Ultrastructural and immunocytochemical evidence of a colonial nervous system in hydroids.

Background: As the sister group to all Bilateria, representatives of the phylum Cnidaria (sea anemones, corals, jellyfishes, and hydroids) possess a recognizable and well-developed nervous system and have attracted considerable attention over the years from neurobiologists and evo-devo researchers. Despite a long history of nervous system investigation in Cnidaria, most studies have been performed on unitary organisms. However, the majority of cnidarians are colonial (modular) organisms with unique and specific features of development and function. Nevertheless, data on the nervous system in colonial cnidarians are scarce. Within hydrozoans (Hydrozoa and Cnidaria), a structurally "simple" nervous system has been described for Hydra and zooids of several colonial species. A more complex organization of the nervous system, closely related to the animals' motile mode of life, has been shown for the medusa stage and a few siphonophores. Direct evidence of a colonial nervous system interconnecting zooids of a hydrozoan colony has been obtained only for two species, while it has been stated that in other studied species, the coenosarc lacks nerves. Methods: In the present study, the presence of a nervous system in the coenosarc of three species of colonial hydroids - the athecate Clava multicornis, and thecate Dynamena pumila and Obelia longissima - was studied based on immunocytochemical and ultrastructural investigations. Results: Confocal scanning laser microscopy revealed a loose system composed of delicate, mostly bipolar, neurons visualized using a combination of anti-tyrosinated and anti-acetylated a-tubulin antibodies, as well as anti-RF-amide antibodies. Only ganglion nerve cells were observed. The neurites were found in the growing stolon tips close to the tip apex. Ultrastructural data confirmed the presence of neurons in the coenosarc epidermis of all the studied species. In the coenosarc, the neurons and their processes were found to settle on the mesoglea, and the muscle processes were found to overlay the nerve cells. Some of the neurites were found to run within the mesoglea. Discussion: Based on the findings, the possible role of the colonial nervous system in sessile hydroids is discussed.

Unusual lophophore innervation in ctenostome Flustrellidra hispida (Bryozoa).

Since ctenostomes are traditionally regarded as an ancestral clade to some other bryozoan groups, the study of additional species may help to clarify questions on bryozoan evolution and phylogeny. One of these questions is the bryozoan lophophore evolution: whether it occurred through simplification or complication. The morphology and innervation of the ctenostome Flustrellidra hispida (Fabricius, 1780) lophophore have been studied with electron microscopy and immunocytochemistry with confocal laser scanning microscopy. Lophophore nervous system of F. hispida consists of several main nerve elements: cerebral ganglion, circumoral nerve ring, and the outer nerve ring. Serotonin-like immunoreactive perikarya, which connect with the circumoral nerve ring, bear the cilium that directs to the abfrontal side of the lophophore and extends between tentacle bases. The circumoral nerve ring gives rise to the intertentacular and frontal tentacle nerves. The outer nerve ring gives rise to the abfrontal neurites, which connect to the outer groups of perikarya and contribute to the formation of the abfrontal tentacle nerve. The outer nerve ring has been described before in other bryozoans, but it never contributes to the innervation of tentacles. The presence of the outer nerve ring participating in the innervation of tentacles makes the F. hispida lophophore nervous system particularly similar to the lophophore nervous system of phoronids. This similarity allows to suggest that organization of the F. hispida lophophore nervous system may reflect the ancestral state for all bryozoans. The possible scenario of evolutionary transformation of the lophophore nervous system within bryozoans is suggested.

Embryonic development of the moon jellyfish Aurelia aurita (Cnidaria, Scyphozoa): another variant on the theme of invagination.

