Search

The capacity to regenerate lost tissues varies significantly among animals. Some phyla, such as the annelids, display substantial regenerating abilities, although little is known about the cellular mechanisms underlying the process. To precisely determine the origin, plasticity and fate of the cells participating in blastema formation and posterior end regeneration after amputation in the annelid Platynereis dumerilii, we developed specific tools to track different cell populations. Using these tools, we find that regeneration is partly promoted by a population of proliferative gut cells whose regenerative potential varies as a function of their position along the antero-posterior axis of the worm. Gut progenitors from anterior differentiated tissues are lineage restricted, whereas gut progenitors from the less differentiated and more proliferative posterior tissues are much more plastic. However, they are unable to regenerate the stem cells responsible for the growth of the worms. Those stem cells are of local origin, deriving from the cells present in the segment abutting the amputation plane, as are most of the blastema cells. Our results favour a hybrid and flexible cellular model for posterior regeneration in Platynereis relying on different degrees of cell plasticity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Animal bodies are composed of cell types with unique expression programs that implement their distinct locations, shapes, structures, and functions. Based on these properties, cell types assemble into specific tissues and organs. To systematically explore the link between cell-type-specific gene expression and morphology, we registered an expression atlas to a whole-body electron microscopy volume of the nereid Platynereis dumerilii. Automated segmentation of cells and nuclei identifies major cell classes and establishes a link between gene activation, chromatin topography, and nuclear size. Clustering of segmented cells according to gene expression reveals spatially coherent tissues. In the brain, genetically defined groups of neurons match ganglionic nuclei with coherent projections. Besides interneurons, we uncover sensory-neurosecretory cells in the nereid mushroom bodies, which thus qualify as sensory organs. They furthermore resemble the vertebrate telencephalon by molecular anatomy. We provide an integrated browser as a Fiji plugin for remote exploration of all available multimodal datasets., Competing Interests: Declaration of interests A.A.W. is the founder and owner of ariadne.ai ag., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondrial genomes are frequently applied in phylogenetic and evolutionary studies across metazoans, yet they are still poorly represented in many groups of invertebrates, including annelids. Here, we report ten mitochondrial genomes from the annelid genus Hydroides (Serpulidae) and compare them with all available annelid mitogenomes. We detected all 13 protein coding genes in Hydroides spp., including the atp8 which was reported as a missing gene in the Christmas Tree worm Spirobranchus giganteus, another annelid of the family Serpulidae. All available mitochondrial genomes of Hydroides show a highly positive GC skew combined with a highly negative AT skew - a feature consistent with that found only in the mitogenome of S. giganteus. In addition, amino acid sequences of the 13 protein-coding genes showed a high genetic distance between the Hydroides clade and S. giganteus, suggesting a fast rate of mitochondrial sequence evolution in Serpulidae. The gene order of protein-coding genes within Hydroides exhibited extensive rearrangements at species level, and were different from the arrangement patterns of other annelids, including S. giganteus. Phylogenetic analyses based on protein-coding genes recovered Hydroides as a monophyletic group sister to Spirobranchus with a long branch, and sister to the fan worm Sabellidae. Yet the Serpulidae + Sabellidae clade was unexpectedly grouped with Sipuncula, suggesting that mitochondrial genomes alone are insufficient to resolve the phylogenetic position of Serpulidae within Annelida due to its high base substitution rates. Overall, our study revealed a high variability in the gene order arrangement of mitochondrial genomes within Serpulidae, provided evidence to question the conserved pattern of the mitochondrial gene order in Annelida and called for caution when applying mitochondrial genes to infer their phylogenetic relationships., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pseudopotamilla occelata is a polychaete worm distributed widely in the northern part of the Pacific coast, having value as fishing bait as well as biological material for some basic research areas, including reproduction. Here we describe methods for handling the gametes and embryos of this worm, focusing on such topics as maintenance of adults, induction of oocyte maturation and fertilization, culture of embryos and larvae, microinjection into oocytes, and calcium (Ca 2+ ) imaging.

Background: Nervous system development is an interplay of many processes: the formation of individual neurons, which depends on whole-body and local patterning processes, and the coordinated growth of neurites and synapse formation. While knowledge of neural patterning in several animal groups is increasing, data on pioneer neurons that create the early axonal scaffold are scarce. Here we studied the first steps of nervous system development in the annelid Malacoceros fuliginosus., Results: We performed a dense expression profiling of a broad set of neural genes. We found that SoxB expression begins at 4 h postfertilization, and shortly later, the neuronal progenitors can be identified at the anterior and the posterior pole by the transient and dynamic expression of proneural genes. At 9 hpf, the first neuronal cells start differentiating, and we provide a detailed description of axonal outgrowth of the pioneer neurons that create the primary neuronal scaffold. Tracing back the clonal origin of the ventral nerve cord pioneer neuron revealed that it is a descendant of the blastomere 2d (2d 221 ), which after 7 cleavages starts expressing Neurogenin, Acheate-Scute and NeuroD., Conclusions: We propose that an anterior and posterior origin of the nervous system is ancestral in annelids. We suggest that closer examination of the first pioneer neurons will be valuable in better understanding of nervous system development in spirally cleaving animals, to determine the potential role of cell-intrinsic properties in neuronal specification and to resolve the evolution of nervous systems.

Background: Diverse architectures of nervous systems (NSs) such as a plexus in cnidarians or a more centralized nervous system (CNS) in insects and vertebrates are present across Metazoa, but it is unclear what selection pressures drove evolution and diversification of NSs. One underlying aspect of this diversity lies in the cellular and molecular mechanisms driving neurogenesis, i.e. generation of neurons from neural precursor cells (NPCs). In cnidarians, vertebrates, and arthropods, homologs of SoxB and bHLH proneural genes control different steps of neurogenesis, suggesting that some neurogenic mechanisms may be conserved. However, data are lacking for spiralian taxa., Results: To that end, we characterized NPCs and their daughters at different stages of neurogenesis in the spiralian annelid Capitella teleta. We assessed cellular division patterns in the neuroectoderm using static and pulse-chase labeling with thymidine analogs (EdU and BrdU), which enabled identification of NPCs that underwent multiple rounds of division. Actively-dividing brain NPCs were found to be apically-localized, whereas actively-dividing NPCs for the ventral nerve cord (VNC) were found apically, basally, and closer to the ventral midline. We used lineage tracing to characterize the changing boundary of the trunk neuroectoderm. Finally, to start to generate a genetic hierarchy, we performed double-fluorescent in-situ hybridization (FISH) and single-FISH plus EdU labeling for neurogenic gene homologs. In the brain and VNC, Ct-soxB1 and Ct-neurogenin were expressed in a large proportion of apically-localized, EdU + NPCs. In contrast, Ct-ash1 was expressed in a small subset of apically-localized, EdU + NPCs and subsurface, EdU - cells, but not in Ct-neuroD + or Ct-elav1 + cells, which also were subsurface., Conclusions: Our data suggest a putative genetic hierarchy with Ct-soxB1 and Ct-neurogenin at the top, followed by Ct-ash1, then Ct-neuroD, and finally Ct-elav1. Comparison of our data with that from Platynereis dumerilii revealed expression of neurogenin homologs in proliferating NPCs in annelids, which appears different than the expression of vertebrate neurogenin homologs in cells that are exiting the cell cycle. Furthermore, differences between neurogenesis in the head versus trunk of C. teleta suggest that these two tissues may be independent developmental modules, possibly with differing evolutionary trajectories.

