Transcription Profiling of Cultured Acropora digitifera Adult Cells Reveals the Existence of Ancestral Genome Regulatory Modules Underlying Pluripotency and Cell Differentiation in Cnidaria.

Due to their pluripotent nature and unlimited cell renewal, stem cells have been proposed as an ideal material for establishing long-term cnidarian cell cultures. However, the lack of unifying principles associated with "stemness" across the phylum complicates stem cells' identification and isolation. Here, we for the first time report gene expression profiles for cultured coral cells, focusing on regulatory gene networks underlying pluripotency and differentiation. Cultures were initiated from Acropora digitifera tip fragments, the fastest growing tissue in Acropora. Overall, in vitro transcription resembled early larvae, overexpressing orthologs of premetazoan and Hydra stem cell markers, and transcripts with roles in cell division, migration, and differentiation. Our results suggest the presence of pluripotent cell types in cultures and indicate the existence of ancestral genome regulatory modules underlying pluripotency and cell differentiation in cnidaria. Cultured cells appear to be synthesizing protein, differentiating, and proliferating.

Phylogenetic analysis of Alphapapillomavirus based on L1, E6 and E7 regions suggests that carcinogenicity and tissue tropism have appeared multiple times during viral evolution.

Members of the Alphapapillomavirus genus are causative agents for cervix cancer and benign lesions in humans. These viruses are classified according to sequence similarities in their L1 region. Yet, viral carcinogenicity has been associated with variations in the proteins encoded by the E6 and E7 genes. In order to relate evolutionary history with origin of carcinogenicity, we performed phylogenetic reconstructions using both nucleotide and predicted amino acid sequences of the L1, E6 and E7 genes. Whilst phylogenetic analysis of L1 reconstructed genus evolutionary history, phylogenies based on E6 and E7 proteins support the idea that mutations at amino acids S/Tx [V/L] (E6) and LxCxE (E7) might be responsible for carcinogenic potential. These findings indicate that virulence within Alphapapillomavirus have appeared multiple times during evolution. Our results reveal that oncogenic potential is not a monophyletic clade-specific adaptation but might be the result of positive selection on random mutations occurring on proteins involved in host infection during viral diversification.

Developmental Progression in the Coral Acropora digitifera Is Controlled by Differential Expression of Distinct Regulatory Gene Networks.

Corals belong to the most basal class of the Phylum Cnidaria, which is considered the sister group of bilaterian animals, and thus have become an emerging model to study the evolution of developmental mechanisms. Although cell renewal, differentiation, and maintenance of pluripotency are cellular events shared by multicellular animals, the cellular basis of these fundamental biological processes are still poorly understood. To understand how changes in gene expression regulate morphogenetic transitions at the base of the eumetazoa, we performed quantitative RNA-seq analysis duringAcropora digitifera's development. We collected embryonic, larval, and adult samples to characterize stage-specific transcription profiles, as well as broad expression patterns. Transcription profiles reconstructed development revealing two main expression clusters. The first cluster grouped blastula and gastrula and the second grouped subsequent developmental time points. Consistently, we observed clear differences in gene expression between early and late developmental transitions, with higher numbers of differentially expressed genes and fold changes around gastrulation. Furthermore, we identified three coexpression clusters that represented discrete gene expression patterns. During early transitions, transcriptional networks seemed to regulate cellular fate and morphogenesis of the larval body. In late transitions, these networks seemed to play important roles preparing planulae for switch in lifestyle and regulation of adult processes. Although developmental progression inA. digitiferais regulated to some extent by differential coexpression of well-defined gene networks, stage-specific transcription profiles appear to be independent entities. While negative regulation of transcription is predominant in early development, cell differentiation was upregulated in larval and adult stages.

The Neuronal Calcium Sensor protein Acrocalcin: a potential target of calmodulin regulation during development in the coral Acropora millepora.

To understand the calcium-mediated signalling pathways underlying settlement and metamorphosis in the Scleractinian coral Acropora millepora, a predicted protein set derived from larval cDNAs was scanned for the presence of EF-hand domains (Pfam Id: PF00036). This approach led to the identification of a canonical calmodulin (AmCaM) protein and an uncharacterised member of the Neuronal Calcium Sensor (NCS) family of proteins known here as Acrocalcin (AmAC). While AmCaM transcripts were present throughout development, AmAC transcripts were not detected prior to gastrulation, after which relatively constant mRNA levels were detected until metamorphosis and settlement. The AmAC protein contains an internal CaM-binding site and was shown to interact in vitro with AmCaM. These results are consistent with the idea that AmAC is a target of AmCaM in vivo, suggesting that this interaction may regulate calcium-dependent processes during the development of Acropora millepora.

