablation experiments, imaging of particular cellular components etc. We thus anticipate that use of transgenesis in Clytia will advance hand in hand with the development and wider application of live imaging approaches. Recent work focusing on jellyfish wound healing(Karam et al. 2017), regeneration[START_REF] Sinigaglia | Pattern regulation in a regenerating jellyfish[END_REF], neurobiology[START_REF] Weissbourd | Functional modules within a distributed neural network control feeding in a model medusa[END_REF] and cell type characterization via scRNAseq[START_REF] Chari | Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types[END_REF]) represent valuable steps forward in this direction, opening many new questions. For instance, what are the functions and organisation of the different neural subtypes of the jellyfish in relation to physiology and behaviour such as swimming, diel migrations, spawning and feeding? How do mechanical forces and transcriptional processes cooperate during wound healing and regeneration in Clytia jellyfish? The power of genomics, imaging, transgenesis and ablation approaches coupled with the transparency, small size and relative complexity of the jellyfish stage, place Clytia in an ideal position to tackle these fundamental questions.

Table 1 Methods and Resources. 1A. METHODS Method References -first citation and updated methods Target life cycle stage Application

 1

	Animal culture	Lechable et al. 2020	Whole life cycle	Planula, polyp and medusa
				culture
	Morpholino antisense oligo Momose and Houliston 2007;	Oocyte to planula	Loss of function
		Amiel et al. 2009
	mRNA microinjection	Momose and Houliston 2007;	Oocyte to planula	Gain of function, protein
		Amiel et al. 2009		localization
	CRISPR/Cas9 gene KO	Momose et al., 2018; Quiroga	Whole life cycle	Loss of function
		Artigas et al. 2018, 2020
	Transgenesis	Weissbourd et al., 2021	Whole life cycle	Gain of function
	Cell type specific ablation	Weissbourd et al., 2021	Whole life cycle	Specific elimination of cells
	Pharmacological treatments	Momose and Houliston 2007;	Whole life cycle	Loss-or Gain-of-function
	(NB target specificity is not	Amiel et al. 2009; Sinigaglia et al.
	guaranteed)	2020
	In situ hybridization	Chevalier et al. 2006; Sinigaglia et	Whole life cycle	Visualizing gene expression
		al., 2018		patterns
	Immuno/phalloidin staining Denker et al. 2008b; Fourrage et	Whole life cycle	Visualizing protein
		al; 2010, Sinigaglia et al. 2020		localization
	Electron microscopy	Roosen-Runge, 1962, Thomas et	Whole life cycle	Ultrastructural descriptions of
		al 1987; Carré & Carré, 2000;		cell types and tissue
		Kraus et al. 2020		organisation.
	Medusa dissection/grafting Kamran et al. 2017; Sinigaglia et	Medusa	Regeneration experiments
		al. 2020
	DIC microscopy for live	Malamy and Shribak 2018	Medusa	Live imaging of epithelial cells
	imaging
	1B. In silico Resources
	Description	References	Database	Details
	Whole genome sequence	Leclère et al. 2019	marimba.obs-vlfr.fr	Improved genome assembly
			NCBI/EBI	(N50 > 1Mb)
	Genome browser	Leclère et al. 2019	marimba.obs-vlfr.fr	Marimba combines genomic
			ensembl.org	and gene expression data
	Transcriptome assembly	Lapebie et al. 2014; Quiroga	marimba.obs-vlfr.fr	Genome-guided
		Artigas et al. 2018; Leclère et al.	NCBI/EBI	transcriptome available
		2019
	Expression profiling	Lapebie et al. 2014; Quiroga	NCBI/EBI	Wnt-manipulated Gastrulae;
	(RNAseq)	Artigas et al. 2018; Condamine et		Ovary tissues; Tentacle bulb
		al. 2019; Leclère et al. 2019,		regions; Life-cycle stages;
		Sinigaglia et al. 2020.		Regenerating manubrium
				time course
	Expressed Sequence Tags	Chevalier et al. 2006; Foret et al.	NCBI/EBI	Mixed stages libraries
	(EST)	2010
	Single cell RNA seq: Data and	Chari et al. 2021	github.com/pachterlab/	Cell types from adult female
	analysis pipelines		CWGFLHGCCHAP_2021	jellyfish, control and starved
	Ontology terms for anatomy	Manuscript in preparation
	and developmental stages

