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Many species of aquatic worms, including members of the phyla Nemertea, Annelida, Platyhelminthes, and Xenacoelomorpha, can regenerate large parts of their body after amputation. In most species, cell proliferation plays key roles in the reconstruction of lost tissues. For example, in annelids and flatworms, inhibition of cell proliferation by irradiation or chemicals prevents regeneration. Cell proliferation also plays crucial roles in growth, body patterning (e.g., segmentation) and asexual reproduction in many groups of aquatic worms. Cell proliferation dynamics in these organisms can be studied using immunohistochemical detection of proteins expressed during proliferation-associated processes or by incorporation and labeling of thymidine analogues during DNA replication. In this chapter, we present protocols for labeling and quantifying cell proliferation by (a) antibody-based detection of either phosphorylated histone H3 during mitosis or proliferating cell nuclear antigen (PCNA) during S-phase, and (b) incorporation of two thymidine analogues, 5'-bromo-2'-deoxyuridine (BrdU) and 5'-ethynyl-2'-deoxyuridine (EdU), detected by immunohistochemistry or inorganic "click" chemistry, respectively. Although these protocols have been developed for whole mounts of small (<2 cm) marine and freshwater worms, they can also be adapted for use in larger specimens or tissue sections.

Animal female and male germ-line cells often form syncytial units termed cysts, clusters, or clones. Within these cysts, the cells remain interconnected by specific cell junctions known as intercellular bridges or ring canals, which enable cytoplasm to be shared and macromolecules and organelles to be exchanged between cells. Numerous analyses have shown that the spatial organization of cysts and their functioning may differ between the sexes and taxa. The vast majority of our knowledge about the formation and functioning of germ-line cysts comes from studies of model species (mainly Drosophila melanogaster); the other systems of the cyst organization and functioning are much less known and are sometimes overlooked. Here, we present the current state of the knowledge of female germ-line cysts in clitellate annelids (Clitellata), which is a monophyletic taxon of segmented worms (Annelida). The organization of germ-line cysts in clitellates differs markedly from that of the fruit fly and vertebrates. In Clitellata, germ cells are not directly connected one to another, but, as a rule, each cell has one ring canal that connects it to an anuclear central cytoplasmic core, a cytophore. Thus, this pattern of cell distribution is similar to the germ-line cysts of Caenorhabditis elegans. The last decade of studies has revealed that although clitellate female germ-line cysts have a strong morphological plasticity, e.g., cysts may contain from 16 to as many as 2500 cells, the oogenesis always shows a meroistic mode, i.e., the interconnected cells take on different fates; a few (sometimes only one) become oocytes, whereas the rest play the role of supporting (nurse) cells and do not continue oogenesis.This is the first comprehensive summary of the current knowledge on the organization and functioning of female germ-line cysts in clitellate annelids.

Background: Spiralians (e.g., annelids, molluscs, and flatworms) possess two sources of mesoderm. One is from endodermal precursors (endomesoderm), which is considered to be the ancestral source in metazoans. The second is from ectoderm (ectomesoderm) and may represent a novel cell type in the Spiralia. In the mollusc Crepidula fornicata, ectomesoderm is derived from micromere daughters within the A and B cell quadrants. Their progeny lie along the anterolateral edges of the blastopore. There they undergo epithelial-mesenchymal transition (EMT), become rounded and undergo delamination/ingression. Subsequently, they assume the mesenchymal phenotype, and migrate beneath the surface ectoderm to differentiate various cell types, including muscles and pigment cells., Results: We examined expression of several genes whose homologs are known to regulate Type 1 EMT in other metazoans. Most of these genes were expressed within spiralian ectomesoderm during EMT., Conclusions: We propose that spiralian ectomesoderm, which exhibits analogous cellular behaviors to other populations of mesenchymal cells, may be controlled by the same genes that drive EMT in other metazoans. Perhaps these genes comprise a conserved metazoan EMT gene regulatory network (GRN). This study represents the first step in elucidating the GRN controlling the development of a novel spiralian cell type (ectomesoderm). Developmental Dynamics 247:1097-1120, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Coral reefs harbor diverse assemblages of organisms yet the majority of this diversity is hidden within the three dimensional structure of the reef and neglected using standard visual surveys. This study uses Autonomous Reef Monitoring Structures (ARMS) and amplicon sequencing methodologies, targeting mitochondrial cytochrome oxidase I and 18S rRNA genes, to investigate changes in the cryptic reef biodiversity. ARMS, deployed at 11 sites across a near- to off-shore gradient in the Red Sea were dominated by Porifera (sessile fraction), Arthropoda and Annelida (mobile fractions). The two primer sets detected different taxa lists, but patterns in community composition and structure were similar. While the microhabitat of the ARMS deployment affected the community structure, a clear cross-shelf gradient was observed for all fractions investigated. The partitioning of beta-diversity revealed that replacement (i.e. the substitution of species) made the highest contribution with richness playing a smaller role. Hence, different reef habitats across the shelf are relevant to regional diversity, as they harbor different communities, a result with clear implications for the design of Marine Protected Areas. ARMS can be vital tools to assess biodiversity patterns in the generally neglected but species-rich cryptic benthos, providing invaluable information for the management and conservation of hard-bottomed habitats over local and global scales.

