Microtubule organization of vertebrate sensory neurons in vivo.

Dorsal root ganglion (DRG) neurons are the predominant cell type that innervates the vertebrate skin. They are typically described as pseudounipolar cells that have central and peripheral axons branching from a single root exiting the cell body. The peripheral axon travels within a nerve to the skin, where free sensory endings can emerge and branch into an arbor that receives and integrates information. In some immature vertebrates, DRG neurons are preceded by Rohon-Beard (RB) neurons. While the sensory endings of RB and DRG neurons function like dendrites, we use live imaging in zebrafish to show that they have axonal plus-end-out microtubule polarity at all stages of maturity. Moreover, we show both cell types have central and peripheral axons with plus-end-out polarity. Surprisingly, in DRG neurons these emerge separately from the cell body, and most cells never acquire the signature pseudounipolar morphology. Like another recently characterized cell type that has multiple plus-end-out neurites, ganglion cells in Nematostella, RB and DRG neurons maintain a somatic microtubule organizing center even when mature. In summary, we characterize key cellular and subcellular features of vertebrate sensory neurons as a foundation for understanding their function and maintenance.

Mesoglea Extracellular Matrix Reorganization during Regenerative Process in Anemonia viridis (Forskål, 1775).

Given the anatomical simplicity and the extraordinary ability to regenerate missing parts of the body, Cnidaria represent an excellent model for the study of the mechanisms regulating regenerative processes. They possess the mesoglea, an amorphous and practically acellular extracellular matrix (ECM) located between the epidermis and the gastrodermis of the body and tentacles and consists of the same molecules present in the ECM of vertebrates, such as collagen, laminin, fibronectin and proteoglycans. This feature makes cnidarians anthozoans valid models for understanding the ECM role during regenerative processes. Indeed, it is now clear that its role in animal tissues is not just tissue support, but instead plays a key role during wound healing and tissue regeneration. This study aims to explore regenerative events after tentacle amputation in the Mediterranean anemone Anemonia viridis, focusing in detail on the reorganization of the ECM mesoglea. In this context, both enzymatic, biometric and histological experiments reveal how this gelatinous connective layer plays a fundamental role in the correct restoration of the original structures by modifying its consistency and stiffness. Indeed, through the deposition of collagen I, it might act as a scaffold and as a guide for the reconstruction of missing tissues and parts, such as amputated tentacles.

Chromatin Dynamics and Gene Expression Response to Heat Exposure in Field-Conditioned versus Laboratory-Cultured Nematostella vectensis.

Organisms' survival is associated with the ability to respond to natural or anthropogenic environmental stressors. Frequently, these responses involve changes in gene regulation and expression, consequently altering physiology, development, or behavior. Here, we present modifications in response to heat exposure that mimics extreme summertime field conditions of lab-cultured and field-conditioned Nematostella vectensis. Using ATAC-seq and RNA-seq data, we found that field-conditioned animals had a more concentrated reaction to short-term thermal stress, expressed as enrichment of the DNA repair mechanism pathway. By contrast, lab animals had a more diffuse reaction that involved a larger number of differentially expressed genes and enriched pathways, including amino acid metabolism. Our results demonstrate that pre-conditioning affects the ability to respond efficiently to heat exposure in terms of both chromatin accessibility and gene expression and reinforces the importance of experimentally addressing ecological questions in the field.

Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development.

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR-expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.

Cnidom variation through distinct developmental stages in the sea anemone Aulactinia marplatensis (Zamponi, 1977) (Cnidaria: Actiniaria).

The cnidae are the exclusive diagnostic structures of phylum Cnidaria. The inventory of all cnidae types of a particular species is called the cnidom. The study of cnidae has been widely addressed in all classes of cnidarians. Particularly in the order Actiniaria (sea anemones), the study of the composition, size and distribution of cnidae is essential to the identification and description of species. In the present study, we examine the cnidom of the sea anemone Aulactinia marplatensis in three different stages of development throughout its life cycle. We found that the composition and abundance patterns are very similar between the adult and juvenile stages, although significant differences in the size capsules were found between both stages and in all cnidae types observed, being bigger those from the adult forms. The planula larvae stage presents a less diverse cnidom in comparison to the juvenile and adult stages; however, it presents an exclusive cnidae type (the mesobasic p-mastigophore) which is the biggest in size of all the cnidae types observed in the species. These results highlight the importance of considering the stage of development when cnidae is used as a diagnostic character, and the particular relevance of the study of the cnidom in larval stages.

