Neuromuscular organization of the benthic ctenophore, Vallicula multiformis.

Ctenophora is the earliest metazoan taxon with neurons and muscles. Recent studies have described genetic, physiological, and cellular characteristics of the neural and muscular systems of this phylogenically important lineage. However, despite the ecological diversity of ctenophore niches, including both pelagic and benthic forms, studies have focused predominantly on pelagic species. In the present study, we describe the neural and muscular architectures of the benthic ctenophore, Vallicula multiformis (Order Platyctenida), employing immunohistochemical analysis using antibodies against amidated neuropeptides with the C-terminal sequences VWYa, NPWa, FGLa, or WTGa to compare it to pelagic species. In V. multiformis, which lacks the characteristic comb rows seen in pelagic ctenophores, neural structures that develop beneath the comb were not detected, whereas the subepithelial and tentacle neural networks showed considerable similarity to those of pelagic species. Despite significant differences in morphology and lifestyle, muscle organization in V. multiformis closely resembles that of pelagic species. Detailed analysis of neurons that express these peptides unveiled a neural architecture composed of various neural subtypes. This included widely distributed subepithelial neural networks (SNNs) and neurosecretory cells located primarily in the peripheral region. The consistent distribution patterns of the VWYa-positive SNN and tentacle nerves between V. multiformis and the pelagic species, Bolinopsis mikado, suggest evolutionarily conserved function of these neurons in the Ctenophora. In contrast, NPWa-positive neurons, which extend neurites connecting the apical organ and comb rows in B. mikado, showed a neurite-less neurosecretory cell morphology in this flattened, sessile species. Evaluation of characteristics and variations in neural and muscular architectures shared by benthic and pelagic ctenophore species may yield valuable insights for unraveling the biology of this rapidly evolving yet enigmatic metazoan lineage. These findings also provide important insight into neural control modalities in early metazoan evolution.

Mass spectrometry of short peptides reveals common features of metazoan peptidergic neurons.

The evolutionary origins of neurons remain unknown. Although recent genome data of extant early-branching animals have shown that neural genes existed in the common ancestor of animals, the physiological and genetic properties of neurons in the early evolutionary phase are still unclear. Here, we performed a mass spectrometry-based comprehensive survey of short peptides from early-branching lineages Cnidaria, Porifera and Ctenophora. We identified a number of mature ctenophore neuropeptides that are expressed in neurons associated with sensory, muscular and digestive systems. The ctenophore peptides are stored in vesicles in cell bodies and neurites, suggesting volume transmission similar to that of cnidarian and bilaterian peptidergic systems. A comparison of genetic characteristics revealed that the peptide-expressing cells of Cnidaria and Ctenophora express the vast majority of genes that have pivotal roles in maturation, secretion and degradation of neuropeptides in Bilateria. Functional analysis of neuropeptides and prediction of receptors with machine learning demonstrated peptide regulation of a wide range of target effector cells, including cells of muscular systems. The striking parallels between the peptidergic neuronal properties of Cnidaria and Bilateria and those of Ctenophora, the most basal neuron-bearing animals, suggest a common evolutionary origin of metazoan peptidergic nervous systems.

Live cell imaging of cell movement and transdifferentiation during regeneration of an amputated multicellular body of the social amoeba Dictyostelium discoideum.

The regeneration of lost body parts is a fascinating phenomenon exhibited by some multicellular organisms. In social amoebae, such as Dictyostelium discoideum, the pseudoplasmodium is a temporary migratory multicellular structure with high regeneration ability. It consists of future stalk cells (prestalk cells) at the anterior end and future spore cells (prespore cells) at the posterior end, and if amputated, the remaining cells can rapidly regenerate the lost portion within several hours. Details of this regeneration event have been extensively documented; however, little is known about the behavior of individual cells involved in this process. In this study, we performed live cell imaging of cell behavior during regeneration of the excised anterior prestalk region. We used cells that specifically express GFP in the prestalk cell lineage to examine how the prestalk region is regenerated after this region is excised. The current model of prestalk regeneration suggests that the progenitors of prestalk cells, known as anterior-like cells (ALCs), which are sparsely distributed in the prespore region, are redistributed to form the new prestalk region. However, we found that the regenerated prestalk region was formed mainly by the transdifferentiation of prespore cells surrounding the excised anterior end, with little clustering of pre-existing ALCs. Furthermore, the movement of randomly distributed labeled cells during regeneration revealed that although the posterior end was deformed and rounded in shape, the relative position of cells along the anterior-posterior axis remained largely unchanged. These results suggest that the original anterior-posterior axis is maintained in posterior bodies and that prespore cells at the anterior side transdifferentiate and regenerate the prestalk region.