Background: Over one third of all animal phyla utilize a mode of early embryogenesis called 'spiral cleavage' to divide the fertilized egg into embryonic cells with different cell fates. This mode is characterized by a series of invariant, stereotypic, asymmetric cell divisions (ACDs) that generates cells of different size and defined position within the early embryo. Astonishingly, very little is known about the underlying molecular machinery to orchestrate these ACDs in spiral-cleaving embryos. Here we identify, for the first time, cohorts of factors that may contribute to early embryonic ACDs in a spiralian embryo., Results: To do so we analyzed stage-specific transcriptome data in eggs and early embryos of the spiralian annelid Platynereis dumerilii for the expression of over 50 candidate genes that are involved in (1) establishing cortical domains such as the partitioning defective (par) genes, (2) directing spindle orientation, (3) conveying polarity cues including crumbs and scribble, and (4) maintaining cell-cell adhesion between embryonic cells. In general, each of these cohorts of genes are co-expressed exhibiting high levels of transcripts in the oocyte and fertilized single-celled embryo, with progressively lower levels at later stages. Interestingly, a small number of key factors within each ACD module show different expression profiles with increased early zygotic expression suggesting distinct regulatory functions. In addition, our analysis discovered several highly co-expressed genes that have been associated with specialized neural cell-cell recognition functions in the nervous system. The high maternal contribution of these 'neural' adhesion complexes indicates novel general adhesion functions during early embryogenesis., Conclusions: Spiralian embryos are champions of ACD generating embryonic cells of different size with astonishing accuracy. Our results suggest that the molecular machinery for ACD is already stored as maternal transcripts in the oocyte. Thus, the spiralian egg can be viewed as a totipotent yet highly specialized cell that evolved to execute fast and precise ACDs during spiral cleaving stages. Our survey identifies cohorts of factors in P. dumerilii that are candidates for these molecular mechanisms and their regulation, and sets the stage for a functional dissection of ACD in a spiral-cleaving embryo.

Evolutionary transitions between feeding and nonfeeding larval development have occurred many times in marine invertebrates, but the developmental changes underlying these frequent and ecologically important transitions are poorly known, especially in spiralians. We use phylogenetic comparative methods to test the hypothesis that evolutionary changes in egg size and larval nutritional mode are associated with parallel changes in allocation of cytoplasm to macromere cell lineages in diverse annelids and molluscs. Our analyses show that embryos of species with large eggs and nonfeeding larvae tend to allocate relatively more embryonic cytoplasm to macromeres at 3rd cleavage than do embryos of species with small eggs and feeding larvae. The association between egg size and allocation to macromeres in these spiralians may be driven by constraints associated with mitotic spindle positioning and size, or may be a result of "adaptation in cleavage" to maintain rapid cell cycles in micromeres, position yolk in cell lineages where it can be most efficiently used, or adjust allocation to ectoderm to accommodate changes in embryonic surface area/volume ratio., (© 2016 Wiley Periodicals, Inc.)

Animals that can reproduce by both asexual agametic reproduction and sexual reproduction must transmit or re-establish their germ line post-embryonically. Although such a dual reproductive mode has evolved repeatedly among animals, how asexually produced individuals establish their germ line remains poorly understood in most groups. We investigated germ line development in the annelid Pristina leidyi, a species that typically reproduces asexually by paratomic fission, intercalating a new tail and head in the middle of the body followed by splitting. We found that in fissioning individuals, gonads occur in anterior segments in the anterior-most individual as well as in new heads forming within fission zones. Homologs of the germ line/multipotency genes piwi, vasa, and nanos are expressed in the gonads, as well as in proliferative tissues including the posterior growth zone, fission zone, and regeneration blastema. In fissioning animals, certain cells on the ventral nerve cord express a homolog of piwi, are abundant near fission zones, and sometimes make contact with gonads. Such cells are typically undetectable near the blastema and posterior growth zone. Time-lapse imaging provides direct evidence that cells on the ventral nerve cord migrate preferentially towards fission zones. Our findings indicate that gonads form routinely in fissioning individuals, that a population of piwi-positive cells on the ventral nerve cord is associated with fission and gonads, and that cells resembling these piwi-positive cells migrate along the ventral nerve cord. We suggest that the piwi-positive ventral cells are germ cells that transmit the germ line across asexually produced individuals via migration along the ventral nerve cord., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The ability to regenerate extensive portions of the body is widespread among the phylum Annelida and this group includes some of the most highly regenerative animals known. Knowledge of the cellular and molecular basis of regeneration in this group is thus important for understanding how regenerative processes have evolved both within the group and across animal phyla. Here, the cellular basis of annelid regeneration is reviewed, with a focus on the earliest steps of regeneration, namely wound-healing and formation of the blastema. Information from a wide range of annelids is compiled in order to identify common and variable elements. There is a large body of valuable older literature on the cellular basis of regeneration in annelids and an effort is made to review this literature in addition to more recent studies. Annelids typically seal the wound through muscular contraction and undergo some autolysis of tissue at the site of the wound. Bodily injury elicits extensive cell migration toward the wound, involving several different types of cells. Some migrating cells form a tissue-clot and phagocytize damaged tissues, whereas others are inferred to contribute to regenerated tissue, specifically mesodermal tissue. In one annelid subgroup, the clitellates, a group of mesodermal cells, sometimes referred to as neoblasts, is inferred to migrate over considerable distances, with cells moving to the wound from several segments away. Epidermis and gut epithelia severed upon amputation typically heal by fusing with like tissue, although not always. After amputation, cellular contacts with the extracellular matrix are disrupted and major changes in cell morphology and adhesion occur within tissues near the wound. Interactions of tissues at the wound appear key for initiating a blastema, with a particularly important role suggested for the ventral nerve cord, although species are variable in this regard; longer-distance effects mediated by the brain are also reported. The anterior-posterior polarity of the blastema can be mis-assigned, leading most commonly to double-headed worms, and the dorsal-ventral polarity of the blastema appears to be induced by the ventral nerve cord. The blastema is thought to arise from contributions of all three tissue layers, with each layer replacing itself in a tissue-specific manner. Blastemal cells originate mostly locally, although some long-distance migration of source-cells is suggested in clitellates. A number of important questions remain about the cellular basis of regeneration in annelids and addressing many of these would be greatly aided by developing approaches to identify and isolate specific cell types and techniques to image and trace cells in vivo., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Background: Planktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data., Results: To compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3β antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the 'apical plate'. We also unraveled a molecular signature of the apical organ--devoid of six3 but expressing foxj, irx, nkx3 and hox--that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla., Conclusions: The similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ--comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells--was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae.