Unipotent progenitors contribute to the generation of sensory cell types in the nervous system of the cnidarian Nematostella vectensis.

Nervous systems often consist of a large number of different types of neurons which are generated from neural stem and progenitor cells by a series of symmetric and asymmetric divisions. The origin and early evolution of these neural progenitor systems is not well understood. Here we use a cnidarian model organism, Nematostella vectensis, to gain insight into the generation of neural cell type diversity in a non-bilaterian animal. We identify NvFoxQ2d as a transcription factor that is expressed in a population of spatially restricted, proliferating ectodermal cells that are derived from NvSoxB(2)-expressing neural progenitor cells. Using a transgenic reporter line we show that the NvFoxQ2d cells undergo a terminal, symmetric division to generate a morphologically homogeneous population of putative sensory cells. The abundance of these cells, but not their proliferation status is affected by treatment with the γ-secretase inhibitor DAPT, suggesting regulation by Notch signalling. Our data suggest that intermediate progenitor cells and symmetric divisions contribute to the formation of the seemingly simple nervous system of a sea anemone.

Temperature-Dependent Growth and Fission Rate Plasticity Drive Seasonal and Geographic Changes in Body Size in a Clonal Sea Anemone.

The temperature-size rule is a commonly observed pattern where adult body size is negatively correlated with developmental temperature. In part, this may occur as a consequence of allometric scaling, where changes in the ratio of surface area to mass limit oxygen diffusion as body size increases. As oxygen demand increases with temperature, a smaller body should be favored as temperature increases. For clonal animals, small changes in growth and/or fission rate can rapidly alter the average body size of clonal descendants. Here I test the hypothesis that the clonal sea anemone Diadumene lineata is able to track an optimal body size through seasonal temperature changes using fission rate plasticity. Individuals from three regions (Florida, Georgia, and Massachusetts) across the species' latitudinal range were grown in a year-long reciprocal common garden experiment mimicking seasonal temperature changes at three sites. Average body size was found to be smaller and fission rates higher in warmer conditions, consistent with the temperature-size rule pattern. However, seasonal size and fission patterns reflect a complex interaction between region-specific thermal reaction norms and the local temperature regime. These details provide insight into both the range of conditions required for oxygen limitation to contribute to a negative correlation between body size and temperature and the role that fission rate plasticity can play in tracking a rapidly changing optimal phenotype.

The impact of the sea anemone Actinothoe sphyrodeta on Mytilus galloprovincialis mussel cultivation (Galicia, Spain).

Marine mussel aggregations act as a substratum and refuge for many fouling species. Mussel cultivation in Galicia, Spain, is carried out on hanging ropes in subtidal systems. The fauna associated with this cultivation includes a large number of invertebrates that compete for space or food with the mussels, or use their clusters as a refuge from predators or water turbulence. Outbreaks of the epibiont anemone Actinothoe sphyrodeta have been reported in cultivated Galician mussels since 2013, but their impact has not been investigated rigorously. Here, the temporal and spatial variability of Actinothoe sphyrodeta on mussel shells throughout one year is presented. Sampling of mussel size, weight and byssus attachment strength allowed mussel tenacity (attachment strength relative to size) to be calculated. A higher presence of Actinothoe sphyrodeta correlated with lower mussel tenacity and greater biomass losses, suggesting that this species could be an economically important biofouling component.

Glypican1/2/4/6 and sulfated glycosaminoglycans regulate the patterning of the primary body axis in the cnidarian Nematostella vectensis.