www.ebi.ac.uk/ols/ontol ogies/clyh Reviewed by the OBO Foundry

Acknowledgements

We thank all the past and present members of our groups who have contributed to the success of Clytia in the laboratory, as well as past and current collaborators including Michael Manuel, Muriel Jager and Eric Quéinnec (Sorbonne University), Ulrich Technau (University of Vienna), Noriyo Takeda (Hiroshima University), Ryusaku Deguchi (Miyagi University of Education), Gáspár Jékely (University of Exeter), Yulia Kraus (Lomonosov Moscow State University), Jaap Kaandorp (Univ. Amsterdam), Jean-René Huynh (Collège de France) and Brady Weissbourd, David Anderson, Tara Chari, Jase Gehring and Lior Pachter (Caltech). We thank Anna Ferraoili for Fig. 2E. Original funding for work from our labs was from the Agence Nationale de la Recherche ANR-17-CE13-0016 (i-MMEJ), ANR-19-CE13-0003 (MYODEVO), the EU Training networks NEPTUNE and EvoCELL, as well as core CNRS and Sorbonne University funding to the LBDV. CM received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 841433 (JOLI), as well as from the "Diversity of Biological Mechanisms'' program of INSB CNRS. We thank the Marine Resources Centre (CRBM and PIV imaging platform) of Institut de la Mer de Villefranche (IMEV), supported by EMBRC-France whose French state funds are managed by the ANR within the Investments of the Future program under reference ANR-10-INBS-02. Clytia resource development was supported by the European Union's Horizon 2020 research and innovation programme under grant agreement No 730984 ASSEMBLE-Plus Joint Research Activity (JRA3) and the CORBEL European Research Infrastructure cluster project.


Fabienne Jézéquel
email: fabienne.jezequel@lip6.fr

Benefits of stochastic arithmetic in high performance simulations and arbitrary precision codes

Keywords: approximate GCD, BLAS, Discrete Stochastic Arithmetic, floating-point arithmetic, Newton method, numerical validation, polynomial roots, rounding errors

DSA (Discrete Stochastic Arithmetic) [START_REF] Vignes | Discrete Stochastic Arithmetic for validating results of numerical software[END_REF] enables one to estimate the rounding error propagation which occurs with floating-point arithmetic. This probabilistic method uses a random rounding mode: at each elementary operation, the result is rounded up or down with the same probability. Therefore, the computer's deterministic arithmetic is replaced by a stochastic arithmetic, where each arithmetic operation is performed several times before the next one is executed. With DSA, temporary results that are actually numerical noise can be discarded and iterative algorithms can be stopped in an optimal way that does not rely on any parameter. DSA can be used to control the accuracy of programs in half, single, double and/or quadruple precision via the CADNA library [CAD, EBFJ15, GJP + 18, JSHH21], and also in arbitrary precision via the SAM library [START_REF]The SAM library[END_REF][START_REF] Graillat | Stochastic arithmetic in multiprecision[END_REF].

We present an algorithm that takes benefits of DSA to efficiently and accurately compute polynomial roots, in particular multiple roots. Thanks to a stochastic version of the polynomial GCD algorithm and the polynomial Euclidean division, the proposed algorithm provides a low-degree polynomial with single roots. Then Newton method can be applied to get fast and accurate approximations of the roots in arbitrary precision [START_REF] Graillat | Computing multiple roots of polynomials in stochastic arithmetic with Newton method and approximate GCD[END_REF].