Like most bilaterian animals, the annelid Platynereis dumerilii generates the majority of its body axis in an anterior to posterior temporal progression with new segments added sequentially. This process relies on a posterior subterminal proliferative body region, known as the "segment addition zone" (SAZ). We explored some of the molecular and cellular aspects of posterior elongation in Platynereis, in particular to test the hypothesis that the SAZ contains a specific set of stem cells dedicated to posterior elongation. We cloned and characterized the developmental expression patterns of orthologs of 17 genes known to be involved in the formation, behavior, or maintenance of stem cells in other metazoan models. These genes encode RNA-binding proteins (e.g., tudor, musashi, pumilio) or transcription factors (e.g., myc, id, runx) widely conserved in eumetazoans. Most of these genes are expressed both in the migrating primordial germ cells and in overlapping ring-like patterns in the SAZ, similar to some previously analyzed genes (piwi, vasa). The SAZ patterns are coincident with the expression of proliferation markers cyclin B and PCNA. EdU pulse and chase experiments suggest that new segments are produced through many rounds of divisions from small populations of teloblast-like posterior stem cells. The shared molecular signature between primordial germ cells and posterior stem cells in Platynereis thus corresponds to an ancestral "stemness" program., (© 2013 Elsevier Inc. All rights reserved.)

Background: For phylogenetic reconstructions, conflict in signal is a potential problem for tree reconstruction. For instance, molecular data from different cellular components, such as the mitochondrion and nucleus, may be inconsistent with each other. Mammalian studies provide one such case of conflict where mitochondrial data, which display compositional biases, support the Marsupionta hypothesis, but nuclear data confirm the Theria hypothesis. Most observations of compositional biases in tree reconstruction have focused on lineages with different composition than the majority of the lineages under analysis. However in some situations, the position of taxa that lack compositional bias may be influenced rather than the position of taxa that possess compositional bias. This situation is due to apparent symplesiomorphic characters and known as "the symplesiomorphy trap"., Results: Herein, we report an example of the sympleisomorphy trap and how to detect it. Worms within Terebelliformia (sensu Rouse & Pleijel 2001) are mainly tube-dwelling annelids comprising five 'families': Alvinellidae, Ampharetidae, Terebellidae, Trichobranchidae and Pectinariidae. Using mitochondrial genomic data, as well as data from the nuclear 18S, 28S rDNA and elongation factor-1α genes, we revealed incongruence between mitochondrial and nuclear data regarding the placement of Trichobranchidae. Mitochondrial data favored a sister relationship between Terebellidae and Trichobranchidae, but nuclear data placed Trichobranchidae as sister to an Ampharetidae/Alvinellidae clade. Both positions have been proposed based on morphological data., Conclusions: Our investigation revealed that mitochondrial data of Ampharetidae and Alvinellidae exhibited strong compositional biases. However, these biases resulted in a misplacement of Trichobranchidae, rather than Alvinellidae and Ampharetidae. Herein, we document that Trichobranchidae was apparently caught in the symplesiomorphy trap suggesting that in certain situations even homologies can be misleading.

Among spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.