We have conducted comparative analysis of the structure of the dorsal lips of the polychaete Eudistylia polymorpha from the family Sabellidae and the obturacula of Oasisia alvinae (Vestimentifera). It has been concluded that the obturacula of Vestimentifera are homologs of the dorsal lips of Polychaete from the family Sabellidae. It has been suggested that the head lobe of siboglinids of the subfamily Frenulata is homologous to the fused obturacula of Vestimentifera.

Background: The annelid anterior central nervous system is often described to consist of a dorsal prostomial brain, consisting of several commissures and connected to the ventral ganglionic nerve cord via circumesophageal connectives. In the light of current molecular phylogenies, our assumptions on the primary design of the nervous system in Annelida has to be reconsidered. For that purpose we provide a detailed investigation of the adult nervous system of Magelonidae - a putatively basally branching annelid family - and studied early stages of the development of the latter., Results: Our comparative investigation using an integrative morphological approach shows that the nervous system of Magelonidae is located inside the epidermis. The brain is composed of an anterior compact neuropil and posteriorly encircles the prostomial coelomic cavities. From the brain two lateral medullary cords branch off which fuse caudally. Prominent brain structures such as nuchal organs, ganglia or mushroom bodies are absent and the entire nervous system is medullary. Our investigations also contradict previous investigations and present an updated view on established assumptions and descriptions., Conclusion: The comprehensive dataset presented herein enables a detailed investigation of the magelonid anterior central nervous system for the first time. The data reveal that early in annelid evolution complexity of brains and anterior sensory structures rises. Polymorphic neurons in clusters and distinct brain parts, as well as lateral organs - all of which are not present in outgroup taxa and in the putative magelonid sister group Oweniidae - already evolved in Magelonidae. Commissures inside the brain, ganglia and nuchal organs, however, most likely evolved in the stem lineage of Amphinomidae + Sipuncula and Pleistoannelida (Errantia+ Sedentaria). The investigation demonstrates the necessity to continuously question established descriptions and interpretations of earlier publications and the need for transparent datasets. Our results also hint towards a stronger inclusion of larval morphology and developmental investigations in order to understand adult morphological features, not only in Annelida.

Spiral cleavage is a mode of embryonic cell division found in species from several Phyla, including molluscs, annelids and flatworms. It reflects a tilting in the direction of spindle orientation and cell division at the 4 to 8-cell stage, which may be dextral or sinistral, and propagates into later organismal asymmetry. Genetic analysis in a small number of gastropod molluscs shows the direction of spiral cleavage is determined by maternal genotype, though whether this is also the case more generally for spiralians, and whether spiral cleavage at the 4-8 cell stage is preceded by earlier internal chirality in any spiralian species, is unknown. Here we study the early cleavage stages of two equal-cleaving spiralians, the dextral annelid Spirobranchus lamarcki and the sinistral mollusc Biomphalaria glabrata, using light sheet microscopy to image subcellular vesicles in live embryos and asking if chirality of movement is identifiable. We observe variability in the early cleavage of S. lamarcki , including a viable 3-cell stage. Image data are analysed by both particle tracking and particle image velocimetry. Neither finds evidence for chiral movement in 1-, 2-, 3-, or 4-cell embryos, nor do we detect consistent differences between the embryos of the dextral and sinistrai species. The methodological and evolutionary implications of this are discussed., (© 2019 Zoological Society of Japan.)

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2'-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Animal bodies comprise diverse arrays of cells. To characterize cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii. We identify five transcriptionally distinct groups of differentiated cells, each expressing a unique set of transcription factors and effector genes that implement cellular phenotypes. Spatial mapping of cells into a cellular expression atlas, and wholemount in situ hybridization of group-specific genes reveals spatially coherent transcriptional domains in the larval body, comprising, for example, apical sensory-neurosecretory cells versus neural/epidermal surface cells. These domains represent new, basic subdivisions of the annelid body based entirely on differential gene expression, and are composed of multiple, transcriptionally similar cell types. They do not represent clonal domains, as revealed by developmental lineage analysis. We propose that the transcriptional domains that subdivide the annelid larval body represent families of related cell types that have arisen by evolutionary diversification. Their possible evolutionary conservation makes them a promising tool for evo-devo research.

Polynoid worm elytra emit light when mechanically or electrically stimulated. Specialized cells, the photocytes, contain light emitting machineries, the photosomes. Successive stimulations induce light intensity variations and show a coupling within and between photosomes. Here, we describe, using electron tomography of cryo-substituted elytra and freeze-fracturing, the structural transition associated to light emission: undulating tubules come closer, organize and their number forming photosomes increases. Two repeating undulating tubules in opposite phase compose the photosome. Undulations are located on three hexagonal layers that regularly repeat and are equally displaced, in x y and z. The tubule membranes within layers merge giving rise to rings that tend to obey to quasi-rhombohedral symmetry. Merging may result either from close-association, hemifusion (one leaflet fusion) or from fusion (two leaflets fusion). Although the resolution of tomograms is not sufficient to distinguish these three cases, freeze-fracturing shows that hemifusion is a frequent process that leads to an reversible anastomosed membrane complex favoring communications, appearing as a major coupling factor of photosome light emission., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Embryonic organizers are signaling centers that coordinate developmental events within an embryo. Localized to either an individual cell or group of cells, embryonic organizing activity induces the specification of other cells in the embryo and can influence formation of body axes. In the spiralian Capitella teleta, previous cell deletion studies have shown that organizing activity is localized to a single cell, 2d, and this cell induces the formation of the dorsal-ventral axis and bilateral symmetry. In this study, we attempt to identify the signaling pathway responsible for the organizing activity of 2d. Embryos at stages when organizing activity is occurring were exposed to various small molecule inhibitors that selectively inhibited either the Activin/Nodal or the BMP branch of the TGF-β signaling pathway. Embryos were then raised to larval stages, and scored for axial anomalies analogous to 2d ablated phenotypes. Our results show that interference with the Activin/Nodal pathway through a short three hour exposure to the inhibitor SB431542 results in larvae that lack bilateral symmetry and a detectable dorsal-ventral axis. However, interference with the BMP signaling pathway through exposure to the inhibitors DMH1 and dorsomorphin dihydrochloride does not appear to play a role in specification by 2d of the dorsal-ventral axis or bilateral symmetry. Our findings highlight species differences in how the molecular architecture of the conserved TGF-β superfamily signaling pathway components was utilized to mediate the organizing activity signal during early spiralian development., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos.