Thanks to DSA, the accuracy estimation and the detection of numerical instabilities can be performed in parallel codes on CPU and on GPU [EBFJ15, EBFJ16, ELB + 18]. However its performance overhead may be large compared with the standard floating-point operations. We show that with perturbed data it is possible to use standard floating-point arithmetic instead of DSA for the purpose of numerical validation. For instance, for codes including matrix multiplications, we can directly utilize the matrix multiplication routine (GEMM) of level-3 BLAS that is performed with standard floating-point arithmetic. Consequently, we can achieve a significant performance improvement by avoiding the performance overhead of DSA operations as well as by exploiting the speed of highly-optimized BLAS implementations [JGM + 20].


Rick C Helmich
email: rick.helmich@radboudumc.nl

Stéphane Lehéricy

Dying-back of ascending noradrenergic projections in

Parkinson's disease is a neurodegenerative disorder characterized by progressive loss of nigrostriatal dopamine neurons. The cardinal motor signs of the disease, particularly bradykinesia and rigidity -and to a lesser extent tremor -have been linked to dopaminergic dysfunction of the basal ganglia. In addition, it has long been known that Parkinson's disease patients also have profound noradrenergic, cholinergic and serotonergic cell loss. Clinically, noradrenergic deficits have been linked to cognitive dysfunction, to specific non-motor symptoms such as REM-sleep behavioural disorder (RBD), and to axial symptoms such as freezing of gait. [START_REF] Prasuhn | Association of locus coeruleus and substantia nigra pathology with cognitive and motor functions in patients with Parkinson disease[END_REF][START_REF] Garcia-Lorenzo | The coeruleus/subcoeruleus complex in rapid eye movement sleep behaviour disorders in Parkinson's disease[END_REF] A key outstanding question is how Parkinson's disease propagates through the brain, and what this pattern tells us about the underlying mechanisms of progressive cell death. The classical post-mortem studies by Braak and colleagues have suggested that there is a bottom-up progression of disease, which may initially start in the gut, before progressing towards caudal and later more rostral parts of the brainstem, and ultimately reaching the cortex.

Other studies point to the role of top-down pathophysiological mechanisms, where increased cortical demands may lead to energetic failure in the highly branched dopaminergic and noradrenergic neurons, causing them to dysfunction and ultimately perish. [START_REF] Foffani | A Cortical Pathogenic Theory of Parkinson's Disease[END_REF] The clinical observation that motor symptoms in Parkinson's disease can start out very localized (e.g. resting tremor of one hand, or bradykinesia of one foot) supports this idea, since this detailed level of somatotopy is observed much more clearly at cortico-striatal than at nigro-striatal projections. [START_REF] Foffani | A Cortical Pathogenic Theory of Parkinson's Disease[END_REF] In addition, several recent studies have shown important inter-individual differences in disease propagation patterns, which are linked to the expression of clinical symptoms such as the presence or absence of RBD. [START_REF] Horsager | Brain-first versus body-first Parkinson's disease: a multimodal imaging case-control study[END_REF][START_REF] Pyatigorskaya | Parkinson Disease Propagation Using MRI Biomarkers and Partial Least Squares Path Modeling[END_REF] In this issue of Brain, Doppler and colleagues address some of these issues, by using neuroimaging techniques to decipher the anatomical pattern of noradrenergic dysfunction in Parkinson's disease. [START_REF] Doppler | Regional locus coeruleus degeneration is uncoupled from noradrenergic terminal loss in Parkinson's disease[END_REF] In a comprehensive study, Doppler et al. used a combination of two imaging techniques to measure different compartments of the noradrenergic system: PET with 11 C-MeNER (Figure 1B), a reboxetine analogue that binds specifically to noradrenaline transporters located at the terminal sites of noradrenergic cells, and neuromelanin-sensitive MRI (Figure 1C), which is a turbo spin-echo (TSE) T1-weighted sequence that is sensitive to the loss of pigmented neurons in the locus coeruleus (LC).