Like most polychaete annelids, Capitella teleta (formerly Capitella sp. I) exhibits a highly stereotypic program of early development known as spiral cleavage. Animals with spiral cleavage have diverse body plans, and homologous embryonic cells can be readily identified among distantly related animals. Spiralian embryos are particularly amenable to studies of fate-mapping, and larval fates of identified cells are conserved among diverse taxa. One cell of particular importance in spiralian development is 2d, or the primary somatoblast, which generates ectoderm of the body posterior to the prototroch. We are interested in the evolution of the primary somatoblast, and thus far, the 2d sublineage has only been analyzed in a few species. In Capitella teleta, 2d generates ectoderm of the segmented trunk and post-segmental pygidium. In this study, development of the 2d lineage was characterized in detail through intracellular injections of DiI, and time-lapse as well as confocal microscopy to analyze cleavage patterns and the fates of larval cells. Analysis of cleavage patterns reveals that the first bilateral division in the 2d sublineage occurs with the division of 2d¹¹², the same 2d daughter cell that first divides bilaterally in the polychaete Platynereis dumerilii. Larval fates of blastomeres 2d¹, 2d², 2d¹¹, 2d¹², 2d¹¹², 2d¹¹²¹, and 2d¹¹²² were determined. All cells show stereotypic descendant clones that are consistent with segregation within sublineages. In the first few divisions of the 2d sublineage, larval-specific structures (neurotroch and telotroch) and pygidial ectoderm are segregated from segmental ectoderm and ventral nerve cord. The daughters of the first bilateral division, 2d¹¹²¹ and 2d¹¹²², generate the right and left halves of the segmental ectoderm and ventral nerve cord respectively, although the clones are consistently asymmetric across the dorsal midline. The pattern of cleavage divisions and the fates of the 2d daughters in Capitella teleta are compared to those in other spiralians with special attention to annelids.

It is proposed here that a biphasic life cycle with partial dedifferentiation of intermediate juvenile or larval stages represents the mainstream developmental mode of metazoans. Developmental plasticity of differentiated cells is considered the essential characteristic of indirect development, rather than the exclusive development of the adult from 'set-aside' cells. Many differentiated larval cells of indirect developers resume proliferation, partially dedifferentiate and contribute to adult tissues. Transcriptional pluripotency of differentiated states has premetazoan origins and seems to be facilitated by histone variant H2A.Z. Developmental plasticity of differentiated states also facilitates the evolution of polyphenism. Uncertainty remains about whether the most recent common ancestor of protostomes and deuterostomes was a direct or an indirect developer, and how the feeding larvae of bilaterians are related to non-feeding larvae of sponges and cnidarians. Feeding ciliated larvae of bilaterians form their primary gut opening by invagination, which seems related to invagination in cnidarians. Formation of the secondary gut opening proceeds by protostomy or deuterostomy, and gene usage suggests serial homology of the mouth and anus. Indirect developers do not use the Hox vector to build their ciliated larvae, but the Hox vector is associated with the construction of the reproductive portion of the animal during feeding-dependent posterior growth. It is further proposed that the original function of the Hox cluster was in gonad formation rather than in anteroposterior diversification.

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.

Dwarf males of the bone-eating worms Osedax (Siboglinidae, Annelida) have been proposed to develop from larvae that settle on females rather than on bone. The apparent arrest in somatic development and resemblance of the males to trochophore larvae has been posited as an example of paedomorphosis. Here, we present the first investigation of the entire muscle and nervous system in dwarf males of Osedax frankpressi, O. roseus, O. rubiplumus, and O. "spiral" analyzed by multistaining and confocal laser scanning microscopy. Sperm shape and spermiogenesis, the sperm duct and internal and external ciliary patterns were likewise visualized. The males of all four species possess morphological traits typical of newly settled siboglinid larvae: a prostomium, a peristomium with a prototroch, one elongate segment and a second shorter segment. Each segment has a ring of eight long-handled hooked chaetae. The longitudinal muscles are distributed as evenly spaced strands forming a grid with the thin outer circular muscles. Oblique protractor and retractor muscles are associated with each of the chaetal sacs. The nervous system comprises a cerebral ganglion, a prototroch nerve ring, paired dorsolateral longitudinal nerves, five ventral longitudinal nerves with paired, posterior ganglia and a terminal commissure, as well as a net of fine peripheral transverse plexuses surrounding the first segment. Internal ciliation occurs as paired ventrolateral bands along the first segment. The bands appear to lead the free mature sperm to a ciliated duct and seminal vesicle lying just behind the prototroch region. A duct then runs from the seminal vesicle into the dorsal part of the prostomium. The similarity of Osedax males to the larvae of Osedax and other siboglinid annelids as well as similarities shown here to the neuromuscular organization seen in other annelid larvae supports the hypothesis of paedomorphosis in males of Osedax.

Ultrastructural characteristics of coelomocytes of the sipunculid Phascolosoma esculenta were studied by transmission electron microscopy. There are several cell types in the coelomic fluid, including three kinds of granulocytes, vesicular cells, germ cells, amoebocytes, phagocytes, and erythrocytes; there are also a new cell complex which is composed of podocytes and granular cells. And several other cell types (erythrocyte and different kinds of granulocytes) gathering together was discovered in the coelomic fluid of P. esculenta. Functional interpretations were provided for these cells using morphological evidence. The coelomocytes from different sipunculid genera and Annelida were compared. The structural diversity of coelomocytes provides both taxonomic characteristics for discriminative identification and phylogenetic markers in Phascolosoma and other sipunculid taxa.