Background: Aurelia aurita (Scyphozoa, Cnidaria) is an emblematic species of the jellyfish. Currently, it is an emerging model of Evo-Devo for studying evolution and molecular regulation of metazoans' complex life cycle, early development, and cell differentiation. For Aurelia, the genome was sequenced, the molecular cascades involved in the life cycle transitions were characterized, and embryogenesis was studied on the level of gross morphology. As a reliable representative of the class Scyphozoa, Aurelia can be used for comparative analysis of embryonic development within Cnidaria and between Cnidaria and Bilateria. One of the intriguing questions that can be posed is whether the invagination occurring during gastrulation of different cnidarians relies on the same cellular mechanisms. To answer this question, a detailed study of the cellular mechanisms underlying the early development of Aurelia is required. Methods: We studied the embryogenesis of A. aurita using the modern methods of light microscopy, immunocytochemistry, confocal laser microscopy, scanning and transmission electron microscopy. Results: In this article, we report a comprehensive study of the early development of A. aurita from the White Sea population. We described in detail the embryonic development of A. aurita from early cleavage up to the planula larva. We focused mainly on the cell morphogenetic movements underlying gastrulation. The dynamics of cell shape changes and cell behavior during invagination of the archenteron (future endoderm) were characterized. That allowed comparing the gastrulation by invagination in two cnidarian species-scyphozoan A. aurita and anthozoan Nematostella vectensis. We described the successive stages of blastopore closure and found that segregation of the germ layers in A. aurita is linked to the 'healing' of the blastopore lip. We followed the developmental origin of the planula body parts and characterized the planula cells' ultrastructure. We also found that the planula endoderm consists of three morphologically distinct compartments along the oral-aboral axis. Conclusions: Epithelial invagination is a fundamental morphogenetic movement that is believed as highly conserved across metazoans. Our data on the cell shaping and behaviours driving invagination in A. aurita contribute to understanding of morphologically similar morphogenesis in different animals. By comparative analysis, we clearly show that invagination may differ at the cellular level between cnidarian species belonging to different classes (Anthozoa and Scyphozoa). The number of cells involved in invagination, the dynamics of the shape of the archenteron cells, the stage of epithelial-mesenchymal transition that these cells can reach, and the fate of blastopore lip cells may vary greatly between species. These results help to gain insight into the evolution of morphogenesis within the Cnidaria and within Metazoa in general.

Studying of Molecular Regulation of Developmental Processes of Lower Metazoans Exemplified by Cnidaria Using High-Throughput Sequencing.

A unique set of features and characteristics of species of the Cnidaria phylum is the one reason that makes them a model for a various studies. The plasticity of a life cycle and the processes of cell differentiation and development of an integral multicellular organism associated with it are of a specific scientific interest. A new stage of development of molecular genetic methods, including methods for high-throughput genome, transcriptome, and epigenome sequencing, both at the level of the whole organism and at the level of individual cells, makes it possible to obtain a detailed picture of the development of these animals. This review examines some modern approaches and advances in the reconstruction of the processes of ontogenesis of cnidarians by studying the regulatory signal transduction pathways and their interactions.

Rehabilitation of Mytilus edulis larvae abnormalities induced by K2Cr2O7 in short-term experiments.

Mytilus edulis embryo-larval development is often used as a bioassay to evaluate the negative impact of contaminants and environmental conditions. The toxicity criteria used in most protocols is the proportions of normal and abnormal larvae. The variety of abnormalities were described and classified, but further development of abnormal larvae remains obscure. This study aimed to reveal the possibility of correction of the morphological abnormalities after short-term exposure (48 h) in a variety of K2Cr2O7 concentrations. For this purpose, abnormal larvae, which developed under the negative influence of the series of K2Cr2O7 concentrations were transferred into clean seawater and studied after further 24 and 48 h. The obtained data, concerning changes in larval morphology, growth and survival rates during washing show that the abnormal larvae have enough capability to recover the normal D-shell structure. Moreover, restoration of the D-shell is possible even after exposure with concentration of the toxicant higher than the average effective one. The present research also pointed out that the development of larval shell (even abnormal one) is positively correlated with ability of the larvae to reconstruct D-shell and their survival rate. High mortality during washing occurs only at toxicant concentrations when no shell was formed within 48 h. Thus, the existence of the shell after 48 h exposure in the toxicant could indicate reversibility of the negative impact and help to distinguish the delay in development from its arrest.

