Extensive conservation of the proneuropeptide and peptide prohormone complement in mollusks.

As one of the most diverse groups of invertebrate animals, mollusks represent powerful models for neurobiological and developmental studies. Neuropeptides and peptide hormones are a heterogeneous class of signalling molecules involved in chemical communication between neurons and in neuroendocrine regulation. Here we present a fine-grained view of the molluscan neuropeptide and peptide hormone toolkit. Our results expand the distribution of several peptide families (e.g., prokineticin, insulin-related peptides, prohormone-4, LFRFamide) within Lophotrochozoa and provide evidence for an early origin of others (e.g., GNXQN/prohormone-2, neuroparsin). We identified a new peptide family broadly distributed among conchiferan mollusks, the PXRX family. We found the Wnt antagonist dickkopf1/2/4 ortholog in lophotrochozoans and nematodes and reveal that the egg-laying hormone family is a DH44 homolog restricted to gastropods. Our data demonstrate that numerous peptides evolved much earlier than previously assumed and that key signalling elements are extensively conserved among extant mollusks.

[Osteoid osteoma in the scaphoid bone as cause of radiocarpal pain in a 15-year-old patient]. / Osteoidosteom im Os scaphoideum als Ursache für radiokarpale Handgelenksbeschwerden bei einem 15-jährigen Patienten.

Osteoid osteomas are benign bone tumors which rarely occur in the hand and impose severe diagnostic problems. The course of the disease is often protracted before the patient receives an adequate surgical treatment. The case of an osteoid osteoma in the scaphoid bone of a 15-year-old patient is presented, who was completely symptom free after a true diagnostic odyssey by resection of the nidus and reconstruction by crest bone graft and spongiosaplasty.

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.

Opsin evolution in the Ambulacraria.

Opsins--G-protein coupled receptors involved in photoreception--have been extensively studied in the animal kingdom. The present work provides new insights into opsin-based photoreception and photoreceptor cell evolution with a first analysis of opsin sequence data for a major deuterostome clade, the Ambulacraria. Systematic data analysis, including for the first time hemichordate opsin sequences and an expanded echinoderm dataset, led to a robust opsin phylogeny for this cornerstone superphylum. Multiple genomic and transcriptomic resources were surveyed to cover each class of Hemichordata and Echinodermata. In total, 119 ambulacrarian opsin sequences were found, 22 new sequences in hemichordates and 97 in echinoderms (including 67 new sequences). We framed the ambulacrarian opsin repertoire within eumetazoan diversity by including selected reference opsins from non-ambulacrarians. Our findings corroborate the presence of all major ancestral bilaterian opsin groups in Ambulacraria. Furthermore, we identified two opsin groups specific to echinoderms. In conclusion, a molecular phylogenetic framework for investigating light-perception and photobiological behaviors in marine deuterostomes has been obtained.

Unexpected co-linearity of Hox gene expression in an aculiferan mollusk.

BACKGROUND: Mollusca is an extremely diverse animal phylum that includes the aculiferans (worm-like aplacophorans and eight-shelled polyplacophorans) and their sister group, the conchiferans, comprising monoplacophorans, bivalves (clams, mussels), gastropods (snails, slugs), scaphopods (tusk shells) and cephalopods (squids, octopuses). Studies on mollusks have revealed an overall number of 11 Hox genes in seven out of eight molluscan "class"-level taxa, but expression data of key developmental regulators such as homeotic genes are only available for three gastropod and two cephalopod species. These show that Hox genes are involved in the formation of specific features including shell, foot, funnel or tentacles and not in antero-posterior body plan patterning as in most other bilaterian animals. The role of Hox genes in non-conchiferan (i.e., aculiferan) mollusks remains entirely unknown. RESULTS: Here we present the first data on the expression of seven Hox genes in apolyplacophoran mollusk, Acanthochitona crinita. In A. crinita the Hox genes Acr-Hox1-5, Hox7 and Post2 are expressed in a co-linear pattern along the antero-posterior axis, but not in molluscan-specific features such as the shell or the foot. The expression pattern is restricted to the post-trochal region and the transcripts are present in ecto-, endo- and mesodermal cell layers. Contrary to the situation in gastropods and cephalopods, we did neither find Hox gene expression in distinct neural subsets of A. crinita, nor in its developing shell plates. CONCLUSIONS: Our analysis and comparison with other lophotrochozoans indicate that the basal role of Hox genes is in antero-posterior axis patterning in mollusks, similar to the vast majority of bilaterian animals, and that this role has been conserved in polyplacophorans, while co-option into patterning of evolutionary novelties emerged either at the base of Conchifera or independently in gastropods and cephalopods. These morphological innovations most likely contributed to the evolutionary success of its representatives, as exemplified by, e.g., the wide ecological range and species richness of gastropods.