Glypicans are members of the heparan sulfate (HS) subfamily of proteoglycans that can function in cell adhesion, cell crosstalk and as modulators of the major developmental signalling pathways in bilaterians. The evolutionary origin of these multiple functions is not well understood. In this study we investigate the role of glypicans in the embryonic and larval development of the sea anemone Nematostella vectensis, a member of the non-bilaterian clade Cnidaria. Nematostella has two glypican (gpc) genes that are expressed in mutually exclusive ectodermal domains, NvGpc1/2/4/6 in a broad aboral domain, and NvGpc3/5 in narrow oral territory. The endosulfatase NvSulf (an extracellular modifier of HS chains) is expressed in a broad oral domain, partially overlapping with both glypicans. Morpholino-mediated knockdown of NvGpc1/2/4/6 leads to an expansion of the expression domains of aboral marker genes and a reduction of oral markers at gastrula stage, strikingly similar to knockdown of the Wnt receptor NvFrizzled5/8. We further show that treatment with sodium chlorate, an inhibitor of glycosaminoglycan (GAG) sulfation, phenocopies knockdown of NvGpc1/2/4/6 at gastrula stage. At planula stage, knockdown of NvGpc1/2/4/6 and sodium chlorate treatment result in alterations in aboral marker gene expression that suggest additional roles in the fine-tuning of patterning within the aboral domain. These results reveal a role for NvGpc1/2/4/6 and sulfated GAGs in the patterning of the primary body axis in Nematostella and suggest an ancient function in regulating Frizzled-mediated Wnt signalling.

Response of bacterial colonization in Nematostella vectensis to development, environment and biogeography.

The establishment of host-bacterial colonization during development is a fundamental process influencing the fitness of many organisms, but the factors controlling community membership and influencing the establishment of the microbial ecosystem during development are poorly understood. The starlet sea anemone Nematostella vectensis serves as a cnidarian model organism due to the availability of laboratory cultures and its high tolerance for broad ranges of salinity and temperature. Here, we show that the anemone's epithelia are colonized by diverse bacterial communities and that the composition of its microbiota is tightly coupled to host development. Environmental variations led to robust adjustments in the microbial composition while still maintaining the ontogenetic core signature. In addition, analysis of bacterial communities of Nematostella polyps from five different populations revealed a strong correlation between host biogeography and bacterial diversity despite years of laboratory culturing. These observed variations in fine-scale community composition following environmental change and for individuals from different geographic origins could represent the microbiome's contribution to host acclimation and potentially adaptation, respectively, and thereby contribute to the maintenance of homeostasis due to environmental changes.

A framework for the establishment of a cnidarian gene regulatory network for "endomesoderm" specification: the inputs of ß-catenin/TCF signaling.

Understanding the functional relationship between intracellular factors and extracellular signals is required for reconstructing gene regulatory networks (GRN) involved in complex biological processes. One of the best-studied bilaterian GRNs describes endomesoderm specification and predicts that both mesoderm and endoderm arose from a common GRN early in animal evolution. Compelling molecular, genomic, developmental, and evolutionary evidence supports the hypothesis that the bifunctional gastrodermis of the cnidarian-bilaterian ancestor is derived from the same evolutionary precursor of both endodermal and mesodermal germ layers in all other triploblastic bilaterian animals. We have begun to establish the framework of a provisional cnidarian "endomesodermal" gene regulatory network in the sea anemone, Nematostella vectensis, by using a genome-wide microarray analysis on embryos in which the canonical Wnt/ß-catenin pathway was ectopically targeted for activation by two distinct pharmaceutical agents (lithium chloride and 1-azakenpaullone) to identify potential targets of endomesoderm specification. We characterized 51 endomesodermally expressed transcription factors and signaling molecule genes (including 18 newly identified) with fine-scale temporal (qPCR) and spatial (in situ) analysis to define distinct co-expression domains within the animal plate of the embryo and clustered genes based on their earliest zygotic expression. Finally, we determined the input of the canonical Wnt/ß-catenin pathway into the cnidarian endomesodermal GRN using morpholino and mRNA overexpression experiments to show that NvTcf/canonical Wnt signaling is required to pattern both the future endomesodermal and ectodermal domains prior to gastrulation, and that both BMP and FGF (but not Notch) pathways play important roles in germ layer specification in this animal. We show both evolutionary conserved as well as profound differences in endomesodermal GRN structure compared to bilaterians that may provide fundamental insight into how GRN subcircuits have been adopted, rewired, or co-opted in various animal lineages that give rise to specialized endomesodermal cell types.

Production of a reference transcriptome and transcriptomic database (EdwardsiellaBase) for the lined sea anemone, Edwardsiella lineata, a parasitic cnidarian.