Intraspecific variability of cell reaggregation during reproduction cycle in sponges.

The ability of sponge cells to reaggregate and reconstruct intact functional organism is known for more than 100 years. This process was studied in numerous species of sponges, and its interspecific variability is well described. However, some data also indicate the existence of a certain intraspecific variability of the cell reaggregation. The present study deals with the cell reaggregation in two demosponges, Halichondria panicea and Halisarca dujardinii, during different periods of their sexual reproduction. In both species, cell reaggregation shows a common pattern at all studied periods of reproduction. However, the course of the reaggregation process significantly depends on the reproduction period of an individual used in the experiment, thus demonstrating pronounced intraspecific variability, which concerns the rate of the cell reaggregation and the final stage of the process. This variability occurs due to tissue rearrangements that accompany reproduction and changes cell composition and amount of available somatic stem cells in sponge tissues, and consequently alters morphogenetic potencies of a cell suspension and multicellular aggregates. In both Halichondria panicea and Halisarca dujardinii, the growth period is the most favorable for the reaggregation process, while the cell reaggregation is depressed during periods of embryogenesis and restoration of somatic tissues after the reproduction. At the same time, the structure of a particular stage of reaggregation and morphogenetic processes underlying the development of multicellular aggregates are always identical, independently from the period of the reproductive cycle.

Correction: Iron metabolic pathways in the processes of sponge plasticity.

[This corrects the article DOI: 10.1371/journal.pone.0228722.].

Iron metabolic pathways in the processes of sponge plasticity.

The ability to regulate oxygen consumption evolved in ancestral animals and is intrinsically linked to iron metabolism. The iron pathways have been intensively studied in mammals, whereas data on distant invertebrates are limited. Sea sponges represent the oldest animal phylum and have unique structural plasticity and capacity to reaggregate after complete dissociation. We studied iron metabolic factors and their expression during reaggregation in the White Sea cold-water sponges Halichondria panicea and Halisarca dujardini. De novo transcriptomes were assembled using RNA-Seq data, and evolutionary trends were analyzed with bioinformatic tools. Differential expression during reaggregation was studied for H. dujardini. Enzymes of the heme biosynthesis pathway and transport globins, neuroglobin (NGB) and androglobin (ADGB), were identified in sponges. The globins mutate at higher evolutionary rates than the heme synthesis enzymes. Highly conserved iron-regulatory protein 1 (IRP1) presumably interacts with the iron-responsive elements (IREs) found in mRNAs of ferritin (FTH1) and a putative transferrin receptor NAALAD2. The reaggregation process is accompanied by increased expression of IRP1, the antiapoptotic factor BCL2, the inflammation factor NFκB (p65), FTH1 and NGB, as well as by an increase in mitochondrial density. Our data indicate a complex mechanism of iron regulation in sponge structural plasticity and help to better understand general mechanisms of morphogenetic processes in multicellular species.

сWnt signaling modulation results in a change of the colony architecture in a hydrozoan.

At the polyp stage, most hydrozoan cnidarians form highly elaborate colonies with a variety of branching patterns, which makes them excellent models for studying the evolutionary mechanisms of body plan diversification. At the same time, molecular mechanisms underlying the robust patterning of the architecturally complex hydrozoan colonies remain unexplored. Using non-model hydrozoan Dynamena pumila we showed that the key components of the Wnt/ß-catenin (cWnt) pathway (ß-catenin, TCF) and the cWnt-responsive gene, brachyury 2, are involved in specification and patterning of the developing colony shoots. Strikingly, pharmacological modulation of the cWnt pathway leads to radical modification of the monopodially branching colony of Dynamena which acquire branching patterns typical for colonies of other hydrozoan species. Our results suggest that modulation of the cWnt signaling is the driving force promoting the evolution of the vast variety of the body plans in hydrozoan colonies and offer an intriguing possibility that the involvement of the cWnt pathway in the regulation of branching morphogenesis might represent an ancestral feature predating the cnidarian-bilaterian split.

