The people behind the papers - Masanori Abe and Reiko Kuroda.

One of the most obvious examples of left-right asymmetry in animal bodies comes from snails: in most species or strains, the shells coil dextrally, but some coil sinistrally. The control of coiling is genetic and begins in the early embryo. Previous work has implicated the formin diaphanous in the regulation of snail shell chirality, and a new paper in Development now decisively proves its involvement, thanks to the first application of CRISPR/Cas9 gene knockouts in molluscs. We caught up with the author team behind the paper: Masanori Abe and his supervisor Reiko Kuroda, Professor at Chubu University in Japan (recently moved from Tokyo University of Science), to find out more.

The evo-devo of molluscs: Insights from a genomic perspective.

Molluscs represent one of ancient and evolutionarily most successful groups of marine invertebrates, with a tremendous diversity of morphology, behavior, and lifestyle. Molluscs are excellent subjects for evo-devo studies; however, understanding of the evo-devo of molluscs has been largely hampered by incomplete fossil records and limited molecular data. Recent advancement of genomics and other technologies has greatly fueled the molluscan "evo-devo" field, and decoding of several molluscan genomes provides unprecedented insights into molluscan biology and evolution. Here, we review the recent progress of molluscan genome sequencing as well as novel insights gained from their genomes, by emphasizing how molluscan genomics enhances our understanding of the evo-devo of molluscs.

A set of simple cell processes is sufficient to model spiral cleavage.

During cleavage, different cellular processes cause the zygote to become partitioned into a set of cells with a specific spatial arrangement. These processes include the orientation of cell division according to: an animal-vegetal gradient; the main axis (Hertwig's rule) of the cell; and the contact areas between cells or the perpendicularity between consecutive cell divisions (Sachs' rule). Cell adhesion and cortical rotation have also been proposed to be involved in spiral cleavage. We use a computational model of cell and tissue biomechanics to account for the different existing hypotheses about how the specific spatial arrangement of cells in spiral cleavage arises during development. Cell polarization by an animal-vegetal gradient, a bias to perpendicularity between consecutive cell divisions (Sachs' rule), cortical rotation and cell adhesion, when combined, reproduce the spiral cleavage, whereas other combinations of processes cannot. Specifically, cortical rotation is necessary at the 8-cell stage to direct all micromeres in the same direction. By varying the relative strength of these processes, we reproduce the spatial arrangement of cells in the blastulae of seven different invertebrate species.

Expression of six3 and otx in Solenogastres (Mollusca) supports an ancestral role in bilaterian anterior-posterior axis patterning.

The homeodomain transcription factors six3 and otx are involved in patterning the anterior body and parts of the central nervous system (CNS) in bilaterians. Their similar expression patterns have been used as an argument for homology of heads, brains, segmentation, and ciliated larvae. We investigated the developmental expression of six3 and otx in the aplacophoran mollusk Wirenia argentea. Six3 is expressed in subepithelial cells delimiting the apical organ of the solenogaster pericalymma larva. Otx is expressed in cells of the prototroch and adjacent regions as well as in posterior extensions of the prototrochal expression domain. Advanced larvae also show pretrochal otx expression in the developing CNS. Comparative analysis of six3 and otx expression in bilaterians argues for an ancestral function in anterior-posterior body axis patterning but, due to its presence in animals lacking a head and/or a brain, not necessarily for the presence of these morphological structures in the last common ancestor (LCA) of bilaterians. Likewise, the hypothesis that the posterior border of otx expression corresponds to the border between the unsegmented head and the segmented trunk of the LCA of protostomes is not supported, since otx is extensively expressed in the trunk in W. argentea and numerous other protostomes.

[Early Peripheral Sensory Neurons in the Development of Trochozoan Animals].