Innervation of bivalve larval catch muscles by serotonergic and FMRFamidergic neurons.

Bivalve larvae use catch muscles for rapid shell closure and maintenance of the closed condition. We used specific antibodies against the muscle proteins together with phalloidin and neuronal markers, FMRFamide and serotonin (5-HT), to analyze mutual distribution of muscle and neuronal elements in larvae of the mussel, Mytilus trossulus, and the oyster, Crassostrea gigas. At trochophore and early veliger stages no anatomical connections between muscular and nervous system were detected. By the pediveliger stage the 5-HT innervation of the anterior adductor developed in oyster only, while rich FMRFa innervation of the adductor muscles developed in both species. Possible roles and mechanisms of FMRFamide and serotonin in the regulation of the catch state are discussed.

Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations.

Among bilaterian invertebrates, cephalopod molluscs (e.g., squids, cuttlefish and octopuses) have a central nervous system (CNS) that rivals in complexity that of the phylogenetically distant vertebrates (e.g., mouse and human). However, this prime example of convergent evolution has rarely been the subject of recent developmental and evolutionary studies, which may partly be due to the lack of suitable neural markers and the large size of cephalopod brains. Here, we demonstrate the usefulness of fluorescence-coupled phalloidin to characterize the CNS of cephalopods using histochemistry combined with confocal laser scanning microscopy. Whole-mount preparations of developmental stages as well as vibratome sections of embryonic and adult brains were analyzed and the benefits of this technique are illustrated. Compared to classical neuroanatomical and antibody-based studies, phalloidin labeling experiments are less time-consuming and allow a high throughput of samples. Besides other advantages summarized here, phalloidin reliably labels the entire neuropil of the CNS of all squids, cuttlefish and octopuses investigated. This facilitates high-resolution in toto reconstructions of the CNS and contributes to a better understanding of the organization of neural networks. Amenable for multi-labeling experiments employing antibodies against neurotransmitters, proteins and enzymes, phalloidin constitutes an excellent neuropil marker for the complex cephalopod CNS.

FMRFamide-like immunoreactivity in the central nervous system of the cephalopod mollusc, Idiosepius notoides.

For more than a century, cephalopod molluscs have been the subject of extensive studies with respect to their complex neuroanatomy and behavior. In comparison to gastropod molluscs surprisingly little work has been carried out on the characterization of neurons in the central nervous system (CNS) of cephalopods with respect to their neurotransmitter phenotypes. This study presents preliminary results on the distribution of FMRFamide-like immunoreactive neurons within the CNS of the pygmy squid Idiosepius notoides. Its gross neuroanatomy resembles that of other cephalopods. FMRFamide-like immunoreactivity was observed in most of the brain lobes. High abundance of FMRFamidergic perikarya was found in the dorsal basal, the central palliovisceral, and the olfactory lobes, whereas none were observed in the middle suboesophageal mass. Single individual perikarya are located within the optic lobes and the vertical lobes. Although certain immunohistochemical traits are shared with other cephalopods, such as a wall-like arrangement of FMRFamide-like immunoreactive cell somata within the dorsal basal lobe, others have so far only been found in Idiosepius. However, future investigations on other species are necessary in order to broaden our knowledge on a common recruitment of certain neurotransmitters in distinct brain lobes of the highly advanced brain of cephalopods.

Comparative lophotrochozoan neurogenesis and larval neuroanatomy: recent advances from previously neglected taxa.

Recently, a number of neurodevelopmental studies of hitherto neglected taxa have become available, contributing to questions relating to the evolution of the nervous system of Lophotrochozoa (Spiralia + Lophophorata). As an example, neurogenesis of echiurans showed that these worm-shaped spiralians, which as adults do not exhibit any signs of segmentation, do show such traits during ontogeny, e.g. by segmentally arranged perikarya and commissures. Similarly, sipunculan worms, which have a single ventral nerve cord in the adult stage, develop this nerve cord by gradual fusion of a paired larval nerve during metamorphosis, and show transitional stages of segmentation. These findings indicate that echiurans, annelids and sipunculans stem from a segmented ancestor. By contrast, no traces of body segmentation are present during neurogenesis of basal molluscs. However, a tetraneurous condition (i.e. one pair of ventral and one pair of lateral nerve cords), as is typical for Mollusca, and a serotonergic larval apical organ that matches the complexity of polyplacophoran apical organs, were found in larval entoprocts, thus strongly supporting a mollusc-entoproct clade. Within the Lophophorata (Ectoprocta + Phoronida + Brachiopoda), data on nervous system development for any of the 3 lophophorate phyla are as of yet too scarce for profound phylogenetic inferences. Taking into account the most recent advances in molecular phylogenetics and developmental neurobiology, a scenario emerges that proposes a clade comprising Sipuncula + Annelida (including Echiura) on the one hand and a monophyletic assemblage of Entoprocta + Mollusca on the other.

