[Show abstract][Hide abstract] ABSTRACT: Summary Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.
[Show abstract][Hide abstract] ABSTRACT: Echinoderms have long served as model organisms for a variety of biological research, especially in the field of developmental biology. Although the genome of the purple sea urchin Strongylocentrotus purpuratus has been sequenced, it is the only echinoderm whose whole genome sequence has been reported. Nevertheless, data is rapidly accumulating on the chromosomes and genomic sequences of all five classes of echinoderms, including the mitochondrial genomes and Hox genes. This blossoming new data will be essential for estimating the phylogenetic relationships among echinoderms, and also to examine the underlying mechanisms by which the diverse morphologies of echinoderms have arisen.
Current Genomics 04/2012; 13(2):134-43. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sea urchin petalloid coelomocytes effectuate the clotting pathway by undergoing a rapid and dynamic cellular transformation that leads to cellular adhesion and wounds closure. We have identified high levels of activity of arylsulfatase (Ars) associated with coelomocytes of the sea urchin Lytechinus variegatus (Lamarck, 1816). Ars activity was extracted from clotted coelomocytes with EDTA and showed high levels of activity up to a 1:100 dilution. Clot formation from isolated coelomic fluid was significantly inhibited by the ARS inhibitor, p-nitrophenyl phosphate. Ars activity was collected by 80% ethanol precipitation, a diagnostic test previously used in Ars isolation. Cellular extraction studies in the presence and absence of the non-ionic detergent Triton X-100 indicated that some Ars activity was present intracellularly, possibly in intracellular membrane-bound compartments, however the majority of Ars activity was extracted from the extracellular coelomocyte membrane. Polyclonal anti-sea urchin embryo Ars antibodies recognized a single protein band with an approximate molecular weight of 75 kDa on western blots. Immunofluorescence using the anti-sea urchin Ars antibody revealed an intracellular and extracellular staining of Ars in both petalloid and filopodial coelomocytes. Taken together, these data indicate that coelomocyte Ars might be involved in cell-to-cell crosslinking of surface sulfated polysaccharides vital for clot formation.
[Show abstract][Hide abstract] ABSTRACT: In mouse Otx2 plays essential roles in anterior-posterior axis formation and head development in anterior visceral endoderm and anterior mesendoderm. The Otx2 expression in these sites is regulated by VE and CM enhancers at the 5' proximal to the translation start site, and we proposed that these enhancers would have been established in ancestral sarcoptergians after divergence from actinopterigians for the use of Otx2 as the head organizer gene (Kurokawa et al., 2010). This would make doubtful an earlier proposal of ours that a 1.1 kb fragment located at +14.4 to +15.5 kb 3' (3'En) of fugu Otx2a gene harbors enhancers phylogenetically and functionally homologous to mouse VE and CM enhancers (Kimura-Yoshida et al., 2007). In the present study, we demonstrate that fugu Otx2a is not expressed in the dorsal margin of blastoderm, shield and early anterior mesendoderm, and that the fugu Otx2a 3'En do not exhibit activities at these sites of fugu embryos. We conclude that the fugu Otx2a 3'En does not harbor an organizer enhancer, but encodes an enhancer for the expression in later anterior mesendodermal tissues. Instead, in fugu embryos Otx2b is expressed in the dorsal margin of blastoderm at blastula stage and shield at 50% epiboly, and this expression is directed by an enhancer, 5'En, located at -1000 to -800 bp, which is uniquely conserved among teleost Otx2b orthologues.
Mechanisms of development 11/2011; 128(11-12):653-61. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Ars insulator is a boundary element identified in the upstream region of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, and possesses the ability to both block enhancer-promoter communications and protect transgenes from silent chromatin. To understand the molecular mechanism of the Ars insulator, we investigated the correlation between chromatin structure, DNA structure and insulator activity. Nuclease digestion of nuclei isolated from sea urchin embryos revealed the presence of a nuclease-hypersensitive site within the Ars insulator. Analysis of micrococcal nuclease-sensitive sites in the Ars insulator, reconstituted with nucleosomes, showed the exclusion of nucleosomes from the central AT-rich region. Furthermore, the central AT-rich region in naked DNA was sensitive to nucleotide base modification by diethylpyrocarbonate (DEPC). These observations suggest that non-B-DNA structures in the central AT-rich region may inhibit nucleosomal formation, which leads to nuclease hypersensitivity. Furthermore, comparison of nucleotide sequences between the HpArs gene and its ortholog in Strongylocentrotus purpuratus revealed that the central AT-rich region of the Ars insulator is conserved, and this conserved region showed significant enhancer blocking activity. These results suggest that the central AT-rich nucleosome-free region plays an important role in the function of the Ars insulator.