Chimeric Mice with Deletion of Cfl2 that Encodes Muscle-Type Cofilin (MCF or Cofilin-2) Results in Defects of Striated Muscles, Both Skeletal and Cardiac Muscles.

Cofilin, a member of the ADF/cofilin family, is an actin-binding protein which is widely distributed among eukaryotic organisms and involved in actin filament dynamics in a variety of cell types. In mammalian striated muscles, muscle-type cofilin (MCF or cofilin-2) is predominantly expressed. Previous investigations have shown that MCF plays an essential role in the regulation of assembly of contractile apparatus in skeletal muscle, but its role in cardiac muscle has remained unclear. In the present study, in order to further clarify the role of MCF in organization of myofibrillar structure in vivo, we generated chimeric mice with a combination of MCF-deficient cells that were generated by Cfl2-knockout (Cfl2-/-) and wild type cells containing MCF, and examined the effect of MCF deficiency on striated muscles, especially on the fine structures of contractile apparatus in cardiac muscle. We found that mice chimeric for MCF deficient cells exhibited structural defects in their skeletal muscles as previously reported. Histological analysis showed that MCF deficiency leads to degradation of myofibers and promotion of muscle regeneration. Electron microscopic observation of cardiac muscle of the chimeric mice showed coexistence of the cells with normal sarcomeres and those with disorganized myofibrils in a chimeric pattern. In these cofilin-deficient cells, myofilaments were scattered in the cytoplasm and myofibrillar structures were severely disrupted. These results provide strong evidence for that MCF plays a critical role in the formation and the maintenance of myofibril structure not only in skeletal muscle but also in cardiac muscle.

Dynamic Transport and Cementation of Skeletal Elements Build Up the Pole-and-Beam Structured Skeleton of Sponges.

Animal bodies are shaped by skeletons, which are built inside the body by biomineralization of condensed mesenchymal cells in vertebrates [1, 2] and echinoderms [3, 4], or outside the body by apical secretion of extracellular matrices by epidermal cell layers in arthropods [5]. In each case, the skeletons' shapes are a direct reflection of the pattern of skeleton-producing cells [6]. Here we report a newly discovered mode of skeleton formation: assembly of sponges' mineralized skeletal elements (spicules) in locations distant from where they were produced. Although it was known that internal skeletons of sponges consist of spicules assembled into large pole-and-beam structures with a variety of morphologies [7-10], the spicule assembly process (i.e., how spicules become held up and connected basically in staggered tandem) and what types of cells act in this process remained unexplored. Here we found that mature spicules are dynamically transported from where they were produced and then pierce through outer epithelia, and their basal ends become fixed to substrate or connected with such fixed spicules. Newly discovered "transport cells" mediate spicule movement and the "pierce" step, and collagen-secreting basal-epithelial cells fix spicules to the substratum, suggesting that the processes of spiculous skeleton construction are mediated separately by specialized cells. Division of labor by manufacturer, transporter, and cementer cells, and iteration of the sequential mechanical reactions of "transport," "pierce," "raise up," and "cementation," allows construction of the spiculous skeleton spicule by spicule as a self-organized biological structure, with the great plasticity in size and shape required for indeterminate growth, and generating the great morphological diversity of individual sponges.

Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation.

BACKGROUND: Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation. RESULTS: We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum. CONCLUSIONS: The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns.

Defects in the synthetic pathway prevent DIF-1 mediated stalk lineage specification cascade in the non-differentiating social amoeba, Acytostelium subglobosum.

Separation of somatic cells from germ-line cells is a crucial event for multicellular organisms, but how this step was achieved during evolution remains elusive. In Dictyostelium discoideum and many other dictyostelid species, solitary amoebae gather and form a multicellular fruiting body in which germ-line spores and somatic stalk cells differentiate, whereas in Acytostelium subglobosum, acellular stalks form and all aggregated amoebae become spores. In this study, because most D. discoideum genes known to be required for stalk cell differentiation have homologs in A. subglobosum, we inferred functional variations in these genes and examined conservation of the stalk cell specification cascade of D. discoideum mediated by the polyketide differentiation-inducing factor-1 (DIF-1) in A. subglobosum. Through heterologous expression of A. subglobosum orthologs of DIF-1 biosynthesis genes in D. discoideum, we confirmed that two of the three genes were functional equivalents, while DIF-methyltransferase (As-dmtA) involved at the final step of DIF-1 synthesis was not. In fact, DIF-1 activity was undetectable in A. subglobosum lysates and amoebae of this species were not responsive to DIF-1, suggesting a lack of DIF-1 production in this species. On the other hand, the molecular function of an A. subglobosum ortholog of DIF-1 responsive transcription factor was equivalent with that of D. discoideum and inhibition of polyketide synthesis caused developmental arrest in A. subglobosum, which could not be rescued by DIF-1 addition. These results suggest that non-DIF-1 polyketide cascades involving downstream transcription factors are required for fruiting body development of A. subglobosum.

