Nucleotide sequence and expression of relaxin-like gonad-stimulating peptide gene in starfish Asterina pectinifera.

Starfish gonad-stimulating substance (GSS) is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Because GSS belongs to the relaxin-like peptide family, we propose renaming for starfish gonadotropic hormone as relaxin-like gonad-stimulating peptide (RGP). This study examined the primary structure and expression regulation of the RGP gene in starfish Asterina pectinifera. RGP consisted of 3896 base pairs (bp) divided over two exons, exon 1 of 208 bp and exon 2 of 2277 bp, and one intron of 1411 bp. Promoter sequences, CAAT and TATA boxes, were present in the 5'-upstream region of the coding DNA sequence of RGP. The transcript was 2485 bases (b) in length. The AAUAAA polyadenylation signal was found in 3'-untranslated region over 2kb away from the stop codon. This showed that only 14% of the RGP mRNA was translated into the peptide, because a size of the open-reading frame was 351 b. Furthermore, an analysis by using real-time quantitative PCR with specific primers for RGP showed that mRNA of RGP was expressed at high levels in the radial nerves. Expression was also observed in the cardiac stomachs, although the level was low, and trace levels were detected in the gonads, pyloric caeca and tube feet. This result suggests that the RGP gene is transcribed mainly in the radial nerves of A. pectinifera.

A method for microinjection of Patiria miniata zygotes.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

Cloning, heterologous expression, and enzymatic characterization of cathepsin L from starfish (Asterina pectinifera).

To determine the role of cathepsin L in Echinoderms, starfish (Asterina pectinifera) cathepsin L (ApCtL) was cloned. The results of RT-PCR analysis indicated that the expression of ApCtL in all of the tissues. The pro-mature enzyme of ApCtL, proApCtL, was expressed in Escherichia coli, and cathepsin activity was detected by cleaving of synthetic fluorogenic peptide substrates and gelatin zymography.

Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation.

Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3'-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.

Modularity of gene-regulatory networks revealed in sea-star development.

Evidence that conserved developmental gene-regulatory networks can change as a unit during deutersostome evolution emerges from a study published in BMC Biology. This shows that genes consistently expressed in anterior brain patterning in hemichordates and chordates are expressed in a similar spatial pattern in another deuterostome, an asteroid echinoderm (sea star), but in a completely different developmental context (the animal-vegetal axis). This observation has implications for hypotheses on the type of development present in the deuterostome common ancestor.

Uncoupling of complex regulatory patterning during evolution of larval development in echinoderms.

BACKGROUND: Conservation of orthologous regulatory gene expression domains, especially along the neuroectodermal anterior-posterior axis, in animals as disparate as flies and vertebrates suggests that common patterning mechanisms have been conserved since the base of Bilateria. The homology of axial patterning is far less clear for the many marine animals that undergo a radical transformation in body plan during metamorphosis. The embryos of these animals are microscopic, feeding within the plankton until they metamorphose into their adult forms. RESULTS: We describe here the localization of 14 transcription factors within the ectoderm during early embryogenesis in Patiria miniata, a sea star with an indirectly developing planktonic bipinnaria larva. We find that the animal-vegetal axis of this very simple embryo is surprisingly well patterned. Furthermore, the patterning that we observe throughout the ectoderm generally corresponds to that of "head/anterior brain" patterning known for hemichordates and vertebrates, which share a common ancestor with the sea star. While we suggest here that aspects of head/anterior brain patterning are generally conserved, we show that another suite of genes involved in retinal determination is absent from the ectoderm of these echinoderms and instead operates within the mesoderm. CONCLUSIONS: Our findings therefore extend, for the first time, evidence of a conserved axial pattering to echinoderm embryos exhibiting maximal indirect development. The dissociation of head/anterior brain patterning from "retinal specification" in echinoderm blastulae might reflect modular changes to a developmental gene regulatory network within the ectoderm that facilitates the evolution of these microscopic larvae.