Neuronal development of the majority of trochozoan animals with biphasic pelago-bentic life cycle starts from transient peripheral neurons, which do not belong to the central nervous system and are mainly located in the apical sensory organ and in the hyposphere. Some of these neurons are pioneer and send neurites that form a scaffold upon which the adult central nervous system later develops. In representative species of molluscs and polychaetes, immunolabelling with the antibodies against neurotransmitters serotonin and FMRFamide, and acetylated α-tubulin revealed that the structure of almost all early peripheral neurons is typical for sensory, most probably chemosensory cells: flask shape, and cilia at the end of the apical dendrite or inside the distal ampoule. Morphology, transmitter specificity, location and projections of the early sensory cells differ in trochophores of different species thus suggesting different origin of these cells. In polychaete larvae, pharmacological inhibition of serotonin synthesis in early peripheral neurons did not affect the development, whereas its increase resulted in developmental arrest and neural malformations, suggesting that early peripheral sensory neurons are involved in developmental regulation.

Comparative transcriptomics enlarges the toolkit of known developmental genes in mollusks.

BACKGROUND: Mollusks display a striking morphological disparity, including, among others, worm-like animals (the aplacophorans), snails and slugs, bivalves, and cephalopods. This phenotypic diversity renders them ideal for studies into animal evolution. Despite being one of the most species-rich phyla, molecular and in silico studies concerning specific key developmental gene families are still scarce, thus hampering deeper insights into the molecular machinery that governs the development and evolution of the various molluscan class-level taxa. RESULTS: Next-generation sequencing was used to retrieve transcriptomes of representatives of seven out of the eight recent class-level taxa of mollusks. Similarity searches, phylogenetic inferences, and a detailed manual curation were used to identify and confirm the orthology of numerous molluscan Hox and ParaHox genes, which resulted in a comprehensive catalog that highlights the evolution of these genes in Mollusca and other metazoans. The identification of a specific molluscan motif in the Hox paralog group 5 and a lophotrochozoan ParaHox motif in the Gsx gene is described. Functional analyses using KEGG and GO tools enabled a detailed description of key developmental genes expressed in important pathways such as Hedgehog, Wnt, and Notch during development of the respective species. The KEGG analysis revealed Wnt8, Wnt11, and Wnt16 as Wnt genes hitherto not reported for mollusks, thereby enlarging the known Wnt complement of the phylum. In addition, novel Hedgehog (Hh)-related genes were identified in the gastropod Lottia cf. kogamogai, demonstrating a more complex gene content in this species than in other mollusks. CONCLUSIONS: The use of de novo transcriptome assembly and well-designed in silico protocols proved to be a robust approach for surveying and mining large sequence data in a wide range of non-model mollusks. The data presented herein constitute only a small fraction of the information retrieved from the analysed molluscan transcriptomes, which can be promptly employed in the identification of novel genes and gene families, phylogenetic inferences, and other studies using molecular tools. As such, our study provides an important framework for understanding some of the underlying molecular mechanisms involved in molluscan body plan diversification and hints towards functions of key developmental genes in molluscan morphogenesis.

Allocation of cytoplasm to macromeres in embryos of annelids and molluscs is positively correlated with egg size.

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.

[Functional role for MAP kinase signaling in cell lineage and dorso-ventral axis specification in the basal gastropod Testudinalia testudinalis (Patellogastropoda, Molluska)].

In Spriralia, the specification of cell lines in the course of development is provided by maternal factors. However, recent studies demonstrated the importance of inductive processes whose significant element is cellular signaling. Our data allow us to speak of the dependent specification of a number of cell lines at the early stages of development of the mollusk Testudinalia testudinalis (Testudinalia tessellate, Patellogastropoda), including the period when the determination of the 3D cell takes place, which is accompanied by a change in the shape and establishing of contacts with animal micromeres by one of the macromeres of the third quartet. It is exactly at this moment that activation of MARK was registered in the 3D blastomere-organizer. An analysis of the influence of the U0126 blocker of the MAP-kinase way on the development of Testudinalia showed that the greatest effect of the inhibitor is observed during the cultivation of embryos until the sixth cycle of fragmentation. It should be noted that a scale of correlation of the degree of defects and increase in concentration exists. Absence of the functioning retractor, disorganization of the muscle system, and abnormal structure of the shell (to the extent of complete absence of the shell), as well as velum, foot, and mantle fold, were observed in a considerable part of larvae after a lengthy upkeep of the objects in the U0126 solution. At the same time, none of the experiments showed a complete disruption of the specification of the dorsoventral axis, which produces a larva with a four-ray radial symmetry. This attests in favor of the existence of various molecular mechanisms of determination of the secondary body axis among the animals from the group Spiralia.