Temporal and non-permanent division of labor during sorocarp formation in the social amoeba Acytostelium subglobosum.

Somatic cell differentiation is crucial for the development of multicellular organisms. While the development of a fruiting body in Dictyostelium discoideum represents a simple model of this process with separation of stalk cells from the spore lineage, that of Acytostelium subglobosum is not accompanied by cell type separation. This species produces acellular stalks and, seemingly, all aggregated amoebae become spores; however, it possesses homologs for the stalk-cell marker genes of D. discoideum. In this study, we analyzed the spatio-temporal expression of A. subglobosum orthologs for D. discoideum stalk- or spore-lineage markers to clarify the developmental process of A. subglobosum. We first found that the prespore vesicles, which contained spore coat proteins, started to accumulate in the tip region and were observed in the entire sorogen throughout later development, confirming that all A. subglobosum cells became spores. The expression of a stalk-lineage gene ortholog, As-ecmA, started at the mound stage and was prominent in the protruding sorogen. Although two spore-lineage gene orthologs, As-cotD1 and -cotD2, were likewise detected shortly after cell aggregation and increased in intensity until tip formation, their expression diminished in the protruding sorogen. Double-fluorescence staining of these prestalk and prespore marker genes revealed that the expression of these marker genes was mutually exclusive and that expression switching occurred in the early tip. Our results indicate that A. subglobosum cells become committed to the spore lineage first, and then, while keeping this commitment intact, participate in stalk formation. Instead of the permanent division of labor observed in D. discoideum, A. subglobosum produces fruiting bodies by all cells contributing to the formation of the stalk as well as forming spores.

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges.

Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA-expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU-incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell-lineage-specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell-lineage-specific markers during archeocyte differentiation into other cell types. Choanocytes are food-entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell-like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.

Toward understanding the morphogenesis of siliceous spicules in freshwater sponge: differential mRNA expression of spicule-type-specific silicatein genes in Ephydatia fluviatilis.

Siliceous spicules of sponges are morphologically diverse and provide good models for understanding the morphogenesis of biomineralized products. The silica deposition enzyme silicatein is a component of siliceous spicules of sponges and is thought to be the key molecule determining the morphology of spicules. Here, we focused on the silicateins of the freshwater sponge Ephydatia fluviatilis, which has two types of morphologically and functionally different spicules, called megascleres and gemmoscleres. We isolated six isoforms of silicateins and examined their mRNA expression in the cells producing megascleres and gemmoscleres. The spicule-type-specific mRNA expression of these isoforms and differential expression during spicule development suggest that the characteristic morphology of spicules is due to the specific properties and combinatory functions of silicatein isoforms.

Caenorhabditis elegans kettin, a large immunoglobulin-like repeat protein, binds to filamentous actin and provides mechanical stability to the contractile apparatuses in body wall muscle.

Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with alpha-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction.

Enhancement of actin-depolymerizing factor/cofilin-dependent actin disassembly by actin-interacting protein 1 is required for organized actin filament assembly in the Caenorhabditis elegans body wall muscle.

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal beta-propeller, whereas one residue is located in the C-terminal beta-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.

The two Caenorhabditis elegans actin-depolymerizing factor/cofilin proteins differently enhance actin filament severing and depolymerization.

Actin-depolymerizing factor (ADF)/cofilin enhances the turnover of actin filaments by two separable activities: filament severing and pointed-end depolymerization. Multicellular organisms express multiple ADF/cofilin isoforms in a tissue-specific manner, and the vertebrate proteins are grouped into ADFs and cofilins on the basis of their biochemical activity. A recent comparative study has shown that ADF has greater severing and depolymerizing activities than cofilin [Chen, H., Bernstein, B. W., Sneider, J. M., Boyle, J. A., Minamide, L. S., and Bamburg, J. R. (2004) Biochemistry 43, 7127-7142]. Here, we show that the two Caenorhabditis elegans ADF/cofilin isoforms exhibit different activities for severing and depolymerizing actin filaments. The ADF-like non-muscle isoform UNC-60A had greater activities to cause net depolymerization and inhibit polymerization than the cofilin-like muscle isoform UNC-60B. Surprisingly, UNC-60B exhibited much stronger severing activity than UNC-60A, which was the opposite of what was observed for vertebrate counterparts. Moreover, UNC-60B induced much faster pointed-end depolymerization of rabbit muscle actin than UNC-60A, while UNC-60A caused slightly faster depolymerization of C. elegans actin than UNC-60B. These results suggest that cofilin-like UNC-60B is kinetically more efficient in enhancing actin turnover than ADF-like UNC-60A, while ADF-like UNC-60A is suitable for maintaining higher concentrations of monomeric actin. These functional differences might be specifically adapted for different actin dynamics in muscle and non-muscle cells.