Involvement of mitogen-activating protein kinase and intracellular pH in the duration of the metaphase I (MI) pause of starfish oocytes after spawning.

The metaphase I (MI) arrest of starfish oocytes is released after spawning. In this study using starfish Asterina pectinifera, the duration of MI after spawning was ~20 min and approximately 30 min in fertilized and unfertilized oocytes, respectively. This prolongation of MI in unfertilized oocytes, referred to as the MI pause, was maintained by mitogen-activating protein kinase (MAPK) as well as low intracellular pH (approximately 7.0). Contrary to previous reports, MI arrest was not maintained by MAPK, since it was inactive in the oocytes arrested at MI in the ovary and activated immediately after spawning. Also, cyclin B was not degraded at pH 6.7 in the cell-free preparation without MAPK activity, whereas it was degraded at pH 7.0, suggesting that MI arrest was solely maintained by lower pH (< 7.0). Normal development occurred when the spawned oocytes were fertilized before the first polar body formation, whereas fertilization after the first polar body formation increased the rate of abnormal development. Thus, due to MI pause and MI arrest, the probability for fertilization before the polar body formation might be increased, leading to normal development.

Transfer of a large gene regulatory apparatus to a new developmental address in echinoid evolution.

Of the five echinoderm classes, only the modern sea urchins (euechinoids) generate a precociously specified embryonic micromere lineage that ingresses before gastrulation and then secretes the biomineral embryonic skeleton. The gene regulatory network (GRN) underlying the specification and differentiation of this lineage is now known. Many of the same differentiation genes as are used in the biomineralization of the embryo skeleton are also used to make the similar biomineral of the spines and test plates of the adult body. Here, we determine the components of the regulatory state upstream of these differentiation genes that are shared between embryonic and adult skeletogenesis. An abrupt "break point" in the micromere GRN is thus revealed, on one side of which most of the regulatory genes are used in both, and on the other side of which the regulatory apparatus is entirely micromere-specific. This reveals the specific linkages of the micromere GRN forged in the evolutionary process by which the skeletogenic gene batteries were caused to be activated in the embryonic micromere lineage. We also show, by comparison with adult skeletogenesis in the sea star, a distant echinoderm outgroup, that the regulatory apparatus responsible for driving the skeletogenic differentiation gene batteries is an ancient pleisiomorphic aspect of the echinoderm-specific regulatory heritage.

Caught in the evolutionary act: precise cis-regulatory basis of difference in the organization of gene networks of sea stars and sea urchins.

The regulatory control of otxbeta1/2 in the sea urchin Strongylocentrotus purpuratus and the sea star Asterina miniata provides an exceptional opportunity to determine the genomic basis of evolutionary change in gene regulatory network (GRN) architectures. Network perturbation analyses in both taxa show that Otx regulates the transcription factors gatae and krox/blimp1 and both of these transcription factors also feed back and regulate otx. The otx gene also autoregulates. This three way interaction is an example of a GRN kernel. It has been conserved for 500 million years since these two taxa last shared a common ancestor. Amid this high level of conservation we show here one significant regulatory change. Tbrain is required for correct otxbeta1/2 expression in the sea star but not in the sea urchin. In sea urchin, tbrain is not co-expressed with otxbeta1/2 and instead has an essential role in specification of the embryonic skeleton. Tbrain in these echinoderms is thus a perfect example of an orthologous gene co-opted for entirely different developmental processes. We isolate and test the sea star otxbeta1/2 cis-regulatory module and demonstrate functional binding sites for each of the predicted inputs, including Tbrain. We compare it to the logic processing operating in the sea urchin otxbeta1/2 cis-regulatory module and present an evolutionary scenario of the change in Tbrain dependence. Finally, inter-specific gene transfer experiments confirm this scenario and demonstrate evolution occurring at the level of sequence changes to the cis-regulatory module.