Developmental expression of a molluscan RXR and evidence for its novel, nongenomic role in growth cone guidance.

It is well known that the vitamin A metabolite, retinoic acid, plays an important role in vertebrate development and regeneration. We have previously shown that the effects of RA in mediating neurite outgrowth, are conserved between vertebrates and invertebrates (Dmetrichuk et al., 2005, 2006) and that RA can induce growth cone turning in regenerating molluscan neurons (Farrar et al., 2009). In this study, we have cloned a retinoid receptor from the mollusc Lymnaea stagnalis (LymRXR) that shares about 80% amino acid identity with the vertebrate RXRalpha. We demonstrate using Western blot analysis that the LymRXR is present in the developing Lymnaea embryo and that treatment of embryos with the putative RXR ligand, 9-cis RA, or a RXR pan-agonist, PA024, significantly disrupts embryogenesis. We also demonstrate cytoplasmic localization of LymRXR in adult central neurons, with a strong localization in the neuritic (or axonal) domains. Using regenerating cultured motor neurons, we show that LymRXR is also present in the growth cones and that application of a RXR pan-agonist produces growth cone turning in isolated neurites (in the absence of the cell body and nucleus). These data support a role for RXR in growth cone guidance and are the first studies to suggest a nongenomic action for RXR in the nervous system.

Clustered Fox genes in lophotrochozoans and the evolution of the bilaterian Fox gene cluster.

FoxC, FoxF, FoxL1 and FoxQ1 genes have been shown to be clustered in some animal genomes, with mesendodermal expression hypothesised as a selective force maintaining cluster integrity. Hypotheses are, however, constrained by a lack of data from the Lophotrochozoa. Here we characterise members of the FoxC, FoxF, FoxL1 and FoxQ1 families from the annelid Capitella teleta and the molluscs Lottia gigantea and Patella vulgata. We cloned FoxC, FoxF, FoxL1 and FoxQ1 genes from C. teleta, and FoxC, FoxF and FoxL1 genes from P. vulgata, and established their expression during development. We also examined their genomic organisation in C. teleta and L. gigantea, and investigated local syntenic relationships. Our results show mesodermal and anterior gut expression is a common feature of these genes in lophotrochozoans. In L. gigantea FoxC, FoxF and FoxL1 are closely linked, while in C. teleta Ct-foxC and Ct-foxL1 are closely linked, with Ct-foxF and Ct-foxQ1 on different scaffolds. Adjacent to these genes there is limited evidence of local synteny. This demonstrates conservation of genomic organisation and expression of these genes can be traced in all three bilaterian Superphyla. These data are evaluated against competing theories for the long-term maintenance of gene clusters.

A novel role for dpp in the shaping of bivalve shells revealed in a conserved molluscan developmental program.

During the molluscan evolution leading to the bivalves, the single dorsal shell was doubled. To elucidate the molecular developmental basis underlying this prominent morphological transition, we described the cell cleavage and expression patterns of three genes, brachyury, engrailed, and dpp in the Japanese spiny oyster Saccostrea kegaki, and examined the function of dpp in this species. The cleavage pattern of the S. kegaki embryo was nearly the same as the previously described pattern of other bivalve species, suggesting that the pattern itself is highly important for the establishment or the maintenance of the bivalve body plan. The expression pattern of a brachyury homolog in S. kegaki (SkBra) was similar to the pattern in gastopods even at the single cell level despite the deep divergence of gastropods and bivalves. Engrailed and dpp were previously found to be expressed around the shell anlagen in gastropods. Like that of gastropods, an engrailed homolog in S. kegaki (SkEn) was found to be expressed around the shell anlagen. However, the dpp homologin S. kegaki (SkDpp) was expressed only in the cells along the dorsal midline. ZfBMP4 treatment experiments revealed the importance of dpp in establishing the characteristic shape of the bivalve shell anlagen.