Different approaches to evolutionary interpretation of ontogenies are compared, with special emphasis on the evolutionary role of morphogenetic mechanisms (construction technologies) substantially affecting the structure of definitive forms: they largely determine the structural characteristics of organs, types of anatomical and histological systems, and specificity of symmetry of organisms and their parts. The role of cellular morphogenesis inherited from protozoic ancestors in the morphogenesis of multicellular organisms is demonstrated. Two main ways of improving morphogeneses are considered, based on epithelial morphogenesis and early determined few-celled primordial. On the one hand, the phylogenetic role of archallaxes and deviations is emphasized, these events often switching evolution to a fundamentally new direction. On the other hand, many characteristics of developmental stages are explainable by rationalization of morphogeneses and do not recapitulate ancestral forms, which should be taken into consideration in phylogenetic interpretation of embryogeneses; in particular, this applies to interpretation of axial relationships.

The simplest animal eyes are eyespots composed of two cells only: a photoreceptor and a shading pigment cell. They resemble Darwin's 'proto-eyes', considered to be the first eyes to appear in animal evolution. Eyespots cannot form images but enable the animal to sense the direction of light. They are characteristic for the zooplankton larvae of marine invertebrates and are thought to mediate larval swimming towards the light. Phototaxis of invertebrate larvae contributes to the vertical migration of marine plankton, which is thought to represent the biggest biomass transport on Earth. Yet, despite its ecological and evolutionary importance, the mechanism by which eyespots regulate phototaxis is poorly understood. Here we show how simple eyespots in marine zooplankton mediate phototactic swimming, using the marine annelid Platynereis dumerilii as a model. We find that the selective illumination of one eyespot changes the beating of adjacent cilia by direct cholinergic innervation resulting in locally reduced water flow. Computer simulations of larval swimming show that these local effects are sufficient to direct the helical swimming trajectories towards the light. The computer model also shows that axial rotation of the larval body is essential for phototaxis and that helical swimming increases the precision of navigation. These results provide, to our knowledge, the first mechanistic understanding of phototaxis in a marine zooplankton larva and show how simple eyespots regulate it. We propose that the underlying direct coupling of light sensing and ciliary locomotor control was a principal feature of the proto-eye and an important landmark in the evolution of animal eyes.

Various types of free-floating cells are found in the coelomic fluid of representatives of several annelid groups. The ultrastructure of these "coelomocytes," however, has been studied to a limited degree. In this study, we used a transmission electron microscope to investigate the coelomocytes in specimens of five species of Naidinae and three species of Rhyacodrilinae (all oligochaetous clitellates within the family Tubificidae). These were compared with each other and with previously described coelomocytes of representatives of other oligochaete taxa. Only one distinguishable coelomocyte type was found in the studied specimens: a round to oblong cell without pseudopodia or other appendages, primarily containing membrane-bound granules of varying electron density, a prominent network of rough endoplasmic reticulum, and free ribosomes. This type differs to a great extent from most of the previously described coelomocytes, but shows similarities to certain types found in members of Enchytraeidae and Megascolecidae. Although we noticed some variation, we did not find any ultrastructural characters in these cells obviously useful for phylogenetic studies within Tubificidae.

Recent molecular phylogenetic analyses suggest a close relationship between two worm-shaped phyla, the nonsegmented Sipuncula (peanut worms) and the segmented Annelida (e.g., earthworms and polychaetes) [1-5]. The striking differences in their bodyplans are exemplified by the annelids' paired, ladder-like ventral nervous system, which contains segmentally arranged ganglia, and the sipunculans' single ventral nerve cord (VNC), which is devoid of any segmental structures [6, 7]. Investigating central nervous system (CNS) formation with serotonin and FMRFamide labeling in a representative sipunculan, Phascolosoma agassizii, we found that neurogenesis initially follows a segmental pattern similar to that of annelids. Starting out with paired FMRFamidergic and serotonergic axons, four pairs of associated serotonergic perikarya and interconnecting commissures form one after another in an anterior-posterior progression. In late-stage larvae, the two serotonergic axons of the VNCs fuse, the commissures disappear, and one additional pair of perikarya is formed. These cells (ten in total) migrate toward one another, eventually forming two clusters of five cells each. These neural-remodeling processes result in the single nonmetameric CNS of the adult sipunculan. Our data confirm the segmental ancestry of Sipuncula and render Phascolosoma a textbook example for the Haeckelian hypothesis of ontogenetic recapitulation of the evolutionary history of a species [8].

Onychophora ("velvet worms") are a key taxon in the discussion of arthropod phylogeny. Studies that analyze neuroanatomical characters against a phylogenetic background have recently provided new insights into this debate. However, to date only a few studies on nervous system organization, particularly in the trunk, are available in Onychophora. To close this gap and to compare the onychophoran nervous system with that of other bilaterians, we have analyzed the pattern of serotonin-like immunoreactivity in Metaperipatus blainvillei (Peripatopsidae). In addition to confirming previous histological observations, our experiments revealed many new aspects of nervous system organization in Onychophora. The serotonergic nervous system of M. blainvillei consists of five longitudinal nerve strands (the paired dorsolateral nerves, the heart nerve, and the paired ventral cords), which are interconnected at regular intervals by ring commissures as well as median commissures. The ring commissures are absent in the leg-bearing regions. In addition to the main nerve tracts, there are several extensive fiber networks innervating the integument, the nephridial organs, and the body musculature. The leg nerves and nephridial nerves represent the only strictly segmental neuronal structures. We conclude that the general architecture of the onychophoran nervous system in the trunk closely resembles the orthogonal organization that is present in various other groups of Bilateria, which suggests that the arthropod nervous system is derived from such an orthogonal pattern. This finding implies that the "rope ladder-like" nervous system may have arisen independently in Panarthropoda and Annelida and does not represent a synapomorphy of these groups., ((c) 2008 Wiley-Liss, Inc.)