BACKGROUND: The lined sea anemone Edwardsiella lineata is an informative model system for evolutionary-developmental studies of parasitism. In this species, it is possible to compare alternate developmental pathways leading from a larva to either a free-living polyp or a vermiform parasite that inhabits the mesoglea of a ctenophore host. Additionally, E. lineata is confamilial with the model cnidarian Nematostella vectensis, providing an opportunity for comparative genomic, molecular and organismal studies. DESCRIPTION: We generated a reference transcriptome for E. lineata via high-throughput sequencing of RNA isolated from five developmental stages (parasite; parasite-to-larva transition; larva; larva-to-adult transition; adult). The transcriptome comprises 90,440 contigs assembled from >15 billion nucleotides of DNA sequence. Using a molecular clock approach, we estimated the divergence between E. lineata and N. vectensis at 215-364 million years ago. Based on gene ontology and metabolic pathway analyses and gene family surveys (bHLH-PAS, deiodinases, Fox genes, LIM homeodomains, minicollagens, nuclear receptors, Sox genes, and Wnts), the transcriptome of E. lineata is comparable in depth and completeness to N. vectensis. Analyses of protein motifs and revealed extensive conservation between the proteins of these two edwardsiid anemones, although we show the NF-κB protein of E. lineata reflects the ancestral structure, while the NF-κB protein of N. vectensis has undergone a split that separates the DNA-binding domain from the inhibitory domain. All contigs have been deposited in a public database (EdwardsiellaBase), where they may be searched according to contig ID, gene ontology, protein family motif (Pfam), enzyme commission number, and BLAST. The alignment of the raw reads to the contigs can also be visualized via JBrowse. CONCLUSIONS: The transcriptomic data and database described here provide a platform for studying the evolutionary developmental genomics of a derived parasitic life cycle. In addition, these data from E. lineata will aid in the interpretation of evolutionary novelties in gene sequence or structure that have been reported for the model cnidarian N. vectensis (e.g., the split NF-κB locus). Finally, we include custom computational tools to facilitate the annotation of a transcriptome based on high-throughput sequencing data obtained from a "non-model system."

Symbiotic state influences life-history strategy of a clonal cnidarian.

Along the North American Pacific coast, the common intertidal sea anemone Anthopleura elegantissima engages in facultative, flexible symbioses with Symbiodinium muscatinei (a dinoflagellate) and Elliptochloris marina (a chlorophyte). Determining how symbiotic state affects host fitness is essential to understanding the ecological significance of engaging in such flexible relationships with diverse symbionts. Fitness consequences of hosting S. muscatinei, E. marina or negligible numbers of either symbiont (aposymbiosis) were investigated by measuring growth, cloning by fission and gonad development after 8.5-11 months of sustained exposure to high, moderate or low irradiance under seasonal environmental conditions. Both symbiotic state and irradiance affected host fitness, leading to divergent life-history strategies. Moderate and high irradiances led to a greater level of gonad development in individuals hosting E. marina, while high irradiance and high summer temperature promoted cloning in individuals hosting S. muscatinei and reduced fitness of aposymbiotic anemones. Associating with S. muscatinei may contribute to the success of A. elegantissima as a spatial competitor on the high shore: (i) by offsetting the costs of living under high temperature and irradiance conditions, and (ii) by promoting a high fission rate and clonal expansion. Our results suggest that basic life-history characteristics of a clonal cnidarian can be affected by the identity of the endosymbionts it hosts.

Characterization of differential transcript abundance through time during Nematostella vectensis development.