The LC is a small nucleus in the dorsal pons that is the primary source of noradrenergic projections in the brain (Figure 1A). In 47 patients with Parkinson's disease and 26 healthy controls, Doppler et al. applied both techniques within a timespan of a few weeks. The authors hypothesized that more severe deficits would be seen at the level of the noradrenergic terminals (PET) than the cell bodies (MRI), in line with findings in the dopaminergic system, where a 'dying-back' mechanism of neurodegeneration has been found in Parkinson's disease. They also tested for regional differences in neurodegeneration within the LC, by comparing rostral, intermediate, and caudal portions of the LC.

There are three main findings. First, and most importantly, the authors found that pronounced axonal damage exceeded somatic damage of the noradrenergic system in patients, as has been found also for the dopaminergic system. This effect was quantified as a smaller ratio of 11 C-MeNER PET to neuromelanin-MRI contrast in Parkinson's disease patients, compared to healthy controls (Figure 1D). The authors refer to this effect as 'uncoupling', which should be understood as disproportionally large noradrenergic deficits at the distal ends of the axonal tree. This effect may be related to the extremely large arborization of both dopaminergic and noradrenergic neurons. [START_REF] Foffani | A Cortical Pathogenic Theory of Parkinson's Disease[END_REF] This anatomical configuration makes these ascending neurotransmitter systems particularly vulnerable to insults.

The findings do not immediately demonstrate where such insults could come from: the 'dyingback' of noradrenergic cells may be the outcome of different pathophysiological processes that can be either bottom-up (e.g. alpha synuclein affecting the cell bodies in the LC), top-down (e.g. altered synaptic demands at projection sites, or cortico-fugal projections to the LC), or a combination of both. Cellular mechanisms such as impaired protein clearance, altered mitochondrial function, inflammation, and management of reactive oxygen species have all been proposed to play a role in cell death in Parkinson's disease. [START_REF] Giguère | On Cell Loss and Selective Vulnerability of Neuronal Populations in Parkinson's Disease[END_REF] Furthermore, recent development of a rodent model exhibiting an age-dependent production of human-like neuromelanin in the substantia nigra has led to the hypothesis that too much intracellular neuromelanin accumulation may compromise neuronal function and trigger a Parkinson's disease-like pathology. [START_REF] Carballo-Carbajal | Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson's disease pathogenesis[END_REF] A second finding by Doppler and colleagues is that there was no correlation between MRI and PET findings, either in healthy controls or in patients with Parkinson's disease. This could be another instance of the 'uncoupling' between noradrenergic terminals and cell bodies. The fact that there was no correlation in control subjects may suggest that the two compartments of the noradrenergic system are independent of each other in the physiological situation. This would be surprising, since the noradrenergic terminals are ultimately connected to the cell bodies in the LC. Alternatively, the lack of correlation could be driven by methodological factors, such as different sensitivity of PET and MRI to the underlying biology, large variability of signal and volume measurements in the LC using MRI, and lack of statistical power owing to a moderate number of patients. In addition, the subjects' RBD status, which was not considered, may also contribute: disease propagation patterns have been found to differ between Parkinsonian patients with and without RBD. [START_REF] Pyatigorskaya | Parkinson Disease Propagation Using MRI Biomarkers and Partial Least Squares Path Modeling[END_REF] A third novel finding reported by Doppler and colleagues is that there were regional differences in the spatial pattern of LC damage in Parkinson's disease, such that the middle and caudal portions of the LC were more affected than the rostral portion of the LC. These data are roughly in line with recent post-mortem investigations in Parkinson's disease, although the differences between LC subregions are relatively small. It would be interesting to study this pattern in relation to the presence of RBD, which is probably linked to the involvement of the locus subcoeruleus located in the caudal part of the LC. [START_REF] Garcia-Lorenzo | The coeruleus/subcoeruleus complex in rapid eye movement sleep behaviour disorders in Parkinson's disease[END_REF] Interestingly, these effects were asymmetric in nature, being more pronounced in the hemisphere contralateral to the clinically most-affected side.