The nervous system in the cyclostome bryozoan Crisia eburnea as revealed by transmission electron and confocal laser scanning microscopy.

INTRODUCTION: Among bryozoans, cyclostome anatomy is the least studied by modern methods. New data on the nervous system fill the gap in our knowledge and make morphological analysis much more fruitful to resolve some questions of bryozoan evolution and phylogeny. RESULTS: The nervous system of cyclostome Crisia eburnea was studied by transmission electron microscopy and confocal laser scanning microscopy. The cerebral ganglion has an upper concavity and a small inner cavity filled with cilia and microvilli, thus exhibiting features of neuroepithelium. The cerebral ganglion is associated with the circumoral nerve ring, the circumpharyngeal nerve ring, and the outer nerve ring. Each tentacle has six longitudinal neurite bundles. The body wall is innervated by thick paired longitudinal nerves. Circular nerves are associated with atrial sphincter. A membranous sac, cardia, and caecum all have nervous plexus. CONCLUSION: The nervous system of the cyclostome C. eburnea combines phylactolaemate and gymnolaemate features. Innervation of tentacles by six neurite bundles is similar of that in Phylactolaemata. The presence of circumpharyngeal nerve ring and outer nerve ring is characteristic of both, Cyclostomata and Gymnolaemata. The structure of the cerebral ganglion may be regarded as a result of transformation of hypothetical ancestral neuroepithelium. Primitive cerebral ganglion and combination of nerve plexus and cords in the nervous system of C. eburnea allows to suggest that the nerve system topography of C. eburnea may represent an ancestral state of nervous system organization in Bryozoa. Several scenarios describing evolution of the cerebral ganglion in different bryozoan groups are proposed.

Embryonic development of thecate hydrozoan Gonothyraea loveni (Allman, 1859).

Progress of Evo-Devo requires broad phylogenetic sampling providing the data for comparative analysis as well as new objects suitable for experimental investigation. Representatives of the early-branching animal phylum Cnidaria and particularly hydrozoans draw great attention due to the high diversity of embryonic and post-embryonic development and life-cycles in general. Most detailed studies on embryonic development in hydrozoans were performed on the species shedding their gametes with subsequent embryo development in the water column. Widely distributed thecate hydrozoan Gonothyraea loveni broods its embryos within reduced medusae attached to the colony until development of a free-swimming metamorphosis competent planula-larva. In the current essay we present a detailed description of G. loveni embryonic development based on in vivo observations, histology, immuno-cytochemistry, and electron microscopy. Starting from early cleavage, the embryo becomes a morula without any sign of blastocoele. Gastrulation proceeds as mixed delamination and ends with parenchymula formation. The first morphological sign of primary body axis appears only in the beginning of parenchymula-preplanula transition. In mature metamorphosis competent planula only the cells of the oral two-thirds of endoderm retain proliferative activity resulting in accumulation of great number of i-cells and nematoblasts, which can be used during metamorphosis accompanied with essential reorganization of larval tissues. G. loveni demonstrates the diversity as well as evolutionary plasticity of hydrozoans development: in brooding hydrozoans embryonic and larval development is highly embryonized in comparison with the spawning species with free-swimming embryos.

Stolonial Movement: A New Type of Whole-Organism Behavior in Porifera.