Journal of Biochemistry 09/2011; 151(1):75-87. · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The stalked crinoid, Metacrinus rotundus, is one of the most basal extant echinoderms. Here, we show the expression patterns of Six3, Pax6, and Otx in the early development of M. rotundus. All three genes are highly expressed in stages from the gastrula to the auricularia larval stage. Ectodermal expression of MrOtx appears to be correlated with development of the ciliary band. These three genes are expressed sequentially along the embryonic body axis in the anterior and middle walls of the archenteron in the order of MrPax6, MrSix3, and MrOtx. The anterior, middle, and posterior parts of the archenteron in the late gastrula differentiate into the axo-hydrocoel, the enteric sac, and somatocoels at later stages, respectively. The three genes are expressed sequentially from the tip of the axo-hydrocoel to the bottom of enteric sac in the order of MrSix3, MrPax6, and MrOtx at the later stages. This suggests that these genes are involved in patterning of the larval endo-mesoderm in stalked crinoids. The present results suggest that radical alterations have occurred in the expression and function of homeobox genes in basal echinoderms.
[Show abstract][Hide abstract] ABSTRACT: phiC31 integrase-based gene delivery has been developed. However, the expression of integrated transgenes is often suppressed by a negative position effect. To improve this system, we constructed a new phiC31 integrase-based expression vector that contains attB, an expression unit placed in reverse orientation with two sea urchin-derived Ars-insulators to avoid position effects. In vitro and in vivo transfection experiments revealed that this new system produces higher levels of transgene expression as well as continued gene expression. Thus, the present gene delivery system will facilitate reverse genetics-based molecular biological studies.
New Biotechnology 11/2010; 28(4):312-9. · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Crinoids have strong regenerative capability and rapidly restore their lost body parts such as arms. We observed the regeneration process of arms of the feather star (stalkless crinoid), Oxycomanthus japonicus, and divided the process into 10 stages. We clarify the position at which the oral and aboral epidermis adhere in wound closure and track the oral-aboral boundary in the regenerate during the entire process of regeneration. We suggest that the concepts of distalization and intercalation, which are proposed to understand animal regeneration integrally, are also applicable to arm regeneration of the feather star. In addition, we clarify that pinnules, appendages extending from the sides of an arm, start to grow in the oral region of the regenerating arm even though a complete pinnule has an oral-aboral axis. The mode of morphogenesis of pinnules in arm regeneration suggests that the oral region functions as the primary patterning tissue for pinnules.
[Show abstract][Hide abstract] ABSTRACT: Convergent evolution of echinoderm pluteus larva was examined from the standpoint of functional evolution of a transcription factor Ets1/2. In sea urchins, Ets1/2 plays a central role in the differentiation of larval skeletogenic mesenchyme cells. In addition, Ets1/2 is suggested to be involved in adult skeletogenesis. Conversely, in starfish, although no skeletogenic cells differentiate during larval development, Ets1/2 is also expressed in the larval mesoderm. Here, we confirmed that the starfish Ets1/2 is indispensable for the differentiation of the larval mesoderm. This result led us to assume that, in the common ancestors of echinoderms, Ets1/2 activates the transcription of distinct gene sets, one for the differentiation of the larval mesoderm and the other for the development of the adult skeleton. Thus, the acquisition of the larval skeleton involved target switching of Ets1/2. Specifically, in the sea urchin lineage, Ets1/2 activated a downstream target gene set for skeletogenesis during larval development in addition to a mesoderm target set. We examined whether this heterochronic activation of the skeletogenic target set was achieved by the molecular evolution of the Ets1/2 transcription factor itself. We tested whether starfish Ets1/2 induced skeletogenesis when injected into sea urchin eggs. We found that, in addition to ectopic induction of mesenchyme cells, starfish Ets1/2 can activate some parts of the skeletogenic pathway in these mesenchyme cells. Thus, we suggest that the nature of the transcription factor Ets1/2 did not change, but rather that some unidentified co-factor(s) for Ets1/2 may distinguish between targets for the larval mesoderm and for skeletogenesis. Identification of the co-factor(s) will be key to understanding the molecular evolution underlying the evolution of the pluteus larvae.