In the deuterostomes and ecdysozoans that have been studied (e.g. chordates and insects), neural fate specification relies on signaling from surrounding cells. However, very little is known about mechanisms of neural specification in the third major bilaterian clade, spiralians. Using blastomere isolation in the annelid Capitella teleta, a spiralian, we studied to what extent extrinsic versus intrinsic signals are involved in early neural specification of the brain and ventral nerve cord. For the first time in any bilaterian, we found that brain neural ectoderm is autonomously specified. This occurs in the daughters of first-quartet micromeres, which also generate anterior neural ectoderm in other spiralians. In contrast, isolation of the animal cap, including the 2d micromere, which makes the trunk ectoderm and ventral nerve cord, blocked ventral nerve cord formation. When the animal cap was isolated with the 2D macromere, the resulting partial larvae had a ventral nerve cord. These data suggest that extrinsic signals from second-quartet macromeres or their daughters, which form mesoderm and endoderm, are required for nerve cord specification in C. teleta and that the 2D macromere or its daughters are sufficient to provide the inductive signal. We propose that autonomous specification of anterior neural ectoderm evolved in spiralians in order to enable them to quickly respond to environmental cues encountered by swimming larvae in the water column. In contrast, a variety of signaling pathways could have been co-opted to conditionally specify the nerve cord. This flexibility of nerve cord development may be linked to the large diversity of trunk nervous systems present in Spiralia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The comparative study of cell types is a powerful approach toward deciphering animal evolution. To avoid selection biases, however, comparisons ideally involve all cell types present in a multicellular organism. Here, we use image registration and a newly developed "Profiling by Signal Probability Mapping" algorithm to generate a cellular resolution 3D expression atlas for an entire animal. We investigate three-segmented young worms of the marine annelid Platynereis dumerilii , with a rich diversity of differentiated cells present in relatively low number. Starting from whole-mount expression images for close to 100 neural specification and differentiation genes, our atlas identifies and molecularly characterizes 605 bilateral pairs of neurons at specific locations in the ventral nerve cord. Among these pairs, we identify sets of neurons expressing similar combinations of transcription factors, located at spatially coherent anterior-posterior, dorsal-ventral, and medial-lateral coordinates that we interpret as cell types. Comparison with motor and interneuron types in the vertebrate neural tube indicates conserved combinations, for example, of cell types cospecified by Gata1/2/3 and Tal transcription factors. These include V2b interneurons and the central spinal fluid-contacting Kolmer-Agduhr cells in the vertebrates, and several neuron types in the intermediate ventral ganglionic mass in the annelid. We propose that Kolmer-Agduhr cell-like mechanosensory neurons formed part of the mucociliary sole in protostome-deuterostome ancestors and diversified independently into several neuron types in annelid and vertebrate descendants., Competing Interests: The authors declare no conflict of interest.

Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.

Similarity and diversity of the phenotype and nucleotide sequences of certain genome loci among the single-celled microalgae isolated from White Sea benthic invertebrates were studied to extend the knowledge of oxygenic photoautotrophs forming microbial communities associated with animals. We compared four Desmodesmus isolates (1Hp86E-2, 1Pm66B, 3Dp86E-1, 2Cl66E) from the sponge Halichondria panicea, trochophore larvae of the polychaete Phyllodoce maculata, and the hydroids Dynamena pumila and Coryne lovenii, respectively. The microalgae appeared to be very similar featuring the phenotypic and genetic traits characteristics of unicellular representatives of the genus Desmodesmus. At the same time, isolates from different animal species displayed certain differences in (i) the epistructure morphology; (ii) type and number of the inclusions such as interthylakoid starch grains and cytoplasmic oil bodies and (iii) fatty acid composition; in Desmodesmus sp. 1Hp86E-2, these differences were most pronounced. Phylogenetic analysis based on ITS1-5.8S rRNA-ITS2 and rbcL sequences showed that all isolates studied differ from known classified representatives of Desmodesmus combining a deletion in the conservative 5.8S rRNA gene and long AC-microsatellite repeats in the ITS1 whereas 1Hp86E-2 represented a distinct branch within this group.

We examine the nature of variation in a hot vent tubeworm, Ridgeia piscesae, to determine how phenotypes are maintained and how reproductive potential is dictated by habitat. This foundation species at northeast Pacific hydrothermal sites occupies a wide habitat range in a highly heterogeneous environment. Where fluids supply high levels of dissolved sulphide for symbionts, the worm grows rapidly in a "short-fat" phenotype characterized by lush gill plumes; when plumes are healthy, sperm package capture is higher. This form can mature within months and has a high fecundity with continuous gamete output and a lifespan of about three years in unstable conditions. Other phenotypes occupy low fluid flux habitats that are more stable and individuals grow very slowly; however, they have low reproductive readiness that is hampered further by small, predator cropped branchiae, thus reducing fertilization and metabolite uptake. Although only the largest worms were measured, only 17% of low flux worms were reproductively competent compared to 91% of high flux worms. A model of reproductive readiness illustrates that tube diameter is a good predictor of reproductive output and that few low flux worms reached critical reproductive size. We postulate that most of the propagules for the vent fields originate from the larger tubeworms that live in small, unstable habitat patches. The large expanses of worms in more stable low flux habitat sustain a small, but long-term, reproductive output. Phenotypic variation is an adaptation that fosters both morphological and physiological responses to differences in chemical milieu and predator pressure. This foundation species forms a metapopulation with variable growth characteristics in a heterogeneous environment where a strategy of phenotypic variation bestows an advantage over specialization.