Molecular and biochemical characterization of kettin in Caenorhabditis elegans.

Kettin is a unique member of the connectin/titin family of muscle elastic proteins, which has repetitive immunoglobulin-like domains that are separated by weakly conserved linker sequences. In striated muscles of insects and crayfish, kettin binds to actin filaments and localizes to the Z-disc and its adjacent region in the I-band. Recent sequence analysis of invertebrate connectin/titin (also known as SLS proteins) has revealed that kettin is a splice variant of connectin/titin. In contrast, in the nematode Caenorhabditis elegans, the kettin gene is independent of the genes for other connectin/titin-related proteins. Immunofluorescent localization of kettin shows that it localizes to the I-bands in the obliquely striated body wall muscle. Therefore, C. elegans is an attractive model system to study specific functions of kettin in muscle cells.

Identification of functional residues on Caenorhabditis elegans actin-interacting protein 1 (UNC-78) for disassembly of actin depolymerizing factor/cofilin-bound actin filaments.

Actin-interacting protein 1 (AIP1) is a WD40 repeat protein that enhances actin filament disassembly in the presence of actin-depolymerizing factor (ADF)/cofilin. AIP1 also caps the barbed end of ADF/cofilin-bound actin filament. However, the mechanism by which AIP1 interacts with ADF/cofilin and actin is not clearly understood. We determined the crystal structure of Caenorhabditis elegans AIP1 (UNC-78), which revealed 14 WD40 modules arranged in two seven-bladed beta-propeller domains. The structure allowed for the mapping of conserved surface residues, and mutagenesis studies identified five residues that affected the ADF/cofilin-dependent actin filament disassembly activity. Mutations of these residues, which reside in blades 3 and 4 in the N-terminal propeller domain, had significant effects on the disassembly activity but did not alter the barbed end capping activity. These data support a model in which this conserved surface of AIP1 plays a direct role in enhancing fragmentation/depolymerization of ADF/cofilin-bound actin filaments but not in barbed end capping.

Microscopic evidence that actin-interacting protein 1 actively disassembles actin-depolymerizing factor/Cofilin-bound actin filaments.

Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. Here, we directly observed the effects of these proteins on filamentous actin by fluorescence microscopy and gained novel insight into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolin-actin complex, or cytochalasin D did not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 was weak and allowed filament elongation, whereas gelsolin or gelsolin-actin complex strongly capped and inhibited elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation.

Actin filament disassembling activity of Caenorhabditis elegans actin-interacting protein 1 (UNC-78) is dependent on filament binding by a specific ADF/cofilin isoform.

Actin-interacting protein 1 (AIP1) is a conserved WD-repeat protein that enhances actin filament disassembly only in the presence of actin depolymerizing factor (ADF)/cofilin. In the nematode Caenorhabditis elegans, an AIP1 ortholog is encoded by the unc-78 gene that is required for organized assembly of muscle actin filaments. We produced bacterially expressed UNC-78 protein and found that it enhances actin filament disassembly preferentially in the presence of a specific ADF/cofilin isoform. Extensive and rapid filament disassembly by UNC-78 was observed in the presence of UNC-60B, a muscle-specific C. elegans ADF/cofilin isoform. UNC-78 also reduced the rate of spontaneous polymerization and enhanced subunit dissociation from filaments in the presence of UNC-60B. However, in the presence of UNC-60A, a non-muscle C. elegans ADF/cofilin isoform, UNC-78 only slightly enhanced filament disassembly. Interestingly, UNC-78 failed to enhance disassembly by mouse muscle-type cofilin. Using mutant forms of UNC-60B, we demonstrated that the F-actin-specific binding site of UNC-60B at the C terminus is required for filament disassembly by UNC-78. UNC-78 was expressed in body wall muscle and co-localized with actin where UNC-60B was also present. Surprisingly, UNC-78 was co-localized with actin in unc-60B null mutants, suggesting that the AIP1-actin interaction is not dependent on ADF/cofilin in muscle. These results suggest that UNC-78 closely collaborates with UNC-60B to regulate actin dynamics in muscle cells.