Complete mitochondrial genome sequences for Crown-of-thorns starfish Acanthaster planci and Acanthaster brevispinus.

BACKGROUND: The crown-of-thorns starfish, Acanthaster planci (L.), has been blamed for coral mortality in a large number of coral reef systems situated in the Indo-Pacific region. Because of its high fecundity and the long duration of the pelagic larval stage, the mechanism of outbreaks may be related to its meta-population dynamics, which should be examined by larval sampling and population genetic analysis. However, A. planci larvae have undistinguished morphological features compared with other asteroid larvae, hence it has been difficult to discriminate A. planci larvae in plankton samples without species-specific markers. Also, no tools are available to reveal the dispersal pathway of A. planci larvae. Therefore the development of highly polymorphic genetic markers has the potential to overcome these difficulties. To obtain genomic information for these purposes, the complete nucleotide sequences of the mitochondrial genome of A. planci and its putative sibling species, A. brevispinus were determined and their characteristics discussed. RESULTS: The complete mtDNA of A. planci and A. brevispinus are 16,234 bp and 16,254 bp in size, respectively. These values fall within the length variation range reported for other metazoan mitochondrial genomes. They contain 13 proteins, 2 rRNA, and 22 tRNA genes and the putative control region in the same order as the asteroid, Asterina pectinifera. The A + T contents of A. planci and A. brevispinus on their L strands that encode the majority of protein-coding genes are 56.3% and 56.4% respectively and are lower than that of A. pectinifera (61.2%). The percent similarity of nucleotide sequences between A. planci and A. brevispinus is found to be highest in the CO2 and CO3 regions (both 90.6%) and lowest in ND2 gene (84.2%) among the 13 protein-coding genes. In the deduced putative amino acid sequences, CO1 is highly conserved (99.2%), and ATP8 apparently evolves faster any of the other protein-coding gene (85.2%). CONCLUSION: The gene arrangement, base composition, codon usage and tRNA structure of A. planci are similar to those of A. brevispinus. However, there are significant variations between A. planci and A. brevispinus. Complete mtDNA sequences are useful for the study of phylogeny, larval detection and population genetics.

Gene-flow patterns in Atlantic and Mediterranean populations of the Lusitanian sea star Asterina gibbosa.

In this study, the population structure of the Lusitanian sea star Asterina gibbosa was assessed using amplified fragment length polymorphism (AFLP). One hundred and twenty-two AFLP loci were analysed in 159 individuals from eight populations from across the species' range and revealed high levels of genetic diversity, with all individuals but two harbouring a unique banding pattern. As reported for other marine invertebrates, we found high levels of genetic differentiation between the Atlantic and Mediterranean basins, suggesting that the Strait of Gibraltar represents a major barrier to dispersal for this sea star. Our assignment studies suggest that, in the Atlantic, a measurable degree of gene flow occurs between populations, which could result in the isolation-by-distance pattern of differentiation observed in this basin. In contrast, no evidence of contemporary gene flow was found in the Mediterranean, suggesting contrasting patterns of dispersal of Asterina gibbosa in the Atlantic and Mediterranean basins.

Expression of AmHNF6, a sea star orthologue of a transcription factor with multiple distinct roles in sea urchin development.

The sea urchin transcription factor SpHNF6 is an early activator of differentiation genes in skeletogenic lineages and regulatory genes in the oral ectoderm. We report here the cloning and the expression of an orthologue of this gene, AmHNF6, from the sea star Asterina miniata. The vertebrate and the echinoderm hnf6 and onecut genes belong to the novel ONECUT homeo domain class of transcription factors. In blastula stage sea star embryos, AmHNF6 is expressed everywhere except around the vegetal pole. As is observed in sea urchin, by the end of gastrulation, the expression of AmHNF6 is distinctly localized to the ciliary bands. This terminal phase of expression has remained unchanged since the divergence of these two taxa half a billion years ago.