Sponges (phylum Porifera) traditionally are represented as inactive, sessile filter-feeding animals devoid of any behavior except filtering activity. However, different time-lapse techniques demonstrate that sponges are able to show a wide range of coordinated but slow whole-organism behavior. The present study concerns a peculiar type of such behavior in the psychrophilic demosponge Amphilectus lobatus: stolonial movement. During stolonial movement, sponges produce outgrowths (stolons) that crawl along a substrate with a speed of 4.4 ± 2.2 µm min-1 and branch, thus forming a complex net covering a considerable area of a substrate. This net is used by sponges to search for new points with appropriate environmental conditions for individual relocation. After such points are found, all cells of the parental sponge migrate through stolons, leaving a naked parental skeleton, forming one or several filial sponges in the new location. Thus, stolonial movement combines traits of crawling along the substrate and asexual reproduction. This behavior relies on massive cell dedifferentiation followed by coordinated cell migration to the point of new sponge body formation and their subsequent differentiation into specialized cell types.

Pharmacological analysis of the transmembrane action potential configuration in myoepithelial cells of the spontaneously beating heart of the ascidian Styela rustica in vitro.

The mechanisms of action potential (AP) generation in the myoepithelial cells of the tunicate heart are not yet well understood. Here, an attempt was made to elucidate these mechanisms by analyzing the effects of specific blockers of K+, Na+ and Ca2+ currents on the configuration of transmembrane APs and their frequency in the spontaneously beating ascidian heart. In addition, an immunocytochemical analysis of heart myoepithelial cells was performed. Staining with anti-FMRF-amide and anti-tubulin antibodies did not reveal any nerve elements within the heart tube. Treatment with 1 mmol l-1 TEA (IK blocker) resulted in depolarization of heart cell sarcolemma by 10 mV, and inhibition of APs generation was recorded after 3 min of exposure. Prior to this moment, the frequency of AP generation in a burst decreased from 16-18 to 2 beats min-1 owing to prolongation of the diastole. After application of ivabradine (3 or 10 µmol l-1), the spontaneous APs generation frequency decreased by 24%. Based on these results and published data, it is concluded that the key role in the automaticity of the ascidian heart is played by the outward K+ currents, Na+ currents, activated hyperpolarization current If and a current of unknown nature IX.

The nervous system of the lophophore in the ctenostome Amathia gracilis provides insight into the morphology of ancestral ectoprocts and the monophyly of the lophophorates.

BACKGROUND: The Bryozoa (=Ectoprocta) is a large group of bilaterians that exhibit great variability in the innervation of tentacles and in the organization of the cerebral ganglion. Investigations of bryozoans from different groups may contribute to the reconstruction of the bryozoan nervous system bauplan. A detailed investigation of the polypide nervous system of the ctenostome bryozoan Amathia gracilis is reported here. RESULTS: The cerebral ganglion displays prominent zonality and has at least three zones: proximal, central, and distal. The proximal zone is the most developed and contains two large perikarya giving rise to the tentacle sheath nerves. The neuroepithelial organization of the cerebral ganglion is revealed. The tiny lumen of the cerebral ganglion is represented by narrow spaces between the apical projections of the perikarya of the central zone. The cerebral ganglion gives rise to five groups of main neurite bundles of the lophophore and the tentacle sheath: the circum-oral nerve ring, the lophophoral dorso-lateral nerves, the pharyngeal and visceral neurite bundles, the outer nerve ring, and the tentacle sheath nerves. Serotonin-like immunoreactive nerve system of polypide includes eight large perikarya located between tentacles bases. There are two analmost and six oralmost perikarya with prominent serotonergic "gap" between them. Based on the characteristics of their innervations, the tentacles can be subdivided into two groups: four that are near the anus and six that are near the mouth. Two longitudinal neurite bundles - medio-frontal and abfrontal - extend along each tentacle. CONCLUSION: The zonality of the cerebral ganglion, the presence of three commissures, and location of the main nerves emanating from each zone might have caused by directive innervation of the various parts of the body: the tentacles sheath, the lophohpore, and the digestive tract. Two alternative scenarios of bryozoan lophophore evolution are discussed. The arrangement of large serotonin-like immunoreactive perikarya differs from the pattern previously described in ctenostome bryozoans. In accordance with its position relative to the same organs (tentacles, anus, and mouth), the lophophore outer nerve ring corresponds to the brachiopod lower brachial nerve and to the phoronid tentacular nerve ring. The presence of the outer nerve ring makes the lophophore innervation within the group (clade) of lophophorates similar and provides additional morphological evidence of the lophophore homology and monophyly of the lophophorates.