Development Genes and Evolution 09/2010; 220(3-4):107-15. · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The large micromeres of the 32-cell stage of sea urchin embryos are autonomously specified and differentiate into primary mesenchyme cells (PMCs), giving rise to the skeletogenic cells. We previously demonstrated that HpEts, an ets-related transcription factor, plays an essential role in the specification of PMCs in sea urchin embryos. In order to clarify the function of HpEts in the gene regulatory network involved in PMC specification, we analyzed the zygotic expression pattern and the cis-regulatory region of HpEts, and examined the activity of the HpEts protein as a transcription factor. Intron-based PCR reveals that zygotic expression of HpEts starts at the cleavage stage, and that the rate of transcription reaches maximum at the unhatched blastula stage. A series of progressive deletions of the fragments from -4.2 kbp to +1206 bp of the HpEts, which directs PMC-specific expression, caused a gradual decrease in the specificity, implying that coordination of several cis-regulatory elements regulates the expression in PMCs. A minimum cis-element required for the temporal expression is located within a 10 bp from -243 bp to -234 bp. The HpEts protein remains in the cytoplasm of entire embryonic cells in the cleavage stage. At the unhatched blastula stage, the HpEts protein translocates into the nucleus in presumptive PMCs. Transactivation assays demonstrate that the HpEts protein activates a promoter of Spicule Matrix Protein 50 (SM50), which is a target of HpEts, which binds to the regulatory region of SM50.
[Show abstract][Hide abstract] ABSTRACT: Cell surface heparan sulfate proteoglycans (HSPGs) play significant roles in the regulation of developmental signaling, including vascular endothelial growth factor (VEGF), fibroblast growth factor, Wnt and bone morphogenetic protein signaling, through modification of their sulfation patterns. Recent studies have revealed that one of the functions of heparan sulfate 6-O-endosulfatase (Sulf) is to remove the sulfate from the 6-O position of HSPGs at the cell surface, thereby regulating the binding activities of heparan sulfate (HS) chains to numerous ligands and receptors in animal species. In this study, we focused on the sea urchin Hemicentrotus pulcherrimus homolog of Sulf (HpSulf), and analyzed its expression pattern and functions during development. HpSulf protein was present throughout development and localized at cell surface of all blastomeres. In addition, the HS-specific epitope 10E4 was detected at the cell surface and partially colocalized with HpSulf. Knockdown of HpSulf using morpholino antisense oligonucleotides (MO) caused abnormal morphogenesis, and the development of MO-injected embryos was arrested before the hatched blastula stage, indicating that HpSulf is necessary for the early developmental process of sea urchin embryos. Furthermore, we found that injection of HpSulf mRNA suppressed the abnormal skeleton induced by overexpression of HpVEGF mRNA, whereas injection of an inactive form of HpSulf mRNA, containing mutated cysteines in the sulfatase domain, did not have this effect. Taken together, these results suggest that HpSulf is involved in the regulation of various signal transductions, including VEGF signaling, during sea urchin development.
Mechanisms of development 04/2010; 127(3-4):235-45. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inherited deficiency for arylsulfatase (Ars) leads to lysosomal storage of sulfated compounds and to serious diseases such as growth retardation, heart failure, and demyelination in the central nervous system. Ars has been regarded as a lysosomal enzyme because of its hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of its enzymatic activity. We previously demonstrated that a large portion of the mammalian arylsulfatase A (ArsA) protein exists on the cell surface of vascular endothelial cells, suggesting that ArsA plays a role in the components of the extracellular matrix. Here we show that ArsA functions as a substrate on which cells adhere and form protrusions. Coating culture plates with recombinant mouse ArsA (rmArsA) stimulates adhesion of human microvascular endothelial cells to the plate followed by the formation of cell protrusions as well as lamellipodia. rmArsA affects the architecture of the cytoskeleton, with a high density of actin filaments localized to peripheral regions of the cells and the extension of bundles of microtubules into the tips of cellular protrusions. rmArsA also affects the distribution pattern of the cell adhesion-associated proteins, integrin α2β1, and paxillin. rmArsA seems to modulate signaling of basic fibroblast growth factor (bFGF) stimulating cytoskeletal rearrangement. We also show that rmArsA tightly binds to sulfated polysaccharides. We suggest that mammalian ArsA plays a role as a novel component of the extracellular matrix. This viewpoint of Ars could be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes.
