m6A Profile Dynamics Indicates Regulation of Oyster Development by m6A-RNA Epitranscriptomes.

The N6-methylation of RNA adenosines (N6-methyladenosine, m6A) is an important regulator of gene expression with critical implications in vertebrate and insect development. However, the developmental significance of epitranscriptomes in lophotrochozoan organisms remains unknown. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq), we generated transcriptome-wide m6A-RNA methylomes covering the whole development of the oyster from oocytes to juveniles. Oyster RNA classes display specific m6A signatures, with messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) exhibiting distinct profiles and being highly methylated compared to transposable element (TE) transcripts. Epitranscriptomes are dynamic and correspond to the chronological steps of development (cleavage, gastrulation, organogenesis, and metamorphosis), with minimal mRNA and lncRNA methylation at the morula stage followed by a global increase. mRNA m6A levels are correlated to transcript levels, and shifts in methylation profiles correspond to expression kinetics. Differentially methylated transcripts cluster according to embryo-larval stages and bear the corresponding developmental functions (cell division, signal transduction, morphogenesis, and cell differentiation). The m6A level of TE transcripts is also regulated and peaks during the gastrulation. We demonstrate that m6A-RNA methylomes are dynamic and associated with gene expression regulation during oyster development. The putative epitranscriptome implication in the cleavage, maternal-to-zygotic transition, and cell differentiation in a lophotrochozoan model brings new insights into the control and evolution of developmental processes.

A functional m6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development.

N6 -methyladenosine (m6 A) is a prevalent epitranscriptomic mark in eukaryotic RNA, with crucial roles for mammalian and ecdysozoan development. Indeed, m6 A-RNA and the related protein machinery are important for splicing, translation, maternal-to-zygotic transition and cell differentiation. However, to date, the presence of an m6 A-RNA pathway remains unknown in more distant animals, questioning the evolution and significance of the epitranscriptomic regulation. Therefore, we investigated the m6 A-RNA pathway in the oyster Crassostrea gigas, a lophotrochozoan model whose development was demonstrated under strong epigenetic influence. Using mass spectrometry and dot blot assays, we demonstrated that m6 A-RNA is actually present in the oyster and displays variations throughout early oyster development, with the lowest levels at the end of cleavage. In parallel, by in silico analyses, we were able to characterize at the molecular level a complete and conserved putative m6 A machinery. The expression levels of the identified putative m6 A writers, erasers and readers were strongly regulated across oyster development. Finally, RNA pull-down coupled to LC-MS/MS allowed us to prove the actual presence of readers able to bind m6 A-RNA and exhibiting specific developmental patterns. Altogether, our results demonstrate the conservation of a complete m6 A-RNA pathway in the oyster and strongly suggest its implication in early developmental processes including MZT. This first demonstration and characterization of an epitranscriptomic regulation in a lophotrochozoan model, potentially involved in the embryogenesis, bring new insights into our understanding of developmental epigenetic processes and their evolution.

Characterization of an evolutionarily conserved calcitonin signalling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas).

In Protostoma, the diuretic hormone 31 (DH31) signalling system was long considered as the orthologue of the chordate calcitonin (CT) signalling system. Using the Pacific oyster (Crassostrea gigas) transcriptomic database GigaTON, we characterized seven G-protein-coupled receptors (GPCRs) named Cragi-CTR1-7 and phylogenetically related to chordate CT receptors (CTRs) and to protostome DH31 receptors. Two CT precursors (Cragi-CTP1 and Cragi-CTP2) containing two CT-type peptides and encoded by two distinct genes with a similar organization were also characterized. These oyster neuropeptides (Cragi-CT1/2) exhibit the two N-terminal paired cysteine residues and, except CTP2-derived peptide (Cragi-CTP2dp), show the C-terminal proline-amide motif typical of deuterostome CT-type peptides. All mature Cragi-CTs except Cragi-CTP2dp were detected in visceral ganglion extracts using mass spectrometry. Cell-based assays revealed that the previously characterized oyster receptors Cg-CT-R and Cragi-CTR2 were specifically activated by Cragi-CT1b and Cragi-CT2, respectively. This activation does not require the co-expression of receptor activity-modifying proteins (RAMPs). Thus, oyster CT signalling appears functionally more closely related to vertebrate CT/CTR signalling than to calcitonin gene-related peptide/calcitonin receptor-like receptor (CGRP/CLR) signalling. Gene expression profiles in different adult tissues and in oysters acclimated to brackish water suggest the potential implication of both Cg-CT-R/Cragi-CT1b and Cragi-CTR2/Cragi-CT2 in water and ionic regulations, although with apparently opposite effects. The present study represents the first comprehensive characterization of a functional CT-type signalling system in a protostome and provides evidence for its evolutionarily ancient origin and its early role in osmotic homeostasis.