Cephalopod Hox genes and the origin of morphological novelties.

Cephalopods are a diverse group of highly derived molluscs, including nautiluses, squids, octopuses and cuttlefish. Evolution of the cephalopod body plan from a monoplacophoran-like ancestor entailed the origin of several key morphological innovations contributing to their impressive evolutionary success. Recruitment of regulatory genes, or even pre-existing regulatory networks, may be a common genetic mechanism for generating new structures. Hox genes encode a family of transcriptional regulatory proteins with a highly conserved role in axial patterning in bilaterians; however, examples highlighting the importance of Hox gene recruitment for new developmental functions are also known. Here we examined developmental expression patterns for eight out of nine Hox genes in the Hawaiian bobtail squid Euprymna scolopes, by whole-mount in situ hybridization. Our data show that Hox orthologues have been recruited multiple times and in many ways in the origin of new cephalopod structures. The manner in which these genes have been co-opted during cephalopod evolution provides insight to the nature of the molecular mechanisms driving morphological change in the Lophotrochozoa, a clade exhibiting the greatest diversity of body plans in the Metazoa.

[The golden age of comparative morphology: laser scanning microscopy and neurogenesis of trochophore animals].

Immunochemical labeling of neuronal elements and laser confocal microscopy have considerably expanded the capacity of comparative morphology and allowed us to monitor the neurogenesis of various trochophore animals at the level of individual identified neurons and their projections. It has been demonstrated that many generally accepted concepts of the larval nervous system and the phylogenetic theories constructed on this basis are incorrect. Comparative analysis has demonstrated that the orthogonal brain is absent at all developmental stages in the representative Lophotrochozoa members. Fundamental differences in the structure and development of the nervous system have been found in the trochophores belonging to different taxonomic groups within Lophotrochozoa; these differences demonstrate that the trochophore larva in these groups are not homologous, while their similarity is most likely a result of convergence. Our results challenge the concept of trochophore as the ancestral form common for all trochophore animals. It is necessary to exclude from phylogenetic discussions the orthogon as a basic plan for the structure of the nervous system and the trochophore as an ancestral form for all Lophtrochozoa.

The publications of embryologist Lev V. Beloussov.

A list of papers and books of the late Lev V. Beloussov was compiled and is available in Word and EndNote Supplements. The breadth of his work is briefly described.

The dynamic nature of mollusc egg surface architecture and its relation to the microtubule network.

Dynamic changes in the surface architecture pattern of embryos of the slipper limpet (Crepidula fornicata, Mollusca) were found in this study to correlate with the dynamic activity and pattern of the underlying mitotic spindle microtubule network, revealed by fluorescent labelling and confocal imaging techniques. Examination of a series of optical sections indicate that this network appears to be spatially co-ordinated together as a whole throughout the embryo. The microtubule pattern also associated with abnormal multipolar spindles resulting from an applied static magnetic field, indicating that the pattern may be generated by a natural endogenous field source. The patterning characteristics of the surface and microtubule network together provide further morphological evidence for a primary morphogenetic or developmental field system which organises the primary body axis and co-ordinates the pattern of cleavage.

Cell-lineage and clonal-contribution map of the trochophore larva of Patella vulgata (mollusca).

Molluscan development is characterised by its extremely regular cleavage pattern. In numerous molluscs the fate of various early-cleavage stage blastomeres has been determined and fate maps have been constructed. On the basis of similarities between these fate maps, a generalised molluscan cell-lineage map has been constructed. Recently, the validity of this map has been challenged. In this study, the cell-lineage of the first-, second-, and third-quartet micromeres and third-generation macromeres of the equally-cleaving gastropod mollusc Patella vulgata was studied by fluorescent cell-lineage tracer injection followed by epifluorescence microscopy and confocal laser scanning microscopy. For the first time, a complete cell-lineage map, in the form of a clonal-contribution map of the trochophore, has been constructed with the use of fluorescent cell-lineage tracers. This map both agrees and differs in a number of respects with the generalised cell-lineage map of molluscs. The most important deviation is that the micromere 2d, formerly referred to as the first somatoblast, is not the only cell that forms the foot and shell gland in Patella.

