Mayuko Hamada

Using the Acropora digitifera genome to understand coral responses to environmental change

Despite the enormous ecological and economic importance of coral reefs, the keystone organisms in their establishment, the scleractinian corals, increasingly face a range of anthropogenic challenges including ocean acidification and seawater temperature rise. To understand better the molecular mechanisms underlying coral biology, here we decoded the approximately 420-megabase genome of Acropora digitifera using next-generation sequencing technology. This genome contains approximately 23,700 gene models. Molecular phylogenetics indicate that the coral and the sea anemone Nematostella vectensis diverged approximately 500 million years ago, considerably earlier than the time over which modern corals are represented in the fossil record (∼240 million years ago). Despite the long evolutionary history of the endosymbiosis, no evidence was found for horizontal transfer of genes from symbiont to host. However, unlike several other corals, Acropora seems to lack an enzyme essential for cysteine biosynthesis, implying dependency of this coral on its symbionts for this amino acid. Corals inhabit environments where they are frequently exposed to high levels of solar radiation, and analysis of the Acropora genome data indicates that the coral host can independently carry out de novo synthesis of mycosporine-like amino acids, which are potent ultraviolet-protective compounds. In addition, the coral innate immunity repertoire is notably more complex than that of the sea anemone, indicating that some of these genes may have roles in symbiosis or coloniality. A number of genes with putative roles in calcification were identified, and several of these are restricted to corals. The coral genome provides a platform for understanding the molecular basis of symbiosis and responses to environmental changes.

The Complex NOD-Like Receptor Repertoire of the Coral Acropora digitifera Includes Novel Domain Combinations

Innate immunity in corals is of special interest not only in the context of self-defense but also in relation to the establishment and collapse of their obligate symbiosis with dinoflagellates of the genus Symbiodinium. In innate immunity system of vertebrates, approximately 20 tripartite nucleotide oligomerization domain (NOD)-like receptor proteins that are defined by the presence of a NAIP, CIIA, HET-E and TP1 (NACHT) domain, a C-terminal leucine-rich repeat (LRR) domain, and one of three types of N-terminal effector domain, are known to function as the primary intracellular pattern recognition molecules. Surveying the coral genome revealed not only a larger number of NACHT- and related domain nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)-encoding loci (~500) than in other metazoans but also surprising diversity of domain combinations among the coral NACHT/NB-ARC-containing proteins; N-terminal effector domains included the apoptosis-related domains caspase recruitment domain (CARD), death effector domain (DED), and Death, and C-terminal repeat domains included LRRs, tetratricopeptide repeats, ankyrin repeats, and WD40 repeats. Many of the predicted coral proteins that contain a NACHT/NB-ARC domain also contain a glycosyl transferase group 1 domain, a novel domain combination first found in metazoans. Phylogenetic analyses suggest that the NACHT/NB-ARC domain inventories of various metazoan lineages, including corals, are largely products of lineage-specific expansions. Many of the NACHT/NB-ARC loci are organized in pairs or triplets in the Acropora genome, suggesting that the large coral NACHT/NB-ARC repertoire has been generated at least in part by tandem duplication. In addition, shuffling of N-terminal effector domains may have occurred after expansions of specific NACHT/NB-ARC-repeat domain types. These results illustrate the extraordinary complexity of the innate immune repertoire of corals, which may in part reflect adaptive evolution to a symbiotic lifestyle in a uniquely complex and challenging environment.

A genomewide analysis of genes for the heat shock protein 70 chaperone system in the ascidian Ciona intestinalis.