Data for evolutive analysis of insulin related peptides in bilaterian species.

In bilaterian species, the amino acid sequence conservation between Insulin related peptides is relatively low except for the cysteine residues involved in the disulphide bonds. In the A chain, the conserved cystein residues are included in a signature motif. Investigating the variations in this motif would give insight into the phylogenetic history of the family. The table presented in this paper contains a large set of insulin-related peptides in bilateral phylogenetic groups (deuterostomian, ecdysozoan, lophotrochozoan). NCBI databases in silico wide screening combined with bibliographic researches provided a framework for identifying and categorising the structural characteristics of these insulin related peptides. The dataset includes NCBI IDs of each sequence with hyperlinks to FASTA format. Moreover, the structural type (α, ß or γ), the A chain motif, the total number of cysteins, the C peptide cleavage mode and the potential additional domains (D or E) are specified for each sequence. The data are associated with the research article "Molecular evolution and functional characterisation of insulin-related peptides in molluscs: contributions of Crassostrea gigas genomic and transcriptomic-wide screening" [1]. The table presented here can be found at http://dx.doi.org/10.17632/w4gr8zcpk5.4#file-21c0f6a5-a3e3-4a15-86e0-e5a696458866.

Molecular evolution and functional characterisation of insulin related peptides in molluscs: Contributions of Crassostrea gigas genomic and transcriptomic-wide screening.

Insulin Related Peptides (IRPs) belong to the insulin superfamily and possess a typical structure with two chains, B and A, linked by disulphide bonds. As the sequence conservation is usually low between members, IRPs are classified according to the number and position of their disulphide bonds. In molluscan species, the first IRPs identified, named Molluscan Insulin-related Peptides (MIPs), exhibit four disulphide bonds. The genomic and transcriptomic data screening in the Pacific oyster Crassostrea gigas (Mollusc, Bivalvia) allowed us to identify six IRP sequences belonging to three structural groups. Cg-MIP1 to 4 have the typical structure of MIPs with four disulphide bonds. Cg-ILP has three disulphide bonds like vertebrate Insulin-Like Peptides (ILPs). The last one, Cg-MILP7 has a significant homology with Drosophila ILP7 (DILP7) associated with two additional cysteines allowing the formation of a fourth disulphide bond. The phylogenetic analysis points out that ILPs may be the most ancestral form. Moreover, it appears that ILP7 orthologs are probably anterior to lophotrochozoa and ecdysozoa segregation. In order to investigate the diversity of physiological functions of the oyster IRPs, we combine in silico expression data, qPCR measurements and in situ hybridization. The Cg-ilp transcript, mainly detected in the digestive gland and in the gonadal area, is potentially involved in the control of digestion and gametogenesis. The expression of Cg-mip4 is mainly associated with the larval development. The Cg-mip transcript shared by the Cg-MIP1, 2 and 3, is mainly expressed in visceral ganglia but its expression was also observed in the gonads of mature males. This pattern suggested the key roles of IRPs in the control of sexual reproduction in molluscan species.

Molecular characterization of an adipokinetic hormone-related neuropeptide (AKH) from a mollusk.

Adipokinetic hormones (AKH) are key regulators of energy mobilization in insects. With the growing number of genome sequence available, the existence of genes encoding AKH related peptides has now been established in protostomes. Here we investigated the occurrence of a mature AKH-like neuropeptide (Cg-AKH) in the oyster Crassostrea gigas. We unambiguously elucidated the primary structure of this neuropeptide by mass spectrometry from peptidic extracts of oyster visceral ganglia. Cg-AKH mature peptide (pQVSFSTNWGS-amide) represents an additional member of the AKH family of peptides. The organization of Cg-AKH encoding gene and its corresponding transcript is also described. Cg-AKH gene was found to be expressed in the nervous system though at extremely low levels compared to other neuropeptide encoding genes such as the oyster GnRH gene. Although both reproduction and feeding are known to affect the energy balance in oysters, no significant differential expression of Cg-AKH gene could be evidenced in relation with the nutritional status or along the reproductive cycle. The possible involvement of Cg-AKH in the regulation of energy balance in oyster remains an open question.