Organiser role of the stem cell of the mesoderm in prototroch patterning in Patella vulgata (Mollusca, Gastropoda).

During early development of the gastropod mollusc Patella vulgata, the stem cell of the mesoderm (3D-macromere) is induced. As a result of this induction, the embryo becomes dorsoventrally organised. At about the same time in development, ciliated cells, so-called trochoblasts, are formed. Later in development, some trochoblasts deciliate and, together with the ciliated trochoblast, form the dorsoventrally organised prototroch, the locomotory organ of the larva. In order to study the role of the 3D-macromere in the specification of trochoblasts and in the induction of the dorsoventral organisation of the prototroch, induction of 3D has been prevented in various ways. it is shown that preventing 3D-induction results in the formation of a radially symmetrical prototroch. The trochoblasts of all four quadrants developed like corresponding trochoblasts of the A-quadrant. Somewhere between 30 and 120 min after fifth cleavage the 3D-macromere induces the formation of specific trochoblasts and organises the dorsoventral pattern of the prototroch. Besides a role of the 3D-macromere, a role of other cells has been demonstrated in the conditionally specified deciliation of trochoblasts.

Purification and characterization of a beta-glucuronidase present during embryogenesis of the mollusk Pomacea sp.

A beta-glucuronidase was purified from Pomacea sp. eggs by ammonium sulfate fractionation, DEAE-BioGel and Heparin-Sepharose chromatographies. This enzyme showed a Mr 180 kDa, with subunits of 90 kDa. The kinetic parameters were: pH 4.0, temperature 60 degrees C, Km 2.7 x 10(-6) and Vmax 15.3 microM/h, activator Mg+2, and inhibitor: lactone of D-saccharic acid. beta-glucuronidase is an exoglucuronidase involved in glycosaminoglycans metabolism with kinetics parameters similar to those found in mammals.

The distribution of cells containing FMRFamide- and 5-HT-related molecules in the embryonic development of Viviparus ater (Mollusca, Gastropoda).

The timing and spatial distribution of cells containing FMRFamide- and 5-HT-related molecules in the embryonic development of the mollusc Viviparus ater are examined using immunohistochemistry. FMRFamide-like molecules emerge in the early stage E8 (8% of embryonic development) before the 5-HT immunoreactivity, and they are not only found during nervous system ontogeny. As the parts of the digestive tract differentiated, the pattern of the diffuse gut endocrine cells, present in adults, start to be established (E20-E30), and both open and closed cell types are immunoreactive to anti-FMRFamide antibody. From their appearance (E20), cells with a 5-HT-like phenotype are distributed in the central nervous ganglia and progressively assembled during embryonic development. The early occurrence of both these molecules in V. ater embryos reinforces the growing view that neurotransmitters play a regulatory role in embryogenic processes. In particular, the very early presence of FMRFamide-related factors suggests an involvement of these molecules in the regulation of basic, not only neuronal, cell behaviours, while 5-HT seems to be a more specific neural development signal.

Changes in membrane properties during in-vitro meiotic maturation of the limpet Patella vulgata.

Changes in the membrane properties of the oocyte of the mollusk, Patella vulgata, were analyzed following the induction of meiosis reinitiation by paleopedial ganglia extract or by the weak base ammonia. During maturation it was possible to distinguish between an early phase characterized by an initial hyperpolarization and a late phase consisting of a depolarization which triggers an action potential with a long-term overshoot (20 minutes) of the membrane potential. Major changes in individual ionic permeabilities were studied using both current and voltage clamp conditions. The depolarizing phase appears to depend on decreases in K+ membrane permeability. Finally we observed that the overshoot did not appear to be directly related to germinal vesicle breakdown (GVBD) since it was absent in Na-deprived artificial sea water and could be elicited in the presence of TEA bromide, which did not induce maturation. This last observation suggests that it may result from a change in specific K+ ion permeability due to the possible activation of stretch channels.