JAMBIO and Its Coastal Organism Joint Surveys: Network of Marine Stations Explores Japanese Coastal Biota.

Marine stations have continued to contribute significantly to understanding the physiology, taxonomy, development, ecology, and evolution of animals. There are more than 50 marine stations of national universities in Japan, and historically their establishments were closely related to the initial stage of zoology in the country. More than 10 years ago, Japanese Association for Marine Biology (JAMBIO) was established to facilitate the collaboration among marine stations in the activities of research, education and administration. One of the successful activities of JAMBIO that contribute to zoology is the JAMBIO Coastal Organism Joint Surveys, in which scientists and students at multiple marine stations, as well as those from research institutes or museums, stay at a marine station for a few days, and collect and make a record of marine organisms. As of 2021, 22 surveys have been performed and new species have been reported from taxa such as Cnidaria, Nematoda, Platyhelminthes, Annelida, Mollusca, Arthropoda, and Echinodermata.

Hydrophilic Shell Matrix Proteins of Nautilus pompilius and the Identification of a Core Set of Conchiferan Domains.

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the basally diverging Nautilids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautilid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we performed a multiomics study on the shell of N. pompilius, by conducting transcriptomics of its mantle tissue and proteomics of its shell matrix. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet and the Euhadra snail) revealed that three proteins and six protein domains were conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six protein domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were probably present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Even though further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be carried out in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Functional shell matrix proteins tentatively identified by asymmetric snail shell morphology.

Molluscan shell matrix proteins (SMPs) are essential in biomineralization. Here, we identify potentially important SMPs by exploiting the asymmetric shell growth in snail, Lymnaea stagnalis. Asymmetric shells require bilaterally asymmetric expression of SMP genes. We examined expression levels of 35,951 transcripts expressed in the left and right sides of mantle tissue of the pond snail, Lymnaea stagnalis. This transcriptome dataset was used to identify 207 SMPs by LC-MS/MS. 32 of the 207 SMP genes show asymmetric expression patterns, which were further verified for 4 of the 32 SMPs using quantitative PCR analysis. Among asymmetrically expressed SMPs in dextral snails, those that are more highly expressed on the left side than the right side are 3 times more abundant than those that are more highly expressed on the right than the left, suggesting potentially inhibitory roles of SMPs in shell formation. The 32 SMPs thus identified have distinctive features, such as conserved domains and low complexity regions, which may be essential in biomineralization.

Identification of TGFß-induced proteins in non-endocrine mouse pituitary cell line TtT/GF by SILAC-assisted quantitative mass spectrometry.

TtT/GF is a mouse cell line derived from a thyrotropic pituitary tumor and has been used as a model of folliculostellate cells. Our previous microarray data indicate that TtT/GF possesses some properties of endothelial cells, pericytes and stem/progenitor cells, along with folliculostellate cells, suggesting its plasticity. We also found that transforming growth factor beta (TGFß) alters cell motility, increases pericyte marker transcripts and attenuates endothelial cell and stem/progenitor cell markers in TtT/GF cells. The present study explores the wide-range effect of TGFß on TtT/GF cells at the protein level and characterizes TGFß-induced proteins and their partnerships using stable isotope labeling of amino acids in cell culture (SILAC)-assisted quantitative mass spectrometry. Comparison between quantified proteins from TGFß-treated cells and those from SB431542 (a selective TGFß receptor I inhibitor)-treated cells revealed 51 upregulated and 112 downregulated proteins (|log2| > 0.6). Gene ontology and STRING analyses revealed that these are related to the actin cytoskeleton, cell adhesion, extracellular matrix and DNA replication. Consistently, TGFß-treated cells showed a distinct actin filament pattern and reduced proliferation compared to vehicle-treated cells; SB431542 blocked the effect of TGFß. Upregulation of many pericyte markers (CSPG4, NES, ACTA, TAGLN, COL1A1, THBS1, TIMP3 and FLNA) supports our previous hypothesis that TGFß reinforces pericyte properties. We also found downregulation of CTSB, EZR and LGALS3, which are induced in several pituitary adenomas. These data provide valuable information about pericyte differentiation as well as the pathological processes in pituitary adenomas.

Insights into the Evolution of Shells and Love Darts of Land Snails Revealed from Their Matrix Proteins.

Over the past decade, many skeletal matrix proteins that are possibly related to calcification have been reported in various calcifying animals. Molluscs are among the most diverse calcifying animals and some gastropods have adapted to terrestrial ecological niches. Although many shell matrix proteins (SMPs) have already been reported in molluscs, most reports have focused on marine molluscs, and the SMPs of terrestrial snails remain unclear. In addition, some terrestrial stylommatophoran snails have evolved an additional unique calcified character, called a "love dart," used for mating behavior. We identified 54 SMPs in the terrestrial snail Euhadra quaesita, and found that they contain specific domains that are widely conserved in molluscan SMPs. However, our results also suggest that some of them possibly have evolved independently by domain shuffling, domain recruitment, or gene co-option. We then identified four dart matrix proteins, and found that two of them are the same proteins as those identified as SMPs. Our results suggest that some dart matrix proteins possibly have evolved by independent gene co-option from SMPs during dart evolution events. These results provide a new perspective on the evolution of SMPs and "love darts" in land snails.