Sponge cell reaggregation: Cellular structure and morphogenetic potencies of multicellular aggregates.

Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions.

Organizer regions in marine colonial hydrozoans.

Organizers are specific tissue regions of developing organisms that provide accuracy and robustness to the body plan formation. Hydrozoan cnidarians (both solitary and colonial) require organizer regions for maintaining the regular body patterning during continuous tissue dynamics during asexual reproduction and growth. While the hypostomal organizer of the solitary Hydra has been studied relatively well, our knowledge of organizers in colonial hydrozoans remains fragmentary and incomplete. As colonial hydrozoans demonstrate an amazing diversity of morphological and life history traits, it is of special interest to investigate the organizers specific for particular ontogenetic stages and particular types of colonies. In the present study we aimed to assess the inductive capacities of several candidate organizer regions in hydroids with different colony organization. We carried out grafting experiments on colonial hydrozoans belonging to Leptothecata and Anthoathecata. We confirmed that the hypostome tip is an organizer in the colonial Anthoathecata as it is in the solitary polyp Hydra. We also found that the posterior tip of the larva is an organizer in hydroids regardless of the peculiarities of their metamorphosis mode and colony structure. We show for the first time that the shoot growing tip, which can be considered a key evolutionary novelty of Leptothecata, is an organizer region. Taken together, our data demonstrate that organizers function throughout the larval and polypoid stages in colonial hydroids.

FMRF-amide immunoreactivity pattern in the planula and colony of the hydroid Gonothyraea loveni.

Gonothyraea loveni (Allman, 1859) is a colonial thecate hydrozoan with a life cycle that lacks a free-swimming medusa stage. The development from zygote to planula occurs within meconidia attached to the female colony. The planula metamorphosis results in the formation of a primary hydranth. The colony then grows by development of new colony elements. In the present work, we studied the temporal pattern of the formation of FMRF-amide-positive cells during embryogenesis, in larvae and during early colony ontogeny. FMRF-amide-positive cells appear in the planula only after its maturation. However, they disappear after planula settlement. For the first time, we show that neural cells are present in the coenosarc of the hydroid colony. We also trace the process of neural net formation during the development of a new shoot internode of the G. loveni colony.

Changes in the patterning of a hydroid colony.

It is a widely held view that colonial hydroids (Cnidaria, Hydroidomedusae) are formed on the basis of a repetition of uniform elements. The dominant opinion is that the equal spatial organisation of the colony exists during all stages of its development except the primary polyp, which develops from the settled larva. However, the complex structure and large dimensions of shoots in certain thecate species (subcl. Leptomedusae) suggest that the organisation of the primary shoot differs strongly from that of established colonies. The present study based on a thorough collection and examination of the collected material allowed to describe the entire sequence of the colony ontogeny in Hydrallmania falcata (Sertulariidae). The established shoots of this species are characterised by relatively large size, spiral arrangement of pinnate branches over the shoot stem, and hydranths arranged in one row along the upper side of branches. We showed that the primary shoot developing from the larva has much smaller dimensions and an alternate arrangement of hydranths. During further colony development the shoot organisation undergoes a gradual transformation ending with the emergence of large shoots with 'characteristic' species-specific features. The discovered sequence of changes in shoot patterning shows certain correlations with alterations of the growing tip dimensions. The dimensions of the growing tip seem to determine the patterning in accordance with the particular spatial location of the tip. This finding implies the necessity of a detailed reinvestigation of the entire colony development in thecate hydroids, which would make a significant contribution to the understanding of the morphogenetic evolution and patterning mechanisms within this group of colonial organisms.