Melatonin, the "hormone of darkness," is a key regulator of vertebrate circadian physiology and behavior. Despite its ubiquitous presence in Metazoa, the function of melatonin signaling outside vertebrates is poorly understood. Here, we investigate the effect of melatonin signaling on circadian swimming behavior in a zooplankton model, the marine annelid Platynereis dumerilii. We find that melatonin is produced in brain photoreceptors with a vertebrate-type opsin-based phototransduction cascade and a light-entrained clock. Melatonin released at night induces rhythmic burst firing of cholinergic neurons that innervate locomotor-ciliated cells. This establishes a nocturnal behavioral state by modulating the length and the frequency of ciliary arrests. Based on our findings, we propose that melatonin signaling plays a role in the circadian control of ciliary swimming to adjust the vertical position of zooplankton in response to ambient light., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Complex tissues, such as the brain, are composed of multiple different cell types, each of which have distinct and important roles, for example in neural function. Moreover, it has recently been appreciated that the cells that make up these sub-cell types themselves harbour significant cell-to-cell heterogeneity, in particular at the level of gene expression. The ability to study this heterogeneity has been revolutionised by advances in experimental technology, such as Wholemount in Situ Hybridizations (WiSH) and single-cell RNA-sequencing. Consequently, it is now possible to study gene expression levels in thousands of cells from the same tissue type. After generating such data one of the key goals is to cluster the cells into groups that correspond to both known and putatively novel cell types. Whilst many clustering algorithms exist, they are typically unable to incorporate information about the spatial dependence between cells within the tissue under study. When such information exists it provides important insights that should be directly included in the clustering scheme. To this end we have developed a clustering method that uses a Hidden Markov Random Field (HMRF) model to exploit both quantitative measures of expression and spatial information. To accurately reflect the underlying biology, we extend current HMRF approaches by allowing the degree of spatial coherency to differ between clusters. We demonstrate the utility of our method using simulated data before applying it to cluster single cell gene expression data generated by applying WiSH to study expression patterns in the brain of the marine annelid Platynereis dumereilii. Our approach allows known cell types to be identified as well as revealing new, previously unexplored cell types within the brain of this important model system.

The bristle worm Platynereis dumerilii displays many interesting biological characteristics. These include its reproductive timing, which is synchronized to the moon phase, its regenerative capacity that is hormonally controlled, and a slow rate of evolution, which permits analyses of ancestral genes and cell types. As a marine annelid, Platynereis is also representative of the marine ecosystem, as well as one of the three large animal subphyla, the Lophotrochozoa. Here, we provide an overview of the molecular resources, functional techniques, and behavioral assays that have recently been established for the bristle worm. This combination of tools now places Platynereis in an excellent position to advance research at the frontiers of neurobiology, chronobiology, evo-devo, and marine biology.

Ever since the origin of the first metazoans over 600 million years ago, cell type diversification has been driven by micro-evolutionary processes at population level, leading to macro-evolution changes above species level. In this review, we introduce the marine annelid Platynereis dumerilii, a member of the lophotrochozoan clade (a key yet most understudied superphylum of bilaterians), as a suitable model system for the simultaneous study, at cellular resolution, of macro-evolutionary processes across phyla and of micro-evolutionary processes across highly polymorphic populations collected worldwide. Recent advances in molecular and experimental techniques, easy maintenance and breeding, and the fast, synchronous and stereotypical development have facilitated the establishment of Platynereis as one of the leading model species in the eco-evo-devo field. Most importantly, Platynereis allows the combination of expression profiling, morphological and physiological characterization at the single cell level. Here, we discuss recent advances in the collection of -omics data for the lab strain and for natural populations collected world-wide that can be integrated with population-specific cellular analyses to result in a cellular atlas integrating genetic, phenotypic and ecological variation. This makes Platynereis a tractable system to begin understanding the interplay between macro- and micro-evolutionary processes and cell type diversity.

Bone-eating worms of the genus Osedax exclusively inhabit sunken vertebrate bones on the seafloor. The unique lifestyle and morphology of Osedax spp. have received much scientific attention, but the whole process of their development has not been observed. We herein report the postembryonic development and settlement of Osedax japonicus Fujikura et al. (Zool Sci 23:733-740, 2006). Fertilised eggs were spawned into the mucus of a female, and the larvae swam out from the mucus at the trochophore stage. Larvae survived for 10 days under laboratory conditions. The larvae settled on bones, elongated their bodies and crawled around on the bones. Then they secreted mucus to create a tube and the palps started to develop. The palps of O. japonicus arose from the prostomium, whereas the anterior appendages of other siboglinids arose from the peristomium. The recruitment of dwarf males was induced by rearing larvae with adult females. Females started to spawn eggs 6 weeks after settlement.

Spiral cleavage is observed in animals that belong to the lophotrochozoa, a large group of marine invertebrates. As characteristic for spiral cleavage, the bulk of mesoderm forms from one cell, the "4d blastomere." This process has not yet been followed in cellular detail in annelids except in the leech, where "mesoteloblasts," a pair of mesodermal stem cells, generate two bands of mesoderm precursor cells in an iterative fashion. It is so far unknown whether such stem cell-like lineage is a general property of 4d-derived mesoderm in spiralian larvae. To address this, we have analyzed the cell lineage of the 4d blastomere in the polychaete annelid Platynereis dumerilii, an emerging model for lophotrochozoan and spiralian embryology (Fischer et al., 2010), by 4D microscopy, a semi-automated cell tracking technique based on differential interference contrast serial imaging (Schnabel et al. '97). Our data reveal that the two daughter cells of the 4d cell undergo seven consecutive rounds of unequal cell divisions. They bud off smaller cells in ventral-vegetal orientation and thus show mesoteloblast- and stem cell-like behavior. Based on these findings, we suggest that mesoteloblast-like mesodermal stem cells that form continuous mesodermal bands are part of the Errantia + Sedentaria ground pattern. In the course of annelid evolution, the number consecutive divisions of these cells would have been low initially with <10 division cycles, giving rise to larval segments only, and then increased up to 35 as observed in clitellates., (Copyright © 2013 Wiley Periodicals, Inc.)

Signalling pathways are essential for the correct development of the central nervous system (CNS) in bilaterian animals. Here we show that in the CNS of the annelid Platynereis dumerilii, neural progenitor cells (NPCs) are located close to the ventral midline and express axin, a negative regulator of the Wnt/β-catenin pathway. Using pharmacological inhibitors, we observe that Wnt/β-catenin is required for the transition between proliferating NPCs and differentiating neurons. We also show that the Rho-associated kinase (Rok) is necessary for neurectoderm morphogenesis and ventral midline formation, and indirectly affects the distribution of the NPCs and the development of axonal scaffolds. Moreover, seven genes belonging to the planar cell polarity (PCP) pathway are expressed in the developing Platynereis neurectoderm, suggesting an involvement in its morphogenesis. When compared with previous studies in vertebrates, our data suggest that the involvement of the Wnt/β-catenin pathway in the control of neural cell proliferation/differentiation is ancestral to bilaterians.