Abstract Molecular chaperones play crucial roles in various aspects of the biogenesis and maintenance of proteins in the cell. The heat shock protein 70 (HSP70) chaperone system, in which HSP70 proteins act as chaperones, is one of the major molecular chaperone systems conserved among a variety of organisms. To shed light on the evolutionary history of the constituents of the chordate HSP70 chaperone system and to identify all of the components of the HSP70 chaperone system in ascidians, we carried out a comprehensive survey for HSP70s and their cochaperones in the genome of Ciona intestinalis. We characterized all members of the Ciona HSP70 superfamily, J-proteins, BAG family, and some other types of cochaperones. The Ciona genome contains 8 members of the HSP70 superfamily, all of which have human and protostome counterparts. Members of the STCH subfamily of the HSP70 family and members of the HSPA14 subfamily of the HSP110 family are conserved between humans and protostomes but were not found in Ciona. The Ciona genome encodes 36 J-proteins, 32 of which belong to groups conserved in humans and protostomes. Three proteins seem to be unique to Ciona. J-proteins of the RBJ group are conserved between humans and Ciona but were not found in protostomes, whereas J-proteins of the DNAJC14, ZCSL3, FLJ13236, and C21orf55 groups are conserved between humans and protostomes but were not found in Ciona. J-proteins of the sacsin group seem to be specific to vertebrates. There is also a J-like protein without a conserved HPD tripeptide motif in the Ciona genome. The Ciona genome encodes 3 types of BAG family proteins, all of which have human and protostome counterparts (BAG1, BAG3, and BAT3). BAG2 group is conserved between humans and protostomes but was not found in Ciona, and BAG4 and BAG5 groups seem to be specific to vertebrates. Members for SIL1, UBQLN, UBADC1, TIMM44, GRPEL, and Magmas groups, which are conserved between humans and protostomes, were also found in Ciona. No Ciona member was retrieved for HSPBP1 group, which is conserved between humans and protostomes. For several groups of the HSP70 superfamily, J-proteins, and other types of cochaperones, multiple members in humans are represented by a single counterpart in Ciona. These results show that genes of the HSP70 chaperone system can be distinguished into groups that are shared by vertebrates, Ciona, and protostomes, ones shared by vertebrates and protostomes, ones shared by vertebrates and Ciona, and ones specific to vertebrates, Ciona, or protostomes. These results also demonstrate that the components of the HSP70 chaperone system in Ciona are similar to but simpler than those in humans and suggest that changes of the genome in the lineage leading to humans after the separation from that leading to Ciona increased the number and diversity of members of the HSP70 chaperone system. Changes of the genome in the lineage leading to Ciona also seem to have made the HSP70 chaperone system in this species slightly simpler than that in the common ancestor of humans and Ciona.

Expression of neuropeptide- and hormone-encoding genes in the Ciona intestinalis larval brain

Despite containing only approximately 330 cells, the central nervous system (CNS) of Ciona intestinalis larvae has an architecture that is similar to the vertebrate CNS. Although only vertebrates have a distinct hypothalamus-the source of numerous neurohormone peptides that play pivotal roles in the development, function, and maintenance of various neuronal and endocrine systems, it is suggested that the Ciona brain contains a region that corresponds to the vertebrate hypothalamus. To identify genes expressed in the brain, we isolated brain vesicles using transgenic embryos carrying Ci-β-tubulin(promoter)::Kaede, which resulted in robust Kaede expression in the larval CNS. The associated transcriptome was investigated using microarray analysis. We identified 565 genes that were preferentially expressed in the larval brain. Among these genes, 11 encoded neurohormone peptides including such hypothalamic peptides as gonadotropin-releasing hormone and oxytocin/vasopressin. Six of the identified peptide genes had not been previously described. We also found that genes encoding receptors for some of the peptides were expressed in the brain. Interestingly, whole-mount in situ hybridization showed that most of the peptide genes were expressed in the ventral brain. This catalog of the genes expressed in the larval brain should help elucidate the evolution, development, and functioning of the chordate brain.

Novel genes involved in canonical Wnt/β-catenin signaling pathway in early Ciona intestinalis embryos

We report here characterization of five genes for novel components of the canonical Wnt/β‐catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss‐of‐function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci‐PGAP1, a Ciona orthologue of human PGAP1, which encodes GPI (glycosylphosphatidylinositol) inositol‐deacylase, Ci‐ZF278, a gene encoding a C2H2 zinc‐finger protein, Ci‐C10orf11, a Ciona orthologue of human C10orf11 that encodes a protein with leucine‐rich repeats, Ci‐Spatial/C4orf17, a single counterpart for two human genes Spatial and C4orf17, and Ci‐FLJ10634, a Ciona orthologue of human FLJ10634 that encodes a member of the J‐protein family. Knockdown of each of the genes mimicked β‐catenin knockdown and resulted in suppression of the expression of β‐catenin downstream genes (Ci‐FoxD, Ci‐Lhx3, Ci‐Otx and Ci‐Fgf9/16/20) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild‐type, of Ci‐β‐catenin. Dosage‐sensitive interactions were found between Ci‐β‐catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci‐β‐catenin in the Wnt/β‐catenin signaling pathway in early Ciona embryos.

How do environmental factors influence life cycles and development? An experimental framework for early‐diverging metazoans

Ecological developmental biology (eco‐devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre‐date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans.