The determination of gene expression patterns in three dimensions with cellular resolution is an important goal in developmental biology. However the most sensitive, efficient, and widely used staining technique for whole-mount in situ hybridization (WMISH), nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase, could not yet be combined with the most precise, high-resolution detection technique, confocal laser-scanning microscopy (CLSM). Here we report the efficient visualization of the NBT/BCIP precipitate using confocal reflection microscopy for WMISH samples of Drosophila, zebrafish, and the marine annelid worm, Platynereis dumerilii. In our simple WMISH protocol for reflection CLSM, NBT/BCIP staining can be combined with fluorescent WMISH, immunostainings, or transgenic green fluorescent protein (GFP) marker lines, allowing double labeling of cell types or of embryological structures of interest. Whole-mount reflection CLSM will thus greatly facilitate large-scale cellular resolution expression profiling in vertebrate and invertebrate model organisms.

Transmission of obligate bacterial symbionts between generations is vital for the survival of the host. Although the larvae of certain hydrothermal vent tubeworms (Vestimentifera, Siboglinidae) are symbiont-free and possess a transient digestive system, these structures are lost during development, resulting in adult animals that are nutritionally dependent on their bacterial symbionts. Thus, each generation of tubeworms must be newly colonized with its specific symbiont. Here we present a model for tubeworm symbiont acquisition and the development of the symbiont-housing organ, the trophosome. Our data indicate that the bacterial symbionts colonize the developing tube of the settled larvae and enter the host through the skin, a process that continues through the early juvenile stages during which the trophosome is established from mesodermal tissue. In later juvenile stages we observed massive apoptosis of host epidermis, muscles and undifferentiated mesodermal tissue, which was coincident with the cessation of the colonization process. Characterizing the symbiont transmission process in this finely tuned mutualistic symbiosis provides another model of symbiont acquisition and additional insights into underlying mechanisms common to both pathogenic infections and beneficial host-symbiont interactions.

Many animals generate new body segments sequentially from a posterior growth zone, and this is generally thought to be the case for the annelids. Most annelids, including polychaetes, have an indirect life cycle and generate their earliest segments during larval life. We have characterized the nature of the growth zone in two polychaetes, Hydroides elegans and Capitella sp. I, during both larval and juvenile stages of segment formation by examining cell division patterns with 5-bromo-2'-deoxyuridine incorporation. Cell division patterns show commonalities between the two species, even though they have distinct body plans and life history characteristics. In both polychaetes, larval segments arise from a field of dividing cells located in lateral regions of the body, rather than from a localized posterior growth zone. Circumferential expansion of the forming segmental tissue is particularly pronounced in Capitella sp. I. Post-metamorphic segments, in contrast, originate from a classical posterior growth zone, with the exception of four posterior thoracic segments of H. elegans, which appear to arise from an area in the middle of the body, indicating plasticity of segment-generating mechanisms present in different annelid life histories. The distinct nature of larval versus juvenile growth zones in H. elegans and Capitella sp. I raises the question of the mechanistic relationship between these two growth zones. The results of this study increase our understanding of the cellular origins of segments in annelids, and serve as a basis for interpretation of molecular expression patterns associated with segment formation in polychaetes.

Functional ultrastructural changes in the rhabdomeric photoreceptors of the cerebral ocelli are described for normal and sexually mature (epitoke) Perinereis brevicirris (Polychaeta, Annelida). With sexual maturation, the cerebral ocelli hypertrophied, increasing in volume to 5.5 times that of ocelli in the normal state, and the thickness of the retinal layer increased up to 10 times. Perinereis ocelli have a pigmented retinal layer consisting of at least two cell types: photoreceptor cell (PR) and pigmented supporting cells (PS). In epitoke ocelli, PR bear well-developed rhabdomeric microvilli, multilamellar bodies, and numerous cytoplasmic membranous structures, including vesicles, smooth endoplasmic reticulum, and secondary lysosomes. Localization of a visual Gq protein in the ocelli was studied with anti-GqC antibody. The antibody strongly labeled not only microvilli and multilamellar bodies throughout the retinal layer, but also secondary lysosomes and vesicles in the cytoplasm of the PR in the epitoke ocelli, although labeling was observed only in the microvilli and multilamellar bodies in normal ocelli. Reverse transcription/polymerase chain reaction analysis revealed that the amount of G protein alpha subunit mRNA in the epitoke head increased by roughly twice that of the normal head. Since Gq protein is essential for phototransduction in Perinereis ocelli, these results suggest that the sites are involved in photoreceptive membrane turnover, which occurs much more extensively in epitoke ocelli. Thus, epitoke ocelli may represent a model system for studying rhabdomeric photoreceptive membrane turnover.