BACKGROUND: Nematostella vectensis, a burrowing sea anemone, has become a popular species for the study of cnidarian development. In previous studies, the expression of a variety of genes has been characterized during N. vectensis development with in situ mRNA hybridization. This has provided detailed spatial resolution and a qualitative perspective on changes in expression. However, little is known about broad transcriptome-level patterns of gene expression through time. Here we examine the expression of N. vectensis genes through the course of development with quantitative RNA-seq. We provide an overview of changes in the transcriptome through development, and examine the maternal to zygotic transition, which has been difficult to investigate with other tools. RESULTS: We measured transcript abundance in N. vectensis with RNA-seq at six time points in development: zygote (2 hours post fertilization (HPF)), early blastula (7 HPF), mid-blastula (12 HPF), gastrula (24 HPF), planula (5 days post fertilization (DPF)) and young polyp (10 DPF). The major wave of zygotic expression appears between 7-12 HPF, though some changes occur between 2-7 HPF. The most dynamic changes in transcript abundance occur between the late blastula and early gastrula stages. More transcripts are upregulated between the gastrula and planula than downregulated, and a comparatively lower number of transcripts significantly change between planula and polyp. Within the maternal to zygotic transition, we identified a subset of maternal factors that decrease early in development, and likely play a role in suppressing zygotic gene expression. Among the first genes to be expressed zygotically are genes whose proteins may be involved in the degradation of maternal RNA. CONCLUSIONS: The approach presented here is highly complementary to prior studies on spatial patterns of gene expression, as it provides a quantitative perspective on a broad set of genes through time but lacks spatial resolution. In addition to addressing the problems identified above, our work provides an annotated matrix that other investigators can use to examine genes and developmental events that we do not examine in detail here.

Calcein labelling and electrophysiology: insights on coral tissue permeability and calcification.

The mechanisms behind the transfer of molecules from the surrounding sea water to the site of coral calcification are not well understood, but are critical for understanding how coral reefs are formed. We conducted experiments with the fluorescent dye calcein, which binds to calcium and is incorporated into growing calcium carbonate crystals, to determine the permeability properties of coral cells and tissues to this molecule, and to determine how it is incorporated into the coral skeleton. We also compared rates of calcein incorporation with rates of calcification measured by the alkalinity anomaly technique. Finally, by an electrophysiological approach, we investigated the electrical resistance of coral tissues in order to better understand the role of tissues in ionic permeability. Our results show that (i) calcein passes through coral tissues by a paracellular pathway, (ii) intercellular junctions control and restrict the diffusion of molecules, (iii) intercellular junctions should have pores of a size higher than 13 Å and lower than 20 nm, and (iv) the resistance of the tissues owing to paracellular junctions has a value of 477 ± 21 Ohm cm(2). We discuss the implication of our results for the transport of calcium involved in the calcification process.

Internal brooding favours pre-metamorphic chimerism in a non-colonial cnidarian, the sea anemone Urticina felina.

The concept of intraorganismal genetic heterogeneity resulting from allogeneic fusion (i.e. chimerism) has almost exclusively been explored in modular organisms that have the capacity to reproduce asexually, such as colonial ascidians and corals. Apart from medical conditions in mammals, the natural development of chimeras across ontogenetic stages has not been investigated in any unitary organism incapable of asexual propagation. Furthermore, chimerism was mainly studied among gregarious settlers to show that clustering of genetically similar individuals upon settlement promotes the occurrence of multi-chimeras exhibiting greater fitness. The possible occurrence of chimeric embryos and larvae prior to settlement has not received any attention. Here we document for the first time the presence of natural chimeras in brooded embryos and larvae of a unitary cnidarian, the sea anemone Urticina felina. Rates of visible bi- and multi-chimerism of up to 3.13 per cent were measured in the broods of 16 females. Apart from these sectorial chimeras, monitored fusion events also yielded homogeneous chimeric entities (mega-larvae) suggesting that the actual rates of natural chimerism in U. felina are greater than predicted by visual assessment. In support of this assumption, the broods of certain individuals comprised a dominant proportion (to 90%) of inexplicably large embryos and larvae (relative to oocyte size). Findings of fusion and chimerism in a unitary organism add a novel dimension to the framework within which the mechanisms and evolutionary significance of genetic heterogeneity in animal taxa can be explored.

Cnidarian hair cell development illuminates an ancient role for the class IV POU transcription factor in defining mechanoreceptor identity.

