Expression of Patella vulgata orthologs of engrailed and dpp-BMP2/4 in adjacent domains during molluscan shell development suggests a conserved compartment boundary mechanism

The engrailed gene is well known from its role in segmentation and central nervous system development in a variety of species. In molluscs, however, engrailed is involved in shell formation. So far, it seemed that engrailed had been co-opted uniquely for this particular process in molluscs. Here, we show that, in the gastropod mollusc Patella vulgata, an engrailed ortholog is expressed in the edge of the embryonic shell and in the anlage of the apical sensory organ. Surprisingly, a dpp-BMP2/4 ortholog is expressed in cells of the ectoderm surrounding, but not overlapping, the engrailed-expressing shell-forming cells. It is also expressed in the anlage of the eyes. Earlier it was shown that a compartment boundary exists between the cells of the embryonic shell and the adjacent ectoderm. We conclude that engrailed and dpp are most likely involved in setting up a compartment boundary between these cells, very similar to the situation in, for example, the developing wing imaginal disc in Drosophila. We suggest that engrailed became involved in shell formation because of its ancestral role, which is to set up compartment boundaries between embryonic domains.     

Developmental role of dpp in the gastropod shell plate and co-option of the dpp signaling pathway in the evolution of the operculum

The operculum is a novel structure in gastropod molluscs. Because the operculum shows notable similarities to the shell plate, we asked whether there were an evolutionary link between these two secretory organs. We found that some of the genes involved in shell-field development are expressed in the operculum, such as dpp and grainyhead, whereas engrailed and Hox1 are not. Specific knockdown of dpp by injection of double-stranded RNA (dsRNA) resulted in malformation of the shell plate. The shell plate was smaller due to failure of activation of cell proliferation in the shell-field margin. The expressions of grainyhead and chitin synthase 1 in the shell field margin were suppressed by dpp-dsRNA. However, matrix secretion was not completely abolished, and the expressions of ferritin, engrailed or Hox1 were not affected by dpp-dsRNA, indicating that dpp is partly involved in the developmental pathway for shell matrix secretion. We also present evidence that dpp performs a key role in operculum development. Indeed, dpp-dsRNA impaired matrix secretion in the operculum as well as expression of grainyhead. Based on these observations that dpp is important for development of both the shell plate and operculum, we conclude that co-option of dpp to the posterior part of the foot contributed to the innovation of the operculum in gastropods.     

The expression of an engrailed protein during embryonic shell formation of the tusk-shell, Antalis entalis (Mollusca, Scaphopoda)

SUMMARY This study presents the first detailed account of the larval and early post-metamorphic development of a scaphopod species, Antalis entalis , since 1883. Special reference is given to the expression pattern of an engrailed protein during the formation of the embryonic (protoconch) and adult shell (teleoconch). We found that in the trochophore-like larva the engrailed protein is expressed in shell-secreting cells at the margin of the protoconch close to the mantle edge. During metamorphosis the growth of the protoconch and expression of the engrailed protein along its margin stop and the teleoconch starts to form. These data suggest a different genetic background regarding protoconch and teleoconch formation in the Scaphopoda and possibly all Conchifera, thus inferring a different evolutionary origin of both organs. The single anlage of the scaphopod protoconch contradicts earlier hypotheses of a monophyletic taxon Diasoma (Scaphopoda + Bivalvia), which has been mainly based on the assumption of a primarily bilobed shell in both taxa. Comparative data on engrailed expression patterns suggest nervous system patterning as the basic function of engrailed in the Bilateria. However, there are several independent gain-of-function events, namely segment compartmentation in the Annelida and Arthropoda, protoconch formation in the Mollusca, skeletogenesis in the Echinodermata, and limb formation in vertebrates. These findings provide further evidence that homologous genes may act in very different pathways of bilaterian body plan formation in various animal phyla.     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.     

Molluscan engrailed expression, serial organization, and shell evolution

Whether the serial features found in some molluscs are ancestral or derived is considered controversial. Here, in situ hybridization and antibody studies show iterated engrailed-gene expression in transverse rows of ectodermal cells bounding plate field development and spicule formation in the chiton, Lepidochitona cavema, as well as in cells surrounding the valves and in the early development of the shell hinge in the clam, Transennella tantilla. Ectodermal expression of engrailed is associated with skeletogenesis across a range of bilaterian phyla, suggesting a single evolutionary origin of invertebrate skeletons. The shared ancestry of bilaterian-invertebrate skeletons may help explain the sudden appearance of shelly fossils in the Cambrian. Our interpretation departs from the consideration of canonical metameres or segments as units of evolutionary analysis. In this interpretation, the shared ancestry of engrailed-gene function in the terminal/posterior addition of serially repeated elements during development explains the iterative expression of engrailed genes in a range of metazoan body plans.     