Characterization of GnRH-related peptides from the Pacific oyster Crassostrea gigas.

Gonadotropin-releasing hormone (GnRH), a key neuropeptide regulating reproduction in vertebrates has now been characterized in a number of non-vertebrate species. Despite the demonstration of its ancestral origin, the structure and the function of this family of peptides remain poorly known in species as distant as lophotrochozoans. In this study, two GnRH-related peptides (Cg-GnRH-a and CgGnRH-G) were characterized by mass spectrometry from extracts of the visceral ganglia of the Pacific oyster Crassostrea gigas. These peptides showed a high degree of sequence identity with GnRHs of other mollusks and annelids and to a lesser extent with those of vertebrates or with AKH and corazonins of insects. Both the mature peptides and the transcript encoding the precursor protein were exclusively expressed in the visceral ganglia. Significant differences in transcriptional activity of Cg-GnRH encoding gene were recorded in the ganglia along the reproductive cycle and according to trophic conditions with a higher level in fed animals compared to starved animals. This suggests the involvement of Cg-GnRHs as synchronizers of nutritional status with energy requirements during reproduction in oyster. Evidence for a role of Cg-GnRHs as neuroregulators and as neuroendocrine factors in bivalve is discussed.

Structural and functional characterizations of an Activin type II receptor orthologue from the pacific oyster Crassostrea gigas.

Members of the Transforming Growth factor beta (TGF-beta) superfamily of cell signalling polypeptides are known to play important roles in cell proliferation and differentiation during development and in various physiological processes of most animal clades. Recent findings in the mollusc Crassostrea gigas demonstrate the occurrence of a diversity of TGF-beta signalling components including various ligands, three type I receptors but only a single type II receptor. This report describes the characterization of Cg-ActRII, a new type II receptor displaying homology with vertebrate and Drosophila Activin type II receptors. The use of zebrafish embryo as a reporter organism revealed that, in a way similar to its zebrafish counterpart, overexpression of Cg-ActRII or its dominant negative acting truncated form resulted in a dose dependent range of dorsoventral defects coupled with anterior disorders. Expression pattern of Cg-ActRII transcripts examined by real time PCR and in situ PCR in C. gigas showed high levels of Cg-ActRII transcripts in early embryonic stages and in the developing larval central nervous system. Except for a high expression in the visceral ganglia, most oyster adult tissues displayed rather low levels of transcripts. Altogether, the data suggest a high degree of conservation at both the structural and functional levels during evolution for this class of receptors.

Characterization of a gonad-specific transforming growth factor-beta superfamily member differentially expressed during the reproductive cycle of the oyster Crassostrea gigas.

Through differential screening between oyster families selected for high and low summer survival, we have characterized a new transforming growth factor-beta (TGF-beta) superfamily member. This novel factor, named oyster-gonadal-TGFbeta-like (og-TGFbeta-like), is synthesized as a 307 amino acid precursor and displays 6 of the 7 characteristic cysteine residues of the C-terminal, mature peptide. Sequence comparison revealed that og-TGFbeta-like has a low percentage of identity with other known TGF-beta superfamily members, suggesting that og-TGFbeta-like is a derived member of this large superfamily. Real-time PCR (RT-PCR) analysis in different oyster tissues showed that og-TGFbeta-like is specifically expressed in both male and female gonads, at distinct levels according to the reproductive stage. Og-TGFbeta-like relative expression was the lowest at the initiation of the reproductive cycle and increased as maturation proceeded to achieve a maximal level in fully mature female and male oysters. In situ hybridisation demonstrated that expression was exclusively detected in the somatic cells surrounding oocytes and spermatocytes. The role of this newly-characterized TGFbeta member in the reproduction of cupped oyster is discussed in regard to the specificity and the localization of its expression, which singularly contrasts with the pleiotropic roles in a variety of physiological processes commonly ascribed to most TGF-beta family members identified so far.