TGFß signaling reinforces pericyte properties of the non-endocrine mouse pituitary cell line TtT/GF.

The non-endocrine TtT/GF mouse pituitary cell line was derived from radiothyroidectomy-induced pituitary adenoma. In addition to morphological characteristics, because the cells are S100ß-positive, they have been accepted as a model of folliculostellate cells. However, our recent microarray analysis indicated that, in contrast to folliculostellate cells, TtT/GF cells might not be terminally differentiated, as they share some properties with stem/progenitor cells, vascular endothelial cells and pericytes. The present study investigates whether transforming growth factor beta (TGFß) can elicit further differentiation of these cells. The results showed that canonical (Tgfbr1 and Tgfbr2) and non-canonical TGFß receptors (Tgfbr3) as well as all TGFß ligands (Tgfb1-3) were present in TtT/GF cells, based on reverse transcription PCR. SMAD2, an intercellular signaling molecule of the TGFß pathway, was localized in the nucleus upon TGFß signaling. Furthermore, TGFß induced cell colony formation, which was completely blocked by a TGFß receptor I inhibitor (SB431542). Real-time PCR analysis indicated that TGFß downregulated stem cell markers (Sox2 and Cd34) and upregulated pericyte markers (Nestin and Ng2). Double immunohistochemistry using mouse pituitary tissue confirmed the presence of NESTIN/NG2 double-positive cells in perivascular areas where pericytes are localized. Our results suggest that TtT/GF cells are responsive to TGFß signaling, which is associated with cell colony formation and pericyte differentiation. As pericytes have been shown to regulate angiogenesis, tumorigenesis and stem/progenitor cells in other tissues, TtT/GF cells could be a useful model to study the role of pituitary pericytes in physiological and pathological processes.

Proteome analysis of shell matrix proteins in the brachiopod Laqueus rubellus.

BACKGROUND: The calcitic brachipod shells contain proteins that play pivotal roles in shell formation and are important in understanding the evolution of biomineralization. Here, we performed a large-scale exploration of shell matrix proteins in the brachiopod Laqueus rubellus. RESULTS: A total of 40 proteins from the shell were identified. Apart from five proteins, i.e., ICP-1, MSP130, a cysteine protease, a superoxide dismutase, and actin, all other proteins identified had no homologues in public databases. Among these unknown proteins, one shell matrix protein was identified with a domain architecture that includes a NAD(P) binding domain, an ABC-type transport system, a transmembrane region, and an aspartic acid rich region, which has not been detected in other biominerals. We also identified pectin lyase-like, trypsin inhibitor, and saposin B functional domains in the amino acid sequences of the shell matrix proteins. The repertoire of brachiopod shell matrix proteins also contains two basic amino acid-rich proteins and proteins that have a variety of repeat sequences. CONCLUSIONS: Our study suggests an independent origin and unique mechanisms for brachiopod shell formation.

The diversity of shell matrix proteins: genome-wide investigation of the pearl oyster, Pinctada fucata.

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

An in-silico genomic survey to annotate genes coding for early development-relevant signaling molecules in the pearl oyster, Pinctada fucata.

The pearl oyster Pinctada fucata has great potential as a model system for lophotrochozoan developmental biology research. Pinctada fucata is an important commercial resource, and a significant body of primary research on this species has emphasized its basic aquaculture biology such as larval biology and growth, aquaculture, pearl formation and quality improvement, shell formation, and biomineralization. Recently, a draft genome sequence of this species was published, and many experimental resources are currently being developed, such as bioinformatics tools, embryo and larva manipulation methods, gene knockdown technique, etc. In this paper, we report the results from our genomic survey pertaining to gene families that encode developmental signaling ligands (Fgf, Hedgehog, PDGF/VEGF, TGFß, and Wnt families). We found most of the representative genes of major signaling pathways involved in axial patterning, as well as copies of the signaling molecule paralogs. Phylogenetic character mapping was used to infer a possible evolutionary scenario of the signaling molecules in the protostomes, and to reconstruct possible copy numbers of signaling molecule-coding genes for the ancestral protostome. Our reconstruction suggests that P. fucata retains the ancestral protostome gene complement, providing further justifications for the use of this taxon as a model organism for developmental genomics research.

A comparative study of the shell matrix protein aspein in pterioid bivalves.

Aspein is one of the unusually acidic shell matrix proteins originally identified from the pearl oyster Pinctada fucata. Aspein is thought to play important roles in the shell formation, especially in calcite precipitation in the prismatic layer. In this study, we identified Aspein homologs from three closely related pterioid species: Pinctada maxima, Isognomon perna, and Pteria penguin. Our immunoassays showed that they are present in the calcitic prismatic layer but not in the aragonitic nacreous layer of the shells. Sequence comparison showed that the Ser-Glu-Pro and the Asp-Ala repeat motifs are conserved among these Aspein homologs, indicating that they are functionally important. All Aspein homologs examined share the Asp-rich D-domain, suggesting that this domain might have a very important function in calcium carbonate formation. However, sequence analyses showed a significantly high level of variation in the arrangement of Asp in the D-domain even among very closely related species. This observation suggests that specific arrangements of Asp are not required for the functions of the D-domain.