Environmental calcium modifies induced defences in snails

Inducible defences are adaptive phenotypes that arise in response to predation threats. Such plasticity incurs costs to individuals, but there has been little interest in how such induced traits in animals may be constrained by environmental factors. Here, we demonstrate that calcium availability interacts with predation cues to modify snail shell growth and form. Small snails increased their growth and were heavier when exposed to fish chemical cues, but this response was calcium limited. There was also an interactive effect of fish cues and calcium on the shell growth of larger snails, but shell strength and aperture narrowness were affected by calcium alone. For small snails, behavioural avoidance was greatest for snails exhibiting least morphological plasticity, suggesting a trade-off. There was no trade-off of somatic growth with plasticity. We suggest that the expression of defensive traits in molluscs can be constrained by calcium availability, which has implications for molluscan ecology and evolution.     

Patterns of engrailed protein in early Drosophila embryos

By the onset of gastrulation during nuclear cycle 14 of Drosophila embryogenesis, the engrailed gene is expressed in fourteen one-cell-wide stripes. Each stripe defines the anlagen of the posterior compartment of a metameric segment. We report here several observations relating to the role and disposition of the engrailed protein during the embryonic stages that precede cellularization. We demonstrate that in embryos mutant for the engrailed gene, there were characteristic morphological abnormalities as early as the 6th cleavage cycle. In addition, the engrailed protein was detected in pre-cycle-9 embryos by Western blot analysis. When localization of engrailed protein begins during cycle 14, engrailed expression was first present in broad anterior and posterior regions before the fourteen-stripe pattern appeared.     

Involvement of Hox genes in shell morphogenesis in the encapsulated development of a top shell gastropod (Gibbula varia L.)

Regulatory gene expression during the patterning of molluscan shells has only recently drawn the attention of scientists. We show that several Hox genes are expressed in association with the shell gland and the mantle in the marine vetigastropod Gibbula varia (L.). The expression of Gva-Hox1, Gva-Post2, and Gva-Post1 is initially detected in the trochophore larval stage in the area of the shell field during formation of embryonic shell. Later, during development, these genes are expressed in the mantle demonstrating their continuous role in larval shell formation and differentiation of mantle edge that secretes the adult shell. Gva-Hox4 is expressed only late during the development of the veliger-like larva and may also be involved in the adult shell morphogenesis. Additionally, this gene also seems to be associated with secretion of another extracellular structure, the operculum. Our data provide further support for association of Hox genes with shell formation which suggest that the molecular mechanisms underlying shell synthesis may consist of numerous conserved pattern-formation genes. In cephalopods, the only other molluscan class in which Hox gene expression has been studied, no involvement of Hox genes in shell formation has been reported. Thus, our results suggest that Hox genes are coopted to various functions in molluscs.     

合浦珠母贝外套膜细胞engrailed、BMP2和Smad3的mRNA表达水平的相关性

软体动物engrailed蛋白和骨形成相关蛋白对胚胎贝壳区域边界形成可能具有重要作用,engrailed还被推测为调节基质蛋白在外套膜组织区域化表达的重要调控因子．因此,弄清调控engrailed在软体动物中特征表达的分子机制有着重要的研究意义．但是,由于贝类基因组测序尚不完整,目前也没有建立获得贝类细胞系,以致于许多预测可能参与调控的基因需要通过克隆来鉴定,而且经典的研究细胞信号通路的方法也很难得到应用．目前,在中国南海广泛养殖的合浦珠母贝中,已获知其BMP2和Smad3的cDNA全长,以该贝的基因组为模板,PCR扩增获得了一段engrailed编码区片段．经软件分析,该片段含有EH4结构域,且与其他物种engrailed蛋白具有很高的同源性．研究的贝中,特别是外套膜组织中,engrailed、BMP2和Smad3三者表达之间的相关性,将有助于我们理解贝壳形成的分子机制．贝壳缺刻后半定量PCR试验结果表明,三者均参与贝壳修复,且在贝壳缺刻后的修复过程中,engrailed和Smad3的mRNA表达变化规律非常相似,提示它们之间可能存在相互影响的联系．用地塞米松(DXM)和过氧化氢(H₂O₂)分别处理原代培养的贝外套膜组织迁出细胞,实时相对定量PCR检测engrailed、BMP2和Smad3的mRNA表达水平,统计分析结果表明,三者具有显著的相关性．上述所有结果为进一步研究贝类生物矿化的发育和信号转导机制提供了新的思路和基础．     