Differential expression of three galaxin-related genes during settlement and metamorphosis in the scleractinian coral Acropora millepora.

BACKGROUND: The coral skeleton consists of CaCO3 deposited upon an organic matrix primarily as aragonite. Currently galaxin, from Galaxea fascicularis, is the only soluble protein component of the organic matrix that has been characterized from a coral. Three genes related to galaxin were identified in the coral Acropora millepora. RESULTS: One of the Acropora genes (Amgalaxin) encodes a clear galaxin ortholog, while the others (Amgalaxin-like 1 and Amgalaxin-like 2) encode larger and more divergent proteins. All three proteins are predicted to be extracellular and share common structural features, most notably the presence of repetitive motifs containing dicysteine residues. In situ hybridization reveals distinct, but partially overlapping, spatial expression of the genes in patterns consistent with distinct roles in calcification. Both of the Amgalaxin-like genes are expressed exclusively in the early stages of calcification, while Amgalaxin continues to be expressed in the adult, consistent with the situation in the coral Galaxea. CONCLUSION: Comparisons with molluscs suggest functional convergence in the two groups; lustrin A/pearlin proteins may be the mollusc counterparts of galaxin, whereas the galaxin-like proteins combine characteristics of two distinct proteins involved in mollusc calcification. Database searches indicate that, although sequences with high similarity to the galaxins are restricted to the Scleractinia, more divergent members of this protein family are present in other cnidarians and some other metazoans. We suggest that ancestral galaxins may have been secondarily recruited to roles in calcification in the Triassic, when the Scleractinia first appeared. Understanding the evolution of the broader galaxin family will require wider sampling and expression analysis in a range of cnidarians and other animals.

Identification and gene expression analysis of a taxonomically restricted cysteine-rich protein family in reef-building corals.

The amount of genomic sequence information continues to grow at an exponential rate, while the identification and characterization of genes without known homologs remains a major challenge. For non-model organisms with limited resources for manipulative studies, high-throughput transcriptomic data combined with bioinformatics methods provide a powerful approach to obtain initial insights into the function of unknown genes. In this study, we report the identification and characterization of a novel family of putatively secreted, small, cysteine-rich proteins herein named Small Cysteine-Rich Proteins (SCRiPs). Their discovery in expressed sequence tag (EST) libraries from the coral Montastraea faveolata required the performance of an iterative search strategy based on BLAST and Hidden-Markov-Model algorithms. While a discernible homolog could neither be identified in the genome of the sea anemone Nematostella vectensis, nor in a large EST dataset from the symbiotic sea anemone Aiptasia pallida, we identified SCRiP sequences in multiple scleractinian coral species. Therefore, we postulate that this gene family is an example of lineage-specific gene expansion in reef-building corals. Previously published gene expression microarray data suggest that a sub-group of SCRiPs is highly responsive to thermal stress. Furthermore, data from microarray experiments investigating developmental gene expression in the coral Acropora millepora suggest that different SCRiPs may play distinct roles in the development of corals. The function of these proteins remains to be elucidated, but our results from in silico, transcriptomic, and phylogenetic analyses provide initial insights into the evolution of SCRiPs, a novel, taxonomically restricted gene family that may be responsible for a lineage-specific trait in scleractinian corals.

Gene expression microarray analysis encompassing metamorphosis and the onset of calcification in the scleractinian coral Montastraea faveolata.

Similar to many marine invertebrates, scleractinian corals experience a dramatic morphological transformation, as well as a habitat switch, upon settlement and metamorphosis. At this time, planula larvae transform from non-calcifying, demersal, motile organisms into sessile, calcifying, benthic juvenile polyps. We performed gene expression microarray analyses between planulae, aposymbiotic primary polyps, and symbiotic adult tissue to elucidate the molecular mechanisms underlying coral metamorphosis and early stages of calcification in the Robust/Short clade scleractinian coral Montastraea faveolata. Among the annotated genes, the most abundant upregulated transcripts in the planula stage are involved in protein synthesis, chromatin assembly and mitochondrial metabolism; the polyp stage, morphogenesis, protein catabolism and organic matrix synthesis; and the adult stage, sexual reproduction, stress response and symbiosis. We also present evidence showing that the planula and adult transcriptomes are more similar to each other than to the polyp transcriptome. Our results also point to a large number of uncharacterized adult coral-specific genes likely involved in coral-specific functions such as symbiosis and calcification.