Connective tissue research 03/2010; 51(5):388-96. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regeneration is a biological phenomenon that occurs in a wide range of animals, and is considered to involve different types of cells including those that are considered to be stem cells. Among the echinoderms, which is a phylum with many regenerating members, crinoids (feather stars and sea lilies) are known to possess high potential of regeneration and are able to regenerate most of their organs. In particular, arm regeneration has been studied using the feather star. During regeneration, coelomocytes and amoebocytes originating from the coelomic canal and the brachial nerve, respectively, migrate to the distal wound area and are involved in the regenerative process. A blastema is formed at the regenerating tip and is derived from migratory amoebocytes. On the other hand, migratory coelomocytes contribute to regenerate the coelomic system. Cells proliferate at the blastema, coelomic canals and brachial nerve. Since the migrating cells differentiate into new structures of the arm, they are considered presumably undifferentiated multipotent stem cells. To deepen our understanding of stem cells in general, we may benefit from an approach from a comparative point of view. Further molecular analyses would increase our knowledge of stem cells in crinoids and allow comparative studies to be possible.
[Show abstract][Hide abstract] ABSTRACT: The study of marine invertebrates is useful in various biological research fields. However, genetic analyses of these animals are limited, mainly due to difficulties in culturing them, and the genetic resources of marine invertebrates have not been organized. Recently, advances have been made in the study of two deuterostomes, an ascidian Ciona intestinalis and a feather star Oxycomanthus japonicus. The draft genome sequence of Ciona intestinalis has been determined, and its compact genome, which has less redundancy of genes compared with vertebrates, provides us with a useful experimental system for analyzing the functions of genes during development. The life cycle of Ciona intestinalis is approximately 2-3 months, and the genetic techniques including a perfect inland culture system, germline transformation with a transposon Minos, enhancer detection and insertional mutagenesis, have been established. The feather star Oxycomanthus japonicus conserves the characteristics of the basic echinoderm body plan with a segmented mesoderm, which is a fascinating characteristic for understanding the evolution of echinoderms. Oxycomanthus japonicus shows strong regeneration ability and is a suitable subject for analysis of the mechanisms of regeneration. In consideration of these features, the National BioResource Project (NBRP) has started to support the supply of wild-types, transgenic lines and inbred lines of Ciona intestinalis and Oxycomanthus japonicus.
[Show abstract][Hide abstract] ABSTRACT: In sea urchin embryos, Notch signaling is required to segregate non-skeletogenic mesoderm from early endomesoderm, and is involved in endoderm development. To further investigate the role of Notch signaling in the endoderm cell lineage, we cloned a cDNA for the Hemicentrotus pulcherrimus ortholog of Suppressor of Hairless (HpSu(H)), which is a major mediator of the Notch signaling pathway, examined the expression during development and performed a functional analysis. HpSu(H) mRNA was ubiquitously expressed up to the unhatched blastula stage, and expression was exclusively detected in the vegetal plate region from the hatched blastula stage and then in the archenteron at the gastrula stage. Perturbation of HpSu(H) by injection of the dominant negative form of HpSu(H) (dn-HpSu(H)) mRNA into fertilized eggs led to the disappearance of secondary mesenchyme cells at the tip of the archenteron in the gastrula and pigment cells in the pluteus larva, confirming that Notch signaling is required for non-skeletogenic me soderm specification. In addition, injection of relatively high amounts of dn-HpSu(H) mRNA caused a defect or atrophy of the foregut in the archenteron at the pluteus stage. This result strongly suggests that Notch signaling is involved in foregut development during sea urchin development.
[Show abstract][Hide abstract] ABSTRACT: Arylsulfatase A (ARSA) and B (ARSB) have been regarded as lysosomal enzymes because of their hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of their enzymatic activity. Using sea urchin embryos, we previously demonstrated that the bulk of ARS is located on the cell surface of the epithelium, colocalizing with sulfated polysaccharides, and that it does not exhibit enzymatic activity. To examine whether ARSA and ARSB exist on the cell surface in mammalian tissues, we raised antibodies against ARSA and ARSB and examined immunohistochemically their localization in the liver using light and electron microscopy. Here we show that mammalian ARSA and ARSB exist on the cell surface of sinusoidal endothelial cells, hepatocytes, and sinusoidal macrophages (Kupffer cells), as well as in the lysosome. They are also colocalized with heparan sulfate proteoglycan. These results suggest that ARSA and ARSB also may function in the cell surface of mammals. This is the first report to show cell-surface localization of ARS in mammalian somatic cells. The extracellular localization of ARS will provide new insight for human ARS deficiency disorders, such as metachromatic leukodystrophy and mucopolysaccharidosis VI.