Vestimentiferan tubeworms are marine invertebrates that inhabit chemosynthetic environments, and although recent molecular phylogenetic analyses have suggested that vestimentiferan tubeworms are derived from polychaete annelids, they show some morphological features that are different from other polychaetes. For example, vestimentiferans lack a digestive tract and have less body segments and comparative neuroanatomy can provide essential insight into the vestimentiferan body plan and its evolution. In the present study, we investigated the adult nervous system in the vestimentiferan Lamellibrachia satsuma using antibodies against synapsin, serotonin, FMRMamide and acetylated α-tubulin. We also examined the expressions of neural marker genes, elav and synaptotagmin to reveal the distribution of neuronal cell bodies. Brain anatomy shows simple organization in Lamellibrachia compared to other polychaetes. This simplification is probably due to the loss of the digestive tract, passing through the body between the brain and the subesophageal ganglion. In contrast, the ventral nerve cord shows a repeated organizational structure as in the other polychaetes, despite the absence of the multiple segmentation of the trunk. These results suggest that the brain anatomy is variable depending on the function and the condition of surrounding tissues, and that the formation of the rope ladder-like nervous system of the ventral nerve cord is independent from segmentation in polychaetes.

The capability of regenerating posterior segments and pygidial structures is ancestral for annelids and has been lost only a few times within this phylum. As one of the three major segmented taxa, annelids enable us to monitor reconstruction of lost tissues and organs. During regeneration, regional identities have to be imprinted onto the newly formed segments. In this study, we show spatial and temporal localization of expression of nine Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii. Hox genes are homeodomain genes encoding transcriptional regulators of axial patterning in bilaterian animals during development. We demonstrate that five Platynereis Hox genes belonging to paralog groups (PG) 1, 4, 5, 6, and 9-14 are expressed in domains of the regenerating nervous system consistent with providing positional information along the anteroposterior axis of the regenerate. We report that expression in regenerating neuromeres is limited to varying subsets of perikarya, called gangliosomes. Four of nine genes analyzed do not appear to be involved in axial patterning. Two genes, Pdu-Hox2 and Pdu-Hox3, are predominantly expressed in the growth zone region. For some Hox genes expression in newly formed coelomic epithelia can be observed. Platynereis Hox genes do not exhibit temporal or spatial colinearity. Although there are some similarities to previously reported expression patterns during larval and postlarval development in Nereididae (Kulakova et al. 2007), expression patterns observed during caudal regeneration also show unique patterns.

Polarized oogenic cysts are clonal syncytia of germ cells in which some of the sister cells (cystocytes) differentiate not as oocytes, but instead as nurse cells: polyploid cells that support oocyte development. The intricate machinery required to establish and maintain divergent cell fates within a syncytium, and the importance of associated oocyte patterning for subsequent embryonic development, have made polarized cysts valuable subjects of study in developmental and cell biology. Nurse cell/oocyte specification is best understood in insects, particularly Drosophila melanogaster. However, polarized cysts have evolved independently in several other animal phyla. We describe the differentiation of female cystocytes in an annelid worm, the polychaete Ophryotrocha labronica. These worms are remarkable for their elegantly simple cysts, which comprise a single oocyte and nurse cell, making them an appealing complement to insects as subjects of study. To elucidate the process of cystocyte differentiation in O. labronica, we have constructed digital 3D models from electron micrographs of serially sectioned ovarian tissue. These models show that 2-cell cysts arise by fragmentation of larger "parental" cysts, rather than as independent units. The parental cysts vary in size and organization, are produced by asynchronous, indeterminate mitotic divisions of progenitor cystoblasts, and lack fusome-like organizing organelles. All of these characteristics represent key cytological differences from "typical" cyst development in insects like D. melanogaster. In light of such differences and the plasticity of female cyst structure among other animals, we suggest that it is time to reassess common views on the conservation of oogenic cysts and the importance of cysts in animal oogenesis generally., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The polychaete family Siboglinidae, which is currently construed as comprising the Frenulata, Monilifera (composed of Sclerolinum), Vestimentifera, and Osedax, has become known for its specialized symbiont-housing organ called the trophosome. This organ replaced the digestive system of the worms and is located in the elongated trunk region in Frenulata, Sclerolinum, and Vestimentifera. Currently two types of trophosomes have been described: in the taxa Frenulata and Sclerolinum the bacteriocytes originate from endoderm, and in Vestimentifera they originate from mesoderm. In Osedax, a trophosome was described as lacking (Rouse et al., 2004), but bacteriocytes are located in Osedax's characteristic root tissue. Here, we argue for a consistent name for the symbiont-housing tissue, namely trophosome, as in other siboglinids. In this study we provide morphological evidence that in Osedax the bacteriocytes are derived from somatic mesoderm. We show that the trophosome in Osedax is an apolar tissue composed of bacteriocytes and nonsymbiotic cells. As in vestimentiferans, a specific cell cycle was identified; however, in this case it is directed from the posterior to the anterior end of the worms instead of from the center toward the periphery. Comparison of all siboglinid trophosomes and re-evaluation of their body regions allows us to discuss whether the trophosomes are homologous and to hypothesize about the organization of the last common ancestor of Siboglinidae.

The trophosome-an organ especially evolved to accommodate symbiotic bacteria-is a key character of the polychaete family Siboglinidae. Astonishingly, the trophosomes vary in organization and origin between the different siboglinid taxa. The trophosome of the small genus Sclerolinum was nearly unknown until now. Here we investigated the trophosome of S. contortum from the Gulf of Mexico, using light and electron microscopy. We show that this organ derives from the visceral mesoderm and propose that the trophosome of the sister clade Vestimentifera and Sclerolinum is a homologous character. Like that of juvenile vestimentiferans, the trophosome of Sclerolinum trophosome is simply organized. This study reveals that the Sclerolinum trophosome exhibits two regions that differ in the organization of host tissue and the size and shape of the symbionts. We suggest that a specific cell cycle within the symbiont-housing organ is directed along the longitudinal body axis, with a region of proliferation anteriorly and a region of degradation posteriorly. Using Raman microspectroscopy we demonstrate that the endosymbionts of S. contortum from the Gulf of Mexico contain sulfur vesicles, and we argue for a chemoautotrophic sulfur-oxidizing metabolism.