The repertoire of chemical defense genes in the coral Acropora digitifera genome.

Scleractinian corals are of fundamental ecological significance in tropical and sub-tropical shallow water. This ecological success is attributed to their ability of formation of obligate endosymbioses with dinoflagellates of the genus Symbiodinium. Nevertheless, approximately one-third of reef-building coral species are critically endangered and the remainder are under threat from the effects of climate change and local impacts. Molecular and cellular mechanisms involved in stress responses and the establishment and collapse of the symbiosis are therefore an urgent subject of research. Metazoans possess large numbers of genes that participate in response to environmental stressors, and chemical defense genes included P450 and other oxidases, various conjugating enzymes, ATP-dependent efflux transporters, oxidative detoxification proteins, as well as transcription factors that regulate these genes. Here we searched those genes in recently decoded the coral Acropora digitifera genome. We found that this genome contains a set of chemical defense genes in numbers comparable with other cnidarians and metazoans and that there are some lineagespecific gene family expansions in the coral genome. These provide information for future research into molecular mechanisms involved in coral stress responses.

Signals from primary mesenchyme cells regulate endoderm differentiation in the sea urchin embryo

Primary mesenchyme cells (PMC), the skeletogenic cells derived from the micromeres of the sea urchin embryo, are involved in the differentiation of the gut. When PMC were deleted from the mesenchyme blastula, both formation of the constrictions in the gut and expression of endoderm‐specific alkaline phosphatase were significantly delayed. Therefore, the correct timing of gut differentiation depends on the existence of PMC, probably via a type of promotive signal. To date, the only role of PMC in other tissue differentiation has been a suppressive signal for the conversion of secondary mesenchyme cells (SMC) into skeletogenic cells. The present experiments using PMC ablation and transplantation showed that both signaling processes occurred in the same short period during gastrulation, but the embryos kept their competence for gut differentiation until a later stage. Further investigations indicated that conversion of SMC did not cause delay in gut differentiation and that SMC did not mediate the PMC signal to the endoderm. Therefore, the effect of PMC on gut differentiation could be a new role that is independent of the suppressive effect for SMC conversion.

Hox10-regulated endodermal cell migration is essential for development of the ascidian intestine

Hox cluster genes play crucial roles in development of the metazoan antero-posterior axis. Functions of Hox genes in patterning the central nervous system and limb buds are well known. They are also expressed in chordate endodermal tissues, where their roles in endodermal development are still poorly understood. In the invertebrate chordate, Ciona intestinalis, endodermal tissues are in a premature state during the larval stage, and they differentiate into the digestive tract during metamorphosis. In this study, we showed that disruption of a Hox gene, Ci-Hox10, prevented intestinal formation. Ci-Hox10-knock-down larvae displayed defective migration of endodermal strand cells. Formation of a protrusion, which is important for cell migration, was disrupted in these cells. The collagen type IX gene is a downstream target of Ci-Hox10, and is negatively regulated by Ci-Hox10 and a matrix metalloproteinase ortholog, prior to endodermal cell migration. Inhibition of this regulation prevented cellular migration. These results suggest that Ci-Hox10 regulates endodermal strand cell migration by forming a protrusion and by reconstructing the extracellular matrix.

Novel genes involved in Ciona intestinalis embryogenesis: Characterization of gene knockdown embryos

The sequenced genome of the urochordate ascidian Ciona intestinalis contains nearly 2,500 genes that have vertebrate homologues, but their functions are as yet unknown. To identify novel genes involved in early chordates embryogenesis, we previously screened 200 Ciona genes by knockdown experiments using specific morpholino oligonucleotides and found that suppression of the translation of 40 genes caused embryonic defects (Yamada et al. [ 2003 ] Development 130:6485–6495). We have since examined an additional 304 genes, that is, screening 504 genes overall, and a total of 111 genes showed morphological defects when gene function was suppressed. We further examined the role of these genes in the differentiation of six major tissues of the embryo: endoderm, muscle, epidermis, neural tissue, mesenchyme, and notochord. Based on the similarity of phenotypes of gene knockdown embryos, genes were categorized into several groups, with the suggestion that the genes within a given group are involved in similar developmental processes. For example, five were shown to be novel genes that are likely involved in β‐catenin–mediated endoderm formation. The type of large‐scale screening used is, therefore, a powerful approach to identify novel genes with significant developmental functions, the details of which will be determined in future studies. Developmental Dynamics 236:1820–1831, 2007.