Medical Molecular Morphology 07/2009; 42(2):63-9. · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arylsulfatases (Arses) have been regarded as lysosomal enzymes because of their hydrolytic activities on synthetic aromatic substrates and their lysosomal localization of their enzymatic activities. Using sea urchin embryos, we previously demonstrated that the bulk of Hemicentrotus Ars (HpArs) does not exhibit enzyme activity and is located on the apical surface of the epithelial cells co-localizing with sulfated polysaccharides. Here we show that HpArs strongly binds to sulfated polysaccharides and that repression of the synthesis by HpArs-morpholino results in retardation of gastrulation in the sea urchin embryo. Accumulation of HpArs protein and sulfated polysaccharides on the apical surface of the epithelial cells in sea urchin larvae is repressed by treatment with beta-aminopropionitrile (BAPN), suggesting that deposition of HpArs and sulfated polysaccharides is dependent on the crosslinking of proteins such as collagen-like molecules. We suggest that HpArs functions by binding to components of the extracellular matrix.
Development Genes and Evolution 06/2009; 219(6):281-8. · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression of the LIM homeobox gene lhx1 (lim1) is specific to the vertebrate gastrula organizer. Lhx1 functions as a transcriptional regulatory core protein to exert ;organizer' activity in Xenopus embryos. Its ancient paralog, lhx3 (lim3), is expressed around the blastopore in amphioxus and ascidian, but not vertebrate, gastrulae. These two genes are thus implicated in organizer evolution, and we addressed the evolutionary origins of their blastoporal expression and organizer activity. Gene expression analysis of organisms ranging from cnidarians to chordates suggests that blastoporal expression has its evolutionary root in or before the ancestral eumetazoan for lhx1, but possibly in the ancestral chordate for lhx3, and that in the ascidian lineage, blastoporal expression of lhx1 ceased, whereas endodermal expression of lhx3 has persisted. Analysis of organizer activity using Xenopus embryos suggests that a co-factor of LIM homeodomain proteins, Ldb, has a conserved function in eumetazoans to activate Lhx1, but that Lhx1 acquired organizer activity in the bilaterian lineage, Lhx3 acquired organizer activity in the deuterostome lineage and ascidian Lhx3 acquired a specific transactivation domain to confer organizer activity on this molecule. Knockdown analysis using cnidarian embryos suggests that Lhx1 is required for chordin expression in the blastoporal region. These data suggest that Lhx1 has been playing fundamental roles in the blastoporal region since the ancestral eumetazoan arose, that it contributed as an 'original organizer gene' to the evolution of the vertebrate gastrula organizer, and that Lhx3 could be involved in the establishment of organizer gene networks.
Development 06/2009; 136(12):2005-14. · 6.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Blastomeres of the 16-cell stage embryos of the sea urchin, Hemicentrotus pulcherrimus, were separated by an elutriator. By differential display, several RNA species that are enriched in micromeres are detected and their cDNA was cloned. One of the cloned cDNA encodes mt 12S rRNA. cDNA for mt 16S rRNA was also cloned from the cDNA library of unfertilized eggs. Two mt rRNAs contain poly(A) tails in their 3′ ends. Both mt rRNAs distribute asymmetrically along a vegetal-animal axis of the 16-cell embryos and are enriched in micromeres, and this is also confirmed by whole mount in situ hybridization as well as electron microscopic in situ hybridization. As development proceeds, these mt rRNAs become more enriched in small micromeres. Results of electron microscopical in situ hybridization reveal both mt rRNAs localize extramitochondrially. Though at present we have no evidence on the role of the extramitochondrial mt rRNAs in sea urchin development, it is speculated considering roles of extramitochondrial mt 16S rRNA in Drosophila development that extramitochondrial mt rRNA may be implicated in development of sea urchin embryos.
[Show abstract][Hide abstract] ABSTRACT: Crinoids, including feather stars, are the most basal group among extant echinoderm classes and share a basic body plan. In spite of their importance for evolutionary developmental study, information on the development of crinoids has been limited, because there are not many species whose spawning season is known, and artificial spawning is impossible. Therefore, it is not easy to obtain fertilized eggs of crinoids. We have observed the spawning and development of the feather star Oxycomanthus japonicus for 7 years. We have established a cultivation system that has enabled us to culture large numbers of O. japonicus from eggs through to sexually mature adults. In the present study, we show that (1) individuals take 2 years to reach sexual maturity; (2) the skeleton of the theca of a stalked juvenile consists of five orals, five basals, five radials, five infrabasals, and an anal plate; and (3) the onset of spawning has shifted by about two weeks since 60 years ago. Our cultivation system can provide enough embryos, larvae, juveniles, and adults for further experiments, extending the possibilities for crinoid research.