We have isolated and characterized the green pigment accumulating in coelomic cells (eleocytes) of the common clam worm Nereis virens by means of RP-HPLC and ESI-tandem mass spectrometry. This pigment is a novel biliverdin-glutathione conjugate in which the glutathione is linked to the biliverdin-backbone via a thioether bond. The yolk precursor vitellogenin, a female-specific high-density lipoprotein (ρ = 1179 kg/m(3)) with a native molecular mass of ∼500 kDa and a subunit mass of ∼150 kDa, is capable of transporting this pigment as well as heme. The sex-independent large discoidal lipoprotein present in the coelomic fluid, which is sequestered by the eleocytes, could also be shown to transport heme. This renders both lipoproteins as heme-lipoproteins. As the vitellogenin is secreted by the eleocytes, this suggests the eleocytes as a metabolic hub linking the uptake of the potent pro-oxidant heme thought to arise from aged hemoglobin via the large discoidal lipoprotein and its conversion to a bile pigment-conjugate. The conjugate is exported from the eleocytes to the oocytes via vitellogenin leading to the accumulation of green yolk protein. In contrast, no such export route exists in male eleocytes resulting in an accumulation of the biliverdin-conjugate in these cells., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Genes of the coe (collier/olfactory/early B-cell factor) family encode Helix-Loop-Helix transcription factors that are widely conserved in metazoans and involved in many developmental processes, neurogenesis in particular. Whereas their functions during vertebrate neural tube formation have been well documented, very little is known about their expression and role during central nervous system (CNS) development in protostomes. Here we characterized the CNS expression of coe genes in the insect Drosophila melanogaster and the polychaete annelid Platynereis dumerilii, which belong to different subgroups of protostomes and show strikingly different modes of development. In the Drosophila ventral nerve cord, we found that the Collier-expressing cells form a subpopulation of interneurons with diverse molecular identities and neurotransmitter phenotypes. We also demonstrate that collier is required for the proper differentiation of some interneurons belonging to the Eve-Lateral cluster. In Platynereis dumerilii, we cloned a single coe gene, Pdu-coe, and found that it is exclusively expressed in post mitotic neural cells. Using an original technique of in silico 3D registration, we show that Pdu-coe is co-expressed with many different neuronal markers and therefore that, like in Drosophila, its expression defines a heterogeneous population of neurons with diverse molecular identities. Our detailed characterization and comparison of coe gene expression in the CNS of two distantly-related protostomes suggest conserved roles of coe genes in neuronal differentiation in this clade. As similar roles have also been observed in vertebrates, this function was probably already established in the last common ancestor of all bilaterians.

A new type of free coelomic cell was isolated from the coelomic fluid of the polychaete Nereis virens. This cell type was present only during sexual maturation and only in males with fully developed spermatozoa. In vivo acridine orange staining and electron microscopy showed that this cell type consists mainly of a large acidic vacuole with a very low proportion of cytoplasm. This cell type stores extremely high concentration of purine nucleosides reaching concentrations of >700 millimolar on a cell weight basis. The nucleoside concentrations are the highest reported from any living cell so far. Inosine (280-711 micromol/g cell mass), guanosine (8-109 micromol/g), deoxyinosine (3-26 micromol/g) and deoxyguanosine (0.05-0.70 micromol/g) were the main nucleosides. The spectrum of nucleosides reflected that of the surrounding coelomic fluid suggesting their exogenous origin. In culture, labeled guanosine was effectively concentrated by the cells. The vacuole is the most likely storage compartment for the nucleosides and its acidic pH provides higher nucleoside solubility and prevents back diffusion by protonation of the nucleosides., (2010 Elsevier Inc. All rights reserved.)

The trophozoites of a novel gregarine apicomplexan, Trichotokara nothriae n. gen. et sp., were isolated and characterized from the intestines of the onuphid tubeworm Nothria conchylega (Polychaeta), collected at 20 m depth from the North-eastern Pacific Coast. The trophozoites were 50-155 microm long with a mid-cell indentation that formed two prominent bulges (anterior bulge, 14-48 microm wide; posterior bulge, 15-55 microm wide). Scanning electron microscopy (SEM) demonstrated that approximately 400 densely packed, longitudinal epicytic folds (5 folds/microm) inscribe the surface of the trophozoites, and a prominently elongated mucron (14-60 microm long and 6-12 microm wide) was covered with hair-like projections (mean length, 1.97 microm; mean width, 0.2 microm at the base). Although a septum occurred at the junction between the cell proper and the mucron in most trophozoites, light microscopy (LM) demonstrated that the cell proper extended into the core of the mucron as a thin prolongation. A spherical nucleus (8-20 microm) was situated in the middle of the trophozoites, and gamonts underwent caudal syzygy. The small subunit (SSU) rDNA sequence and molecular phylogenetic position of T. nothriae was also characterized. The sequence from this species was the most divergent of all SSU rDNA sequences currently known from gregarines and formed a weakly supported clade with Lecudina polymorpha, which also possesses densely packed epicyctic folds (3-5 folds/mum) and a prominently elongated mucron., ((c) 2010 Elsevier Inc. All rights reserved.)

The effect of high hydrostatic pressures on the ecotoxicological profile of pollutants is an unexplored research area. Using Pomatoceros lamarcki as a surrogate organism for this eco-barotoxicological study, it was found that in a 48 h larval bioassay with water accommodated fractions (WAF) of crude oil of up to 15.1 mg L(-1) (total hydrocarbon content) and hydrostatic pressures up to 300 bar (3000 m), an additive response was found (p < 0.001) rather than any synergism (p = 0.881). Comprehensive cytogenetic analysis of 6-h (15 degrees C) embryos exposed to WAF (0.19 mg L(-1)) at 100 bar showed no effects on mitotic fidelity or cell division rate over the 1 bar treatment. However, embryo's treated with the clastogen mitomycin-c at 100 bar exhibited a significant increase in mitotic aberrations over 1 bar treated as was the case with hypo/hypersaline treatments (p < 0.05). Conversely, an increase in hydrostatic pressure actually reduced the effects of spindle inhibition by the aneugen colchicine (p < 0.05)., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The spectacular escalation in complexity in early bilaterian evolution correlates with a strong increase in the number of microRNAs. To explore the link between the birth of ancient microRNAs and body plan evolution, we set out to determine the ancient sites of activity of conserved bilaterian microRNA families in a comparative approach. We reason that any specific localization shared between protostomes and deuterostomes (the two major superphyla of bilaterian animals) should probably reflect an ancient specificity of that microRNA in their last common ancestor. Here, we investigate the expression of conserved bilaterian microRNAs in Platynereis dumerilii, a protostome retaining ancestral bilaterian features, in Capitella, another marine annelid, in the sea urchin Strongylocentrotus, a deuterostome, and in sea anemone Nematostella, representing an outgroup to the bilaterians. Our comparative data indicate that the oldest known animal microRNA, miR-100, and the related miR-125 and let-7 were initially active in neurosecretory cells located around the mouth. Other sets of ancient microRNAs were first present in locomotor ciliated cells, specific brain centres, or, more broadly, one of four major organ systems: central nervous system, sensory tissue, musculature and gut. These findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution. Also, they outline a minimum set of cell types and tissues that existed in the protostome-deuterostome ancestor.