Although specialized mechanosensory cells are found across animal phylogeny, early evolutionary histories of mechanoreceptor development remain enigmatic. Cnidaria (e.g. sea anemones and jellyfishes) is the sister group to well-studied Bilateria (e.g. flies and vertebrates), and has two mechanosensory cell types - a lineage-specific sensory effector known as the cnidocyte, and a classical mechanosensory neuron referred to as the hair cell. While developmental genetics of cnidocytes is increasingly understood, genes essential for cnidarian hair cell development are unknown. Here, we show that the class IV POU homeodomain transcription factor (POU-IV) - an indispensable regulator of mechanosensory cell differentiation in Bilateria and cnidocyte differentiation in Cnidaria - controls hair cell development in the sea anemone cnidarian Nematostella vectensis. N. vectensis POU-IV is postmitotically expressed in tentacular hair cells, and is necessary for development of the apical mechanosensory apparatus, but not of neurites, in hair cells. Moreover, it binds to deeply conserved DNA recognition elements, and turns on a unique set of effector genes - including the transmembrane receptor-encoding gene polycystin 1 - specifically in hair cells. Our results suggest that POU-IV directs differentiation of cnidarian hair cells and cnidocytes via distinct gene regulatory mechanisms, and support an evolutionarily ancient role for POU-IV in defining the mature state of mechanosensory neurons.

Application of a behavioural and biochemical endpoint in ecotoxicity testing with Exaiptasia pallida.

Exaiptasia pallida has been applied as a cnidarian model to assess the toxicity of various contaminants using endpoints related to growth, reproduction and mortality. However, increasingly accepted behavioural and biochemical endpoints are underrepresented in ecotoxicity testing with cnidarian species. The aim of this study was to assess the suitability of tentacle retraction and superoxide dismutase activity as behavioural and biochemical endpoints for ecotoxicity testing with E. pallida. A concentration-dependent, tentacle retraction response was found in sub-lethal toxicity testing for anemones exposed to 1-65 µg L-1 Cu and 2-630 µg L-1 Zn for 24 and 96 h. Semi-quantitative and quantitative approaches to tentacle retraction analysis showed a difference in response sensitivity, however, both methods resulted in similar 24- and 96-h EC50 values for Cu and Zn. Additionally, tentacle retraction analysis provided the benefit of identifying recovery in anemones previously exposed to 359 µg L-1 Zn following a 96-h recovery period. Conversely, no significant difference in superoxide dismutase activity was detected in anemones exposed to the Cu and Zn solutions compared with controls, after either 24- or 96-h exposures. These findings support the ease of application and sensitivity of tentacle retraction as an endpoint in ecotoxicity testing with E. pallida and recommend its suitability for use in acute, sub-lethal toxicity testing. Moreover, evidence of recovery in E. pallida following exposure suggests that recovery should be incorporated into future toxicity assessments.

Generating Transgenic Reporter Lines for Studying Nervous System Development in the Cnidarian Nematostella vectensis.

Neurons often display complex morphologies with long and fine processes that can be difficult to visualize, in particular in living animals. Transgenic reporter lines in which fluorescent proteins are expressed in defined populations of neurons are important tools that can overcome these difficulties. By using membrane-attached fluorescent proteins, such reporter transgenes can identify the complete outline of subsets of neurons or they can highlight the subcellular localization of fusion proteins, for example at pre- or postsynaptic sites. The relative stability of fluorescent proteins furthermore allows the tracing of the progeny of cells over time and can therefore provide information about potential roles of the gene whose regulatory elements are controlling the expression of the fluorescent protein. Here we describe the generation of transgenic reporter lines in the sea anemone Nematostella vectensis, a cnidarian model organism for studying the evolution of developmental processes. We also provide an overview of existing transgenic Nematostella lines that have been used to study conserved and derived aspects of nervous system development.

Hedgehog signaling is required for endomesodermal patterning and germ cell development in the sea anemone Nematostella vectensis.

Two distinct mechanisms for primordial germ cell (PGC) specification are observed within Bilatera: early determination by maternal factors or late induction by zygotic cues. Here we investigate the molecular basis for PGC specification in Nematostella, a representative pre-bilaterian animal where PGCs arise as paired endomesodermal cell clusters during early development. We first present evidence that the putative PGCs delaminate from the endomesoderm upon feeding, migrate into the gonad primordia, and mature into germ cells. We then show that the PGC clusters arise at the interface between hedgehog1 and patched domains in the developing mesenteries and use gene knockdown, knockout and inhibitor experiments to demonstrate that Hh signaling is required for both PGC specification and general endomesodermal patterning. These results provide evidence that the Nematostella germline is specified by inductive signals rather than maternal factors, and support the existence of zygotically-induced PGCs in the eumetazoan common ancestor.