The eggs of Urechis unicinctus Von Drasche, an echiuroid, are arrested at P-I stage in meiosis. The meiosis is reinitiated by fertilization. Immunoblotting analysis using anti-ERK2 and anti-phospho-MAPK antibodies revealed a 44 kDa MAP kinase species that was constantly expressed in U. unicinctus eggs, quickly phosphorylated after fertilization, and dephosphorylated slowly before the completion of meiosis I. Phosphorylation of the protein was not depressed by protein synthesis inhibitor Cycloheximide (CHX), but was depressed by the MEK1 inhibitor PD98059. Under PD98059 treatment, polar body extrusion was suppressed and the function of centrosome and spindle was abnormal though GVBD was not affected, indicating that MAP kinase cascade was important for meiotic division of U. unicinctus eggs. Other discovery includes: A23187 and OA could parthenogenetically activate U. unicinctus eggs and phosphorylated 44 kDa MAP kinase species, indicating that the effect of fertilization on reinitiating meiosis and phosphorylation of 44 kDa MAP kinase specie is mediated by raising intracellular free calcium and by phosphorylation of some proteins, and that phosphotase(s) sensitive to OA is responsible for arresting U. unicinctus eggs in prophase I. diC8, an activator of PKC, accelerated the process of U. unicinctus egg meiotic division after fertilization and accelerated the dephosphorylation of 44 kDa MAP kinase specie, which implied that the acceleration effect of PKC on meiotic division was mediated by inactivation of MAP kinase cascade. Elevating cAMP/PKA level in U. unicinctus eggs had no effect on meiotic division of the eggs., (Copyright (c) 2005 Wiley-Liss, Inc.)

Microscope techniques, light microscope (LM), transmission electron microscope (TEM), scanning electron microscope (SEM) were employed to describe and classify coelomocytes of the oligochaete Dendrobaena veneta. Three main cell types were distinguished in the coelomic fluid: eleocytes, amoebocytes and granulocytes. Eleocytes are large, oval cells containing characteristic granules called chloragosomes. Amoebocytes are most numerous coelomocytes and have been divided into two types (I and II). Both amoebocytes of the types I and II often form aggregations of a few to about a dozen cells. Granulocytes are oval cells with spherical nuclei and cytoplasm containing polymorphic, electron dense granules. Contrary to the amoebocytes, the granulocytes do not form aggregations. Morphology and ultrastructure of coelomocytes are presented on micrographs: similarities and differences are compared to coelomocytes of related species.

The Ets family includes numerous proteins with a highly conserved DNA-binding domain of 85 amino acids named the ETS domain. Phylogenetic analyses from ETS domains revealed that this family could be divided into 13 groups, among them are ETS and ERG. The ets genes are present in the Metazoan kingdom and we have previously characterized the Nd ets and Nd erg genes in the polychaete annelid Hediste diversicolor. Here, we isolated a fragment encoding the ETS domain from Nd Ets, by genomic library screening. By Northern blot analysis, we showed that this gene was transcribed as one major mRNA of 2.6 kb and one minor mRNA of 3.2 kb. By in situ hybridization, we observed that Nd ets was expressed in the intestine and oocytes and that Nd erg was expressed in cellular clumps present in the coelomic cavity, in an area of proliferating cells situated between the last metamere and the pygidium. Finally, we showed that Nd erg shared the expression pattern of Nd ets in oocytes. Molecular modeling studies have revealed that the spatial structure of ETS domain of Nd Ets and Nd Erg was conserved, in comparison to the murine Ets-1 and human Fli-1 proteins, respectively., (Copyright 2002 Elsevier Science Inc.)

Ectodermal segmentation in the oligochaete annelid Tubifex is a process of separation of 50-microm-wide blocks of cells from the initially continuous ectodermal germ band (GB), a cell sheet consisting of four bandlets of blast cells derived from ectoteloblasts (N, O, P and Q). In this study, using intracellular lineage tracers, we characterized the morphogenetic processes that give rise to formation of these ectodermal segments. The formation of ectodermal segments began with formation of fissures, first on the ventral side and then on the dorsal side of the GB; the unification of these fissures gave rise to separation of a 50-microm-wide block of approximately 30 cells from the ectodermal GB. A set of experiments in which individual ectoteloblasts were labeled showed that as development proceeded, an initially linear array of blast cells in each ectodermal bandlet gradually changed its shape and that its contour became indented in a lineage-specific manner. These morphogenetic changes resulted in the formation of distinct cell clumps, which were separated from the bandlet to serve as segmental elements (SEs). SEs in the N and Q lineages were each comprised of clones of two consecutive primary blast cells. In contrast, in the O and P lineages, individual blast cell clones were distributed across SE boundaries; each SE was a mixture of a part of a more anterior clone and a part of the next more posterior clone. Morphogenetic events, including segmentation, in an ectodermal bandlet proceeded normally in the absence of neighboring ectodermal bandlets. Without the underlying mesoderm, separated SEs failed to space themselves at regular intervals along the anteroposterior axis. We suggest that ectodermal segmentation in Tubifex consists of two stages, autonomous morphogenesis of each bandlet leading to generation of SEs and the ensuing mesoderm-dependent alignment of separated SEs.