Possible implication of Hox genes<Emphasis Type="Italic"> Abdominal-B</Emphasis> and<Emphasis Type="Italic"> abdominal-A</Emphasis> in the specification of genital and abdominal segments in cirripedes

The crustaceans cirripedes (barnacles) are characterised by the lack of fully developed abdominal segments at any stage of their life cycle. However, in nauplius larvae of the cirripede Sacculina carcini, we detected five small engrailed stripes in a postero-dorsal region behind the sixth thoracic segment, that we interpreted as a vestigial abdomen. Here, we present additional morphological and genetic data on Sacculina to further characterise this structure. Scanning electron microscopy analysis confirms the existence of a segmented region in this part of the naupliar body. However, at the late naupliar stage, this structure stops its development and degenerates. This region expresses the Hox gene Abdominal-B, which may indicate that it actually corresponds to the posterior-most part of the Sacculina trunk. In addition, Abdominal-B expression differentiates two types of larvae that probably correspond to male and female larvae, respectively. In contrast, no abdominal-A expression can be detected in the vestigial abdomen. We discuss the possible implication of the loss or divergence of the Abdominal-A protein in the impaired development of abdominal segments in cirripedes.     

The late Cenozoic Thiaridae (Mollusca, Gastropoda, Cerithioidea) of the Albertine Rift Valley (Uganda-Congo) and their bearing on the origin and evolution of the Tanganyikan thalassoid malacofauna

The recent cerithioid malacofauna of meromictic Lake Tanganyika is unique in its degree of thalassoid convergence with marine molluscs. This is generally considered the result of a long-lasting intensive escalation and cladogenesis caused by a coevolutionary prey–predator interaction in a freshwater ecosystem with sea-like characteristics, i.e. exceptional longevity (ca. 7–12 Ma) and vast dimensions (present surface area: 32900 km², present maximum depth: 1470 m). In the Albertine Basin, ca 300 km north of the Tanganyika Trough, Palaeolake Obweruka existed during Mio-Pliocene times. In many aspects, it can be considered as a 'sister lake' of Lake Tanganyika, being also long-lived (from ca 7.5 to 2.5 Ma), extensive (surface: 27000 km²) and meromictic. Like Lake Tanganyika it belonged to the Congo catchment. Although thalassoid molluscs have been known from the Albertine deposits since the beginning of the 20th Century, previous researchers recognised only four polymorphic thiarid species, two of which were thalassoid. Detailed in situ collecting shows that the perceived low species diversity and high variability of the Obwerukan thalassoids is a phenomenon of the museum drawer. In the field the thiarid species and other molluscs are not extremely polymorphic, as formerly thought, but tend to occur in discrete morphometric packages according to stratigraphic level and geographic area. The species richness of the Palaeolake Obweruka thiarids has thus been severely underestimated, as is also the case for the Recent Tanganyikan thiarids. The present authors discern 35 species and 7 genera in the Albertine deposits (2 of which are new to science), and consider this to be a conservative estimate. In their degree of diversification and morphological escalation, the Obwerukan thiarids are thus comparable to the Tanganyika thalassoids. Arguments are presented that the thalassoids of these lakes are polyphyletic, that they are derived from the same genera (mainly Potadoma, Potadomoides and Pseudocleopatra) and that the tempo and mode of their intralacustrine evolution must have been largely similar. As for the tempo of evolution, the Albertine fossil record shows that escalation in shell morphology and ornamentation in the Thiaridae was extremely rapid, as in the Ampullariidae and Viviparidae (Van Damme & Pickford, 1995, 1999). In the Thiaridae heavily ornate shells evolved from an unornamented ancestor in two consecutive bursts, each lasting less than 0.1 million years over a total period of ca. 1 million years. The punctuated escalation in the thiarids was initiated millions of years after the lake was formed. This implies that the degree of escalation is not related to age of a lake and that the Tanganyikan thalassoids or part of them may not be particularly old, even though Lake Tanganyika itself is. As to the mode of evolution, it appears that escalation is a coevolutionary reaction to predation but that this selective pressure only triggers a reaction under specific conditions and in synergy with other environmental factors. The Obwerukan thiarid fossil record also indicates that iterative evolution of molluscan shells may be of frequent occurrence. What is considered to be a single polymorphic chronospecies, static over a great length of time, may actually be a sequence of several species that branched at different moments from the same ancestral phenotype. Finally, there is the inference that bottle-necking may be, geologically speaking, so common that it forms an essential force in macroevolution.     