Characterization of a defensin from the oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial activities, and gene expression.

In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized alpha-beta motif (CS-alphabeta) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.

Structural and functional evidence for a singular repertoire of BMP receptor signal transducing proteins in the lophotrochozoan Crassostrea gigas suggests a shared ancestral BMP/activin pathway.

The transforming growth factor beta (TGF-beta) superfamily includes bone morphogenetic proteins, activins and TGF-betasensu stricto (s.s). These ligands, which transduce their signal through a heteromeric complex of type I and type II receptors, have been shown to play a key role in numerous biological processes including early embryonic development in both deuterostomes and ecdyzozoans. Lophochotrozoans, the third major group of bilaterian animals, have remained in the background of the molecular survey of metazoan development. We report the cloning and functional study of the central part of the BMP pathway machinery in the bivalve mollusc Crassostrea gigas (Cg-BMPR1 type I receptor and Cg-TGFbetasfR2 type II receptor), showing an unusual functional mode of signal transduction for this superfamily. The use of the zebrafish embryo as a reporter organism revealed that Cg-BMPR1, Cg-TGFbetasfR2, Cg-ALR I, an activin Type I receptor or their dominant negative acting truncated forms, when overexpressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning, due to strong modulations of ventrolateral mesoderm patterning. The results suggest that Cg-BMPR1, and to a certain degree Cg-TGFbetasfR2 proteins, function in C. gigas in a similar way to their zebrafish orthologues. Finally, based on phylogenetic analyses, we propose an evolutionary model within the complete TGF-beta superfamily. Thus, evidence provided by this study argues for a possible conserved endomesoderm/ectomesoderm inductive mechanism in spiralians through an ancestral BMP/activin pathway in which the singular, promiscuous and probably unique Cg-TGFbetasfR2 would be the shared type II receptor interface for both BMP and activin ligands.

Transforming growth factor-beta-related proteins: an ancestral and widespread superfamily of cytokines in metazoans.

Members of the transforming growth factor beta (TGF-beta) superfamily of cell signalling polypeptides have attracted much attention because of their ability, from nematodes to mammals, to control cellular functions that in turn, regulate embryo development and tissue homeostasis. On the basis of structure similarities, the TGF-beta members (ligands, receptors and Smads) are subdivided into TGF-beta sensu stricto, bone morphogenetic proteins (BMP) and activins. Although BMP is the best characterized pathway in metazoans, recent findings in molluscs and non-bilateria as well as the analysis of nematode and arthropod genomes, demonstrate the early origin of these distinct subfamilies of ligands, receptors and Smads. This report analyses the large diversity of ligands, receptors and Smads in metazoans from cnidarians and molluscs to mammals. The contribution of new data, mainly from the lophochotrozoan Crassostrea gigas and other organisms on the fringe of the 'branded model organisms', will help us to demonstrate that TGF-betas are probably the most ancestral active cytokines characterized at the molecular level in both Protostome and Deuterostome lineages.

Gene structure and expression of cg-ALR1, a type I activin-like receptor from the bivalve mollusc Crassostrea gigas.

Members of the transforming growth factor beta superfamily of cell signaling polypeptides have attracted much attention because of their ability, from nematodes to mammals, to control cellular functions that in turn, regulate embryo development and tissue homeostasis (the transforming growth factors betas 95 (1990) 419). To understand the divergent evolution of the structures and functions of the transforming growth factor beta receptors (superfamily) we report here the cloning and characterization of an activin-like type I receptor gene from the oyster Crassostrea gigas (cgALR1). This 6 Kb gene encodes a 534 amino acid long protein consisting of a signal peptide, an extracellular ligand binding domain, a transmembrane region and an intracellular domain. The intracellular domain contains sequence motifs such as the GS box and EIF/V and RIKKTL boxes that are thought to be hallmarks of activin type I receptors. The protein sequence shares 67% amino acid identity with other serine/threonine kinase receptors in the most conserved kinase domain and 47-49% similarity with vertebrate type I receptors. The temporal expression pattern of cgALR1 transcripts was examined during early larval developmental stages. To gain insight into evolutionary diversification, phylogenetic analysis as well as an investigation of the genomic structure, including the promoter region of the cgALR1 gene were carried out.