Annelids are strongly segmented animals that display a high degree of metamerism in their body plan. The embryonic origin of metameric segmentation was examined in an oligochaete annelid Tubifex using lineage tracers. Segmental organization arises sequentially in the anterior-to-posterior direction along the longitudinal axis of the mesodermal germ band, a coherent column of primary blast cells that are produced from the mesodermal teloblast. Shortly after its birth, each primary blast cell undergoes a spatiotemporally stereotyped sequence of cell divisions to generate three classes of cells (in terms of cell size), which together give rise to a distinct cell cluster. Each cluster is composed of descendants of a single primary blast cell; there is no intermingling of cells between adjacent clusters. Relatively small-sized cells in each cluster become localized at its periphery, and they form coelomic walls including an intersegmental septum to establish individuality of segments. A set of cell ablation experiments showed that these features of mesodermal segmentation are not affected by the absence of the overlying ectodermal germ band. These results suggest that each primary blast cell serves as a founder cell of each mesodermal segment and that the boundary between segments is determined autonomously. It is concluded that the metameric body plan of Tubifex arises from an initially simple organization (i.e., a linear series) of segmental founder cells.

Coelomocytes of Nereis diversicolor synthesize and secrete vitellogenin in vitro. Using a monoclonal antibody which specifically recognized vitellogenin, we showed by immunocytochemistry that among the coelomocytes only a subpopulation, called eleocytes, contained vitellogenin. These results assert that eleocytes are the vitellogenin producing cells in nereids.

Annelids are capable of cellular and humoral defence reactions against foreign antigens. The main aim of this study was to characterise the antigen-binding properties of coelomocytes of Eisenia foetida by means of quantitative autoradiography and direct measurement of radioactivity. It was found that the antigen-binding capacity was significantly increased after antigen stimulation. Furthermore, the preincubation of coelomocytes with non-labelled proteins reduced the binding of radiolabelled antigen. The highest level of inhibition was found when the same protein was used for preincubation. These results indicate that antigen-binding properties are to some extent specific.

Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80--120 A, the distance between two particles (centre to centre) is 150--250 A. Additionally striking rows of large particles (long diameter 150--200 A). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100--150 A, centre to centre distance approx. 250 A) whereas the particles of the extracellular face (EF, mean diameter 200-250 A) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.

The endothelia of Tubifex tubifex Müller consist of myoendothelial cells, chloragocytes, or podocytes. The latter seem to occur only as windows on the ventral vessel which has an endothelium of myoendothelial cells elsewhere. The podocytes are large cells, with several processes on the inner side which ramify into several pedicels. These are aligned upon the outside of the basement membrane which lines the inside of the endothelium. The gaps between adjacent pedicels are about 40 nm wide. In capillaries fenestrated endothelia occur with irregular spacings measuring up to 0.4-1 micron. A diaphragm in podocytes or capillary fenestrations do not seem to exist. The basement membrane is the only continuous layer lining the blood vessels and capillaries of Tubifex with a rather uniform diameter in the range of 50nm. It is the only permeability barrier between blood and coelomic fluid.

Sheep and goat fast migrating IgG molecules and their F(ab')2 fragments react with L. terrestris and E. foetida coelomic fluid proteins. The reaction was not mediated by antibody binding site but by some other part of the IgG molecule, since affinity purified antibodies of different antigen specificities as well as IgG preparations from normal sheep sera reacted with the same coelomic fluid proteins. The presence of at least two reacting proteins was detected by immunoelectrophoresis and immunoblotting analysis. Molecular weights of these proteins were 47 and 50 kD in L. terrestris and 42 kD in E. foetida. Sheep antibodies and IgG molecules also bind to the Sheep antibodies and IgG molecules also bind to the surface of about 2% of coelomic leukocytes and the immunoblotting analysis showed that the IgG binding proteins in coelomic fluids and in cell lysates were similar.

We investigated the nature of the asymmetric positioning and attachment of Chaetopterus oocyte meiotic spindles to the animal pole cortex by micromanipulation. The manipulated spindle's behavior was analyzed in clarified oocyte fragments using video-enhanced polarized light microscopy. As the spindle was drawn towards the cell interior with a microneedle, the cell surface dimpled inwards adjacent to the outer spindle pole. As the spindle was pulled further inwards, the dimple suddenly receded indicating a rupture of a mechanical link between the cell cortex and outer spindle pole. The spindle paused briefly when released from the microneedle; then it spontaneously migrated back to the original attachment site and reassociated with the cell cortex. Positive birefringent astral fibers were seen running between the outer spindle pole and the cortex during the migration. The velocity of the spindle during its migration tended to increase as it came closer to the cortex. Velocities as high as 1.25 micron/sec. were measured. If removed too far from the attachment site cortex (greater than 35 micron), the spindle remained stationary until pushed closer to the original attachment site. Spindles, inverted by micromanipulation, migrated and reattached to the cortical site by their former inner pole; thus either spindle pole can seek out and migrate to the original attachment site. However, spindle poles pushed against other cortical regions did not attach demonstrating that there is only one unique, localized attachment site for spindle attachment.