The authors did not report correlations between noradrenergic deficits and clinical symptoms. This is partly due to the fact that different cohorts were combined, and unfortunately there was no homogeneous evaluation of non-motor symptoms across all individuals. Surprisingly, there was no correlation between noradrenergic dysfunction and progression markers of Parkinson's disease, such as disease duration, Hoehn & Yahr stage, and total Unified Parkinson's Disease Rating Scale (UPDRS) score. On the other hand, these findings are in line with another recent neuromelanin-sensitive MRI study in Parkinson's disease, which showed that motor severity correlated only with neuromelanin in the substantia nigra, while cognitive measures only correlated with neuromelanin signal in the LC. [START_REF] Prasuhn | Association of locus coeruleus and substantia nigra pathology with cognitive and motor functions in patients with Parkinson disease[END_REF] Doppler and colleagues measured performance on the Montreal Cognitive Assessment (MoCA), which was included as a covariate of no interest in the analyses. While the MoCA is a useful screening tool, it is not sensitive enough to detect subtle cognitive deficits in Parkinson's disease, and it is not specific enough to localize these deficits to anatomical circuits. [START_REF] Prasuhn | Association of locus coeruleus and substantia nigra pathology with cognitive and motor functions in patients with Parkinson disease[END_REF] It would be interesting to test if the dying-back of the noradrenergic system is associated with specific patterns of cognitive deficits in Parkinson's disease. Within the motor domain, there may be a relationship between noradrenergic deficits and tremor severity. 11 C-MeNER PET studies have shown that the noradrenergic system is relatively spared in tremordominant Parkinson's disease patients, who also have relatively spared cognitive function.

Furthermore, noradrenergic excitation of the thalamus has recently been linked to the increase of Parkinson's tremor during cognitive stress, which is a well-known clinical observation. [START_REF] Dirkx | Cognitive load amplifies Parkinson's tremor through excitatory network influences onto the thalamus[END_REF] More specifically, while tremor is likely generated by dopamine-dependent changes in the basal ganglia and the cerebello-thalamo-cortical loop, relatively preserved noradrenergic projections to the thalamus modulate tremor amplitude during arousal. [START_REF] Dirkx | Cognitive load amplifies Parkinson's tremor through excitatory network influences onto the thalamus[END_REF] Hence, both a spared and a degenerated noradrenergic system may contribute to clinical symptoms in Parkinson's disease. This shows that the pathophysiology of Parkinson's disease involves a complex cocktail of neurotransmitter changes, where the whole is greater than the sum of the parts. The absence of correlations between clinical measures of Parkinson's disease duration and the effects reported here is perhaps not so surprising, given emerging evidence that different subgroups of patients have different disease propagation routes. The question of how Parkinson's disease travels through the brain can then only be answered in large datasets, where detailed clinical and cognitive phenotyping along multiple axes is available. The current study provides an excellent point of departure to start unravelling these issues.

Funding R.C.H. is supported by the Netherlands Organization for Scientific Research and the Michael J. Fox Foundation. S.L. received grants from 'Investissements d'avenir' [grant number ANR-10-IAIHU-06 and ANR-11-INBS-0006] and research grant from BIOGEN Inc.

Competing interests

The authors report no competing interests. . [START_REF] Doppler | Regional locus coeruleus degeneration is uncoupled from noradrenergic terminal loss in Parkinson's disease[END_REF] (C) The structural integrity of the LC (indicated in red), as compared to a control region (pons), using neuromelanin-sensitive MRI (taken from Fig 1). [START_REF] Doppler | Regional locus coeruleus degeneration is uncoupled from noradrenergic terminal loss in Parkinson's disease[END_REF] (D) The ratio between PET and MRI signal shown for the hypothalamus (taken from Fig 7). [START_REF] Doppler | Regional locus coeruleus degeneration is uncoupled from noradrenergic terminal loss in Parkinson's disease[END_REF] Data were normalized to the average in healthy controls. In patients, the significantly lower ratio means that noradrenergic dysfunction of the terminals (PET) exceeded that of the cell bodies (MRI). DVR, distribution volume ratio; LC, locus coeruleus.

Figure legend