Development of the muscle system and contractile activity in the mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Mollusca,Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and fluorescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 μm in both mussel larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing video workstation. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult molluscs.     

Sound reasons for silence: why do molluscs not communicate acoustically?

Many adaptively beneficial states of form, behaviour and physiology are absent in large parts of the evolutionary tree of life. Although the causes of these absences can never be fully known, insights into the possibilities and limitations of adaptive evolution can be gained by examining the conditions that would be necessary for the forbidden phenotypes to evolve. Here, the case of acoustic communication in molluscs is considered. The production of sound as a warning to predators or as a means to attract mates is widespread among arthropods and vertebrates, both on land and in water, but is unknown among molluscs, even though many derived clades of gastropods and cephalopods are characterized by internal fertilization and by the evolution of long‐distance visual and chemical signalling. Many molluscs possess suitable hard parts – shell, operculum and jaws – for producing sound, but most shell‐bearing molluscs lack the agility or aggression necessary to cope with high‐activity enemies attracted to an acoustic beacon. Their evolutionary background, arising from the generally passive adaptations of molluscs and other animals with low metabolic rates, prevents selection favouring communication by sound, and indeed favours silence. Several clades of shell‐bearing gastropods and cephalopods were identified in which sound production has the greatest potential to arise or to be discovered. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 485–493.     

Negative regulation ofUltrabithorax expression byengrailed is required for proper specification of wing development inDrosophila melanogaster

In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such asDrosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modified into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity geneengrailed (en) is critical to keep the homeotic selector geneUltrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repressUbx, and (iii) the repression ofUbx byen is independent of Hedgehog signalling through which the long-range signalling ofen is mediated during wing development. Finally we provide evidence for a possible mechanism by whichen repressesUbx. On the basis of these results we propose thaten has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keepingUbx repressed.     

Heterochrony in Shell Sculpture Development within the Ostracode Genus Hemicythere

The evolutionary issues of taxonomic significance of the shell sculpture in Ostracoda are addressed. This is a comparative ontogenetic study of three types of lateral sculpture (fossa-mural reticulation, punctation, and foveolation), as well as flanges, ridges, and tubercles is made in different age stages and morphological variations of three ostracode species with different strengths of the expression of sculpture: Hemicythere borealis (Brady, 1868), H. villosa (Sars, 1866), and H. nana (Schornikov, 1974). It is shown that the development of each sculpture type proceeds relatively independently and represents a mosaic; developmental stages are accompanied by numerous heterochronies. This results in many parallelisms, and even the trend of the evolution of a structure can change. The pathways of morphological evolution of Hemicythere species are discussed.     

Wandel der Vorstellungen von der Frühevolution der Mollusken,besonders der Gastropoda und Cephalopoda

The ancestors of the molluscs are still unknown. Arguments in favour of flatworm-relationship are just as valid as those proposing segmented annelid-like worms as their closest relatives. The earliest molluscs lived before the onset of the Cambrian; but only at the end of this period the now existing classes of conchifers made their first appearance. Early and Middle Cambrian molluscan fossils are problematic. The hyoliths, for example, show relations to such fossils asSalterella andVolborthella, while closest living relatives can be found among tube-building annelids. Scars produced by tissue attached to the shell are of no use in the reconstruction of molluscan phylogeny, but a very useful tool in the analysis of the function regarding the body in interaction with its shell. The ontogenesis of the shell and muscles attached to it in recentFissurella (archaeogastropod) is presented as an example. Well known ontogenies in recent molluscs can aid to the reconstruction of fossils, demonstrated by some fossil gastropods. The interaction of soft and hard tissues, the function and structure of the shell of recent molluscs enables us to interpret the fossil forms, as for example the Lower Devonian coleoid cephalopods from the Hunsrück Schiefer. Reconstruction of the course of evolution is only possible, if information on living molluscs is integrated into historical data provided by the fossils.