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

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 engrailedin 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.

Development of the musculature in the limpet Patella (Mollusca, Patellogastropoda).

Whole-mount technique using fluorescent-labelled phalloidin for actin staining and confocal laser scanning microscopy as well as semi-thin serial sectioning, scanning and transmission electron microscopy were applied to investigate the ontogeny of the various muscular systems during larval development in the limpets Patella vulgata L. and P. caerulea L. In contrast to earlier studies, which described a single or two larval shell muscles, the pretorsional trochophore-like larva shows no less than four different muscle systems, namely the asymmetrical main head/foot larval retractor muscle, an accessory larval retractor with distinct insertion area, a circular prototroch/velar system, and a plexus-like pedal muscle system. In both Patella species only posttorsional larvae are able to retract into the shell and to close the aperture by means of the operculum. Shortly after torsion the two adult shell muscles originate independently in lateral positions, starting with two fine muscle fibres which insert at the operculum and laterally at the shell. During late larval development the main larval retractor and the accessory larval retractor become reduced and the velar muscle system is shed. In contrast, the paired adult shell muscles and the pedal muscle plexus increase in volume, and a new mantle musculature, the tentacular muscle system, and the buccal musculature arise. Because the adult shell muscles are entirely independent from the various larval muscular systems, several current hypotheses on the ontogeny and phylogeny of the early gastropod muscle system have to be reconsidered.

The development of the dentigerous bones and teeth in the hemiramphid fish Dermogenys pusillus (Atheriniformes, Teleostei).

The development of the dentition and dentigerous bones was studied in the hemiraphid Dermogenys pusillus using histological sections, scanning electron microscopy, and cleared and stained specimens. Five days after birth, the toothless tip of the lower jaw begins to grow longer than the tip of the upper jaw. The growth originates from small cartilaginous triangles connected with Meckel's cartilage. The peri- and enchondrial ossification of the growing cartilaginous bars advances rostrad. The pharyngeal tooth plates are formed by fusion of the slat-like dentigerous dermal bones with the bony fractions of the gill branches. Hence, the tooth plates are composite bones. The ventral tooth plate ist formed by the two ceratobranchials V and the basibranchial IV together with the respective dermal bones. The paired pharyngobranchials III and IV are fused with the dorsal tooth plate, and the pharyngobranchials II is fused with the two respective lateral tooth plates. Mineralization starts after birth in elements of the pharyngeal tooth plates and their teeth. There are no indications that the pedicel on which the tooth is established is formed by the enamel organ, which is covered by pulpal cells. The enamel organ originates from the stratum basale of the oropharyngeal epithelium and moves within the jaw from labial toward lingual, the site of the establishment of the tooth. The anlage of the tooth on the tooth plates of the pharynx lies at the level of the tooth base.

Dentigerous bones and dentition in the hemiramphid fish Dermogenys pusillus (Atheriniformes, Teleostei).

Structure and arrangement of the teeth were studied in the hemiramphid Dermogenys pusillus, using scanning electron microscopy as well as cleared and stained specimens. The teeth of the jaws are small, monocuspid, and tilted towards the esophagus. They are arranged along the lateral edges of the premaxillas and dentaries. Each premaxilla bears additional teeth on an osseous bar extending from rostro-lateral to medio-lingual. The dentition of both dentaries curves slightly within the cavity of the mouth and gently tapers off laterorostrad just beyond the tip of the upper jaw. The part of the lower jaw, which typically protrudes beyond the upper jaw, is without teeth. One dorsal and two lateral tooth-bearing bony plates (tooth plates) are found in the pharyngeal region. Their teeth are largely irregular in arrangement. The teeth on the two lateral plates are small and monocuspid, whereas the dorsal and the ventral tooth plate possess additional strong bi- and tricuspid teeth. The teeth of the jaws and of the pharyngeal region obviously have a bony pedicel ("attachment bone") which is asymmetric in all teeth. An elastic suture connects the cone of dentine with the bony pedicel. The special construction of the teeth and their arrangement on the various dentigerous bones will be discussed with respect to their function in catching prey.