Aspects of muscle development are still widely neglected in studies on invertebrate ontogeny, which is probably at least partly due to the inherent complexity of animal myoanatomical bodyplans. This has resulted in significant gaps in our understanding of the evolutionary and ontogenetic origin of this crucial mesoderm-derived organ system, particularly in indirect developing representatives of the Lophotrochozoa. Here, we document the temporal and spatial patterns of muscle formation and cell proliferation in the polychaete Sabellaria alveolata during planktotrophic larval development in order to assess the presumed generation of muscle units and segments from a posterior growth zone. In addition, we address the question whether the three primary segments differ in their mode of myogenesis from the subsequently forming segments. We found that in the first three segments the ventral transverse muscles differentiate from anterior to posterior, whereas the ventral oblique muscles develop simultaneously. Hence, subsequent and simultaneous developmental processes of specific muscle groups are possibly regulated in different ways, thus emphasizing the plasticity of the formation of metamerically arranged organ systems in polychaetes. The occurrence of three clusters of proliferating cells in the trunk region of the metatrochophore indicates synchronous subdivision of the larval mesoderm in the first three segments. Assuming a polychaete-like ancestor at the base of the annelid tree, comparative analysis suggests that the bodywall of the last common annelid ancestor might have been devoid of circular muscles and consisted of four separate longitudinal muscle strands that develop from anterior to posterior.

Despite the wide variety of larval forms among polychaetes, most are clearly derived from the canonical spiralian trochophore. Within the genus Owenia (family Oweniidae), however, the mitraria larva lacks the characteristic ciliary bands of the trochophore, and those it has are monociliated, typically a deuterostome characteristic. Adult Owenia spp. also possess a monociliated epidermis and deuterostome-like nephridia. This study is the first detailed account of early embryology for any member of the Oweniidae. Light, confocal, and scanning electron microscopy were used to investigate organogenesis from fertilization through metamorphosis in Owenia collaris. Equal spiral cleavage yields an embryo with an unusually large blastocoel for a spiralian. The embryo undergoes gastrulation by invagination, and begins swimming 24 h after fertilization. Three important events deviate markedly from stereotypical polychaete embryogenesis. First, at the 8-cell stage the micromeres are larger than the macromeres, as in nemerteans. Second, the blastopore becomes the anus, as in some deuterostomes, while the stomodeum may form secondarily. Third, the cells that would form the prototroch in a canonical spiralian trochophore (1q(2) descendants) never undergo cleavage arrest, and the primary ciliated band of the mitraria never contains large, multiciliated cells. The mitraria larva thus represents a mixture of protostome and deuterostome developmental traits, suggesting that spiralian development is not so rigidly constrained as it might appear.

Polarized oogenic cysts are clonal syncytia in which cellular compartments (cystocytes) differentiate as either oocytes or nurse cells-supportive germ cells that transport material to the oocyte through cytoplasmic bridges. Although polarized cysts have evolved independently in several animal phyla, most research on this phenomenon has focused on insects, particularly Drosophila melanogaster. To provide a foundation for comparative studies of cyst polarization, we have examined the coelomic development of polarized cysts in an annelid worm, Ophryotrocha labronica, using a variety of microscopic techniques. This species has elegantly simple cysts, each consisting of two interconnected cells (one oocyte and one nurse cell) free of somatic follicular tissue. These cysts arise from segmental ovaries, but are released to the coelom early in oogenesis, where they carry out most of their development. Although the oocyte remains transcriptionally active through oogenesis, nurse cells in this species are robust biosynthetic factories, with highly polyploid nuclei, enormous nucleoli, numerous mitochondria, and abundant rough endoplasmic reticulum. Ultimately, the nurse cell dumps its cytoplasm into oocyte, detaches, and undergoes programmed cell death. Although in insects, and possibly some polychaetes, the overall polarity of the cyst is linked to the specification of future embryonic axes in the oocyte, in Ophryotrocha this appears not to be the case. Our findings provide a detailed analysis of cyst development, addressing earlier misconceptions in the literature and adding new insights. This foundation shows the potential of Ophryotrocha labronica as a model system for further experimental analysis of nurse cell-oocyte differentiation.

This study deals with neurohistologic analysis of the common nerve plexus of the body wall of the polychaete Nephthys ciliata. The cell composition and international relations are shown in the nerve plexus subepidermal and intramuscular areas. Morphological data are presented about a possible origin, on the basis if differentiating primary sensory bipolars, of typical associative and presumably motor neurons located outside the abdominal brain. In the nerve plexus, both axo-axonal and axo-dendritic as well axo-somatic international contacts are shown. A characteristic feature of the studied peripheral nerve plexus of the Nephthys ciliata body wall is emphasized: a sufficiently pronounced development of population of associative neurons. This provides the structural basis for peripheral integration of nervous processes in the central nervous system of the whole animal. This work also describes small groups of sensory and associative neurons; they also seem to serve for a relative autonomization of the peripheral chain of the central nervous system of Nephthys ciliata. This also can be facilitated by single presumed motor neurons of the plexus. In the studied nerve plexus there are actually absent the typical associative-motor neurons that are so characteristic of the abdominal brain of polychaetes, oligochaetes, and leeches.

Hypotaurine (HT) has been proposed to reduce sulfide toxicity in some deep-sea invertebrates by scavenging free radicals produced from sulfide oxidation or by scavenging sulfide via the reaction of HT with sulfide, forming thiotaurine (ThT). We tested whether HT or several antioxidants could reduce the total dissolved sulfide concentration in buffered seawater exposed to H(2)S, and whether HT, ThT or antioxidants could increase the viability of Glycera dibranchiata erythrocytes exposed to H(2)S in vitro. We found that 5 and 50 mmol l(-1) HT reduced the dissolved sulfide in cell-free buffer exposed to H(2)S by up to 80% whereas the antioxidants glutathione ethyl ester (GEE), N-acetylcysteine (NAC), L-ascorbic acid (ASC), Tempol and Trolox had no consistent effect. Exposure of erythrocytes to 0.10%-3.2% H(2)S (producing 0.18-4.8 mmol l(-1) sulfide) decreased the fraction of viable cells, as evidenced by loss of plasma membrane integrity, with virtually no cells remaining viable at 1.0% or more H(2)S. Addition of HT (0.5-50 mmol l(-1)) significantly increased the fraction of viable cells (e.g. from 0.01 to 0.84 at 0.32% H(2)S) whereas ThT (0.5 and 5 mmol l(-1)) decreased cell viability. GEE (0.03-3 mmol l(-1)) and NAC (0.001-1 mmol l(-1)), which contain sulfhydryl groups, increased cell viability during H(2)S exposure but to a lesser extent than HT whereas ASC, Tempol and Trolox, which do not contain sulfhydryl groups, decreased viability or had no effect. These data show that HT can protect cells from sulfide in vitro and suggest that sulfide scavenging, rather than free radical scavenging, is the most important mechanism of protection.

