Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus)

A systematic search in the available scaffolds of the Strongylocentrotus purpuratus genome has revealed that this sea urchin has 11 members of the ets gene family. A phylogenetic analysis of these genes showed that almost all vertebrate ets subfamilies, with the exception of one, so far found only in mammals, are each represented by one orthologous sea urchin gene. The temporal and spatial expression of the identified ETS factors was also analyzed during embryogenesis. Five ets genes (Sp-Ets1/2, Sp-Tel, Sp-Pea, Sp-Ets4, Sp-Erf) are also maternally expressed. Three genes (Sp-Elk, Sp-Elf, Sp-Erf) are ubiquitously expressed during embryogenesis, while two others (Sp-Gabp, Sp-Pu.1) are not transcribed until late larval stages. Remarkably, five of the nine sea urchin ets genes expressed during embryogenesis are exclusively (Sp-Ets1/2, Sp-Erg, Sp-Ese) or additionally (Sp-Tel, Sp-Pea) expressed in mesenchyme cells and/or their progenitors. Functional analysis of Sp-Ets1/2 has previously demonstrated an essential role of this gene in the specification of the skeletogenic mesenchyme lineage. The dynamic, and in some cases overlapping and/or unique, developmental expression pattern of the latter five genes suggests a complex, non-redundant function for ETS factors in sea urchin mesenchyme formation and differentiation.     

Opsins and clusters of sensory G-protein-coupled receptors in the sea urchin genome

Rhodopsin-type G-protein-coupled receptors (GPCRs) contribute the majority of sensory receptors in vertebrates. With 979 members, they form the largest GPCR family in the sequenced sea urchin genome, constituting more than 3% of all predicted genes. The sea urchin genome encodes at least six Opsin proteins. Of these, one rhabdomeric, one ciliary and two G(o)-type Opsins can be assigned to ancient bilaterian Opsin subfamilies. Moreover, we identified four greatly expanded subfamilies of rhodopsin-type GPCRs that we call sea urchin specific rapidly expanded lineages of GPCRs (surreal-GPCRs). Our analysis of two of these groups revealed genomic clustering and single-exon gene structures similar to the most expanded group of vertebrate rhodopsin-type GPCRs, the olfactory receptors. We hypothesize that these genes arose by rapid duplication in the echinoid lineage and act as chemosensory receptors of the animal. In support of this, group B surreal-GPCRs are most prominently expressed in distinct classes of pedicellariae and tube feet of the adult sea urchin, structures that have previously been shown to react to chemical stimuli and to harbor sensory neurons in echinoderms. Notably, these structures also express different opsins, indicating that sea urchins possess an intricate molecular set-up to sense their environment.     

Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin

Sea urchin eggs secrete a serine protease activity, CGSP1, at fertilization that is essential for the block to polyspermy. Several targets of this proteolytic activity on the plasma membrane were identified here using a cell surface biotinylation approach. Amino acid microsequencing of one of these proteins led to the identification of a 4.75-kb cDNA clone from a Strongylocentrotus purpuratus ovary cDNA library that encodes a 160-kDa protein called p160. This protein contains five CUB domains and a putative transmembrane domain suggesting that p160 is an integral membrane protein with protein-protein interaction motifs facing the extracellular matrix of the egg. Whole-mount immunolocalization studies demonstrate that p160 is on the surface of the egg, enriched at the tips of microvilli. The protein is removed at fertilization in a protease-dependent manner, and functional assays suggest that p160 serves to link the plasma membrane to the vitelline layer until fertilization. Thus, p160 is a key candidate for a vitelline-layer linker protein, the selective proteolysis of which functions in the block to polyspermy in the sea urchin egg.     

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus

Summary Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunore-active to anti-actin at about 60 h (12–14° C). Initially, eachmyoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.     

Innate immune response in the sea urchin Echinometra lucunter (Echinodermata)

Echinometra lucunter, (Pindá) is a sea urchin encountered in the Brazilian coast and exposed to high and low temperatures related to low and high tides. Despite their great distribution and importance, few studies have been done on the biological function of their coelomocytes. Thus, Echinometra lucunter perivisceral coelomocytes were characterized under optical and transmission electron microscopy. Phagocytic amoebocytes in the perivisceral coelom were labelled by injecting ferritin, and ferritin labelled phagocytic amoebocytes were found in the peristomial connective tissue after injecting India ink into the tissue, indicating the amoebocytes ability to respond to an inflammatory stimulus. Results showed that the phagocytic amoebocytes were the main inflammatory cells found in the innate immune response of E. lucunter. While other works have recorded these phenomena in sea urchins found in moderate and constant temperature, this study reports on these same phenomena in a tropical sea urchin under great variation of temperature, thus providing new data to inflammatory studies in invertebrate pathology.     

Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism.     

dbEST-derived SSR markers in sea urchin (Hemicentrotus pulcherrimus)

Sea urchin is an important model organism for evolutionary biology, embryology, and gene regulation study. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Strongylocentrotus purpuratus and Hemicentrotus pulcherrimus. Characteristics of nine EST-SSR loci were investigated using 41 Hemicentrotus pulcherrimus individuals. The number of alleles per locus ranged from two to five. The observed heterozygosity ranged from 0.122 to 0.7805, while the expected heterozygosity ranged from 0.4472 to 0.7696. These loci and markers will be useful for population genetics and systemic evolution among species of sea urchin.     

R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.     

DNA variations within the sea urchin Otx gene enhancer

We performed both intra- and interspecific comparisons of the Otx gene in the sea urchin to investigate DNA variations within the enhancer elements. Intraspecific comparisons within Hemicentrotus pulcherrimus showed that indel variations were rare within the Otx enhancer, whereas SNP variations were found uniformly within the whole test region. A similar pattern of DNA variation was observed in comparisons between closely related species. On the other hand, both nucleotide substitution and indel variations were at high levels between distant species. Additionally, the regions corresponding to the Otx enhancer in two related species showed substantial activities during development. Our results suggest the possibility that a stabilizing selection has occurred during the evolution of the Otx gene enhancer in the sea urchin that maintains its expression pattern.