Sulfide oxidation in the lugworm, Arenicola marina (L.), is most likely localized in the mitochondria, which can either produce ATP with sulfide as a substrate or detoxify it via an alternative oxidase. The present study identified selective activators of the energy-conserving and the detoxifying sulfide oxidation pathways respectively. In the presence of the ROS scavengers glutathione (GSH) and ascorbate, isolated lugworm mitochondria rapidly oxidized up to 100 micromoll(-1) sulfide with maximal oxygen consumption rates but did not produce any ATP in the process. Under these conditions, salicylhydroxamic acid (SHAM), which is an inhibitor of the alternative oxidase of plant mitochondria, completely blocked oxygen consumption whereas inhibitors of complex III and IV had hardly any effect. By contrast, dehydroascorbate (DHA) enabled the mitochondria to gain ATP from sulfide oxidation even if the sulfide concentration far exceeded the threshold for inhibition of cytochrome oxidase. In the presence of dehydroascorbate, respiratory rates were independent of sulfide concentrations, with a respiratory control ratio of 2.1+/-0.2, and both oxygen consumption and ATP production were completely inhibited by myxothiazol and sodium azide but only marginally by SHAM. The present data indicate that a redox mechanism may contribute to the regulation of sulfide oxidation in lugworm mitochondria in vivo. Thus, mitochondria are presumably much more sulfide resistant in a cellular context than previously thought.

The marine environment is becoming increasingly contaminated by environmental pollutants with the potential to damage DNA, with marine sediments acting as a sink for many of these contaminants. Understanding genotoxic responses in sediment-dwelling marine organisms, such as polychaetes, is therefore of increasing importance. This study is an exploration of species-specific and cell-specific differences in cell sensitivities to DNA-damaging agents in polychaete worms, aimed at increasing fundamental knowledge of their responses to genotoxic damage. The sensitivities of coelomocytes from three polychaetes species of high ecological relevance, i.e. the lugworm Arenicola marina, the harbour ragworm Nereis diversicolor and the king ragworm Nereis virens to genotoxic damage are compared, and differences in sensitivities of their different coelomic cell types determined by use of the comet assay. A. marina was found to be the most sensitive to genotoxic damage induced by the direct-acting mutagen methyl methanesulfonate (MMS), and showed dose-dependent responses to MMS and the polycyclic aromatic hydrocarbon benzo(a)pyrene. Significant differences in sensitivity were also measured for the different types of coelomocyte. Eleocytes were more sensitive to induction of DNA damage than amoebocytes in both N. virens and N. diversicolor. Spermatozoa from A. marina showed significant DNA damage following in vitro exposure to MMS, but were less sensitive to DNA damage than coelomocytes. This investigation has clearly demonstrated that different cell types within the same species and different species within the polychaetes show significantly different responses to genotoxic insult. These findings are discussed in terms of the relationship between cell function and sensitivity and their implications for the use of polychaetes in environmental genotoxicity studies.

The regulation of larval development by starved adults occurs in both freshwater snails, Helisoma trivolvis and marine polychaetes, Platynereis dumerilii. Serotonin (5-HT) links this environmental signal which is detected by larval apical sensory neurons to changes in larval development. A profile of the stage-dependent expression of 5-HT receptors and coupled G proteins is essential in this regulatory mechanism. The final effect on development depends on the modulation of the activity of the larval digestive system.

In response to Wnt signaling during animal development, beta-catenin accumulates in nuclei to mediate the transcriptional activation of target genes. Here, we show that a highly conserved beta-catenin in the annelid Platynereis dumerilii exhibits a reiterative, nearly universal embryonic pattern of nuclear accumulation remarkably similar to that observed in the nematode Caenorhabditis elegans. Platynereis exhibits beta-catenin sister-cell asymmetries after all cell divisions that occur along the animal/vegetal axis beginning early in embryogenesis, but not after two transverse divisions that establish bilateral symmetry in the trunk. Moreover, ectopic activation of nuclear beta-catenin accumulation in Platynereis causes animal-pole sister cells, which normally have low nuclear beta-catenin levels, to adopt the fate of their vegetal-pole sisters, which normally have high nuclear beta-catenin levels. The presence of reiterative and functionally important beta-catenin asymmetries in two distantly related animal phyla suggests an ancient metazoan origin of a beta-catenin-mediated binary cell-fate specification module.

The embryos of indirect developers generate an intermediate larval stage that nourishes the proliferation of undifferentiated multipotent cell precursors in charge of postembryonic adult formation. Multipotency affects the regulation of many genes and seems to be mediated in part by chromatin modification. Chromatin transcriptional properties are regulated by histone modification and by incorporation of peculiar histone variants. The histone variant H2A.Z is associated with transcriptionally competent chromatin and silent genes primed for activation or permanent repression. However, despite the extensive mechanistic characterizations in unicellular eukaryotes, the essential role of the highly conserved H2A.Z variant during animal embryogenesis remains obscure. We show that the expression of H2A.Z in the larvae of two distant indirectly developing marine invertebrates, a polychaete and a sea urchin, remains high in all their embryonic and postembryonic developmentally competent cell precursors, and declines during their differentiation. In particular, the expression in undifferentiated multipotent adult precursors during feeding larval stages in both organisms provides unique insight about its general association with developmental potential. Our experiments confirm previous reports indicating that the expression of H2A.Z is proliferation (DNA synthesis) independent, in contrast with the DNA synthesis dependence of "mainstream" histones. We suggest that similar H2A.Z transcriptional functions previously identified in unicellular organisms also help to maintain an open chromatin state competent for transcriptional-regulatory transactions during metazoan development.

During frog and fish development, convergent extension movements transform the spherical gastrula into an elongated neurula. Such transformation of a ball- into a worm-shaped embryo is an ancestral and fundamental feature of bilaterian development, yet this is modified or absent in the protostome model organisms Caenorhabditis or Drosophila. In the polychaete annelid Platynereis dumerilii, early embryonic and larval stages resemble a sphere that subsequently elongates into worm shape. Cellular and molecular mechanisms of polychaete body elongation are yet unknown. Our in vivo time-lapse analysis of Platynereis axis elongation reveals that the polychaete neuroectoderm converges and extends by mediolateral cell intercalation. This occurs on both sides of the neural midline, the line of fusion of the slit-like blastopore. Convergent extension moves apart mouth and anus that are both derived from the blastopore. Tissue elongation is actin-dependent but microtubule-independent. Dependence on JNK activity and spatially restricted expression of strabismus indicates involvement of the noncanonical Wnt pathway. We detect a morphogenetic boundary between the converging and extending trunk neuroectoderm and the anterior otx-expressing head neuroectoderm that does not elongate. Our comparative analysis uncovers striking similarities but also differences between convergent extension in the polychaete and in the frog (the classical vertebrate model for convergent extension). Based on these findings, we propose that convergent extension movements of the trunk neuroectoderm represent an ancestral feature of bilaterian development that triggered the separation of mouth and anus along the elongating trunk.