Molecular heterotopy in the expression of <Emphasis Type="Italic">Brachyury</Emphasis> orthologs in order Clypeasteroida (irregular sea urchins) and order Echinoida (regular sea urchins)

The expression patterns of Brachyury (Bra) orthologs in the development of four species of sand dollars (order: Clypeasteroida), including a direct-developing species, and of a sea urchin species (order: Echinoida) were investigated during the period from blastula to the pluteus stage, with special attention paid to the relationship between the expression pattern and the mode of gastrulation. The sand dollar species shared two expression domains of the Bra orthologs with the Echinoida species, in the vegetal ring (the first domain) and the oral ectoderm (the second domain). The following heterotopic changes in the expression of the Bra genes were found among the sand dollar species and between the sand dollars and the Echinoida species. (1) The vegetal ring expressing Bra in the sand dollars was much wider and was located at a higher position along the AV axis, compared with that in the Echinoida species. The characteristic Bra expression in the vegetal ring of the sand dollar embryos was thought to be involved in the mode of gastrulation, in which involution continues from the beginning of invagination until the end of gastrulation. (2) Two of the three indirect-developing sand dollar species that were examined exhibited a third domain, in which Bra was expressed on the oral side of the archenteron. (3) In the direct-developing sand dollar embryos, Bra was expressed with an oral-aboral asymmetry in the vegetal ring and with a left-right asymmetry in the oral ectoderm. In the Echinoida species, Bra was expressed in the vestibule at the six-armed pluteus stage.Edited by N. Satoh     

Molecular phylogeny of echinometrid sea urchins: more species of Heliocidaris with derived modes of reproduction

Abstract. Pachechinus bajulus is an endemic Australian sea urchin with an unusual mode of brooded larval development. We used mitochondrial and nuclear gene sequences to estimate the phylogenetic relationships among Pachechinus and other Echinometridae, including well‐studied species of Heliocidaris with planktonic development. We found strong evidence for the planktotrophic species Heliocidaris tuberculata as the sister group to a clade of three closely related species in which development is known (Heliocidaris erythrogramma, P. bajulus) or suspected (Pachechinus australiae) to be lecithotrophic. Clade support values and likelihood ratio tests rejected monophyly of Heliocidaris species. The sister group to H. erythrogramma is most likely the two Pachechinus species. We resolve the paraphyly problem by reassigning the Pachechinus species to the genus Heliocidaris (the senior synonym), which has six extant species including Heliocidaris australiae and Heliocidaris bajulus. The phylogeny has potentially important implications for comparative studies of developmental morphology and genetics that have assumed a close sister‐group relationship between H. erythrogramma and H. tuberculata, and highlights the need for such data from H. bajulus and other Heliocidaris species.     

Embryonic expression of engrailed in sea urchins

Neural patterning genes that are expressed along the anterior-posterior axis of deuterostomes are expressed late in larval development in echinoderms and are thought to function in establishing the highly-derived, adult body plan. We have used genomic resources to clone an engrailed gene (SpEn) from Strongylocentrotus purpuratus, and with this we have developed an antibody specific for SpEn. SpEn is expressed late in embryogenesis in the developing larval nervous system. At the prism stage, a small number of neuroblasts in the oral ectoderm on the edge of the larval mouth begin expressing SpEn. The cells are in bilaterally symmetric positions. The expression of SpEn precedes the expression of the neural markers, synaptotagmin and serotonin in the SpEn immunoreactive cells. The SpEn cells are located on the margin of the domain of cells expressing SpNK2.1, but they do not have nuclear SpNK2.1. Expression of engrailed in a pair of bilateral neural structures in early development appears to be a shared feature of bilaterians.     

Evolutionary reorganizations of ontogenesis in sea urchins

The data published during recent 15-20 years on comparative, experimental and molecular embryology of unusually developing sea urchins have been reviewed. These animals are characterized by large lipidrich eggs, highly modified embryogenesis, and the absence of a planktotrophic larva. Such a type of development is evolutionary advanced and arose independently in various phylogenetic lineages of the sea urchins.     

Nuclear activity during oogenesis in sea urchins

Summary Early meiotic stages of Arbacia punctulata oocytes have revealed the presence of synaptinemal complexes in the chromosomes, which persist through zygotene-pachytene. The synaptinemal complexes conform broadly to the usual tripartite structures found in other higher forms. In addition, nuclei at these stages consist of a small nucleolus and dense bodies of varying sizes. The nucleolus is fibrillar in texture throughout and does not seem to incorporate Uridine-5-³H after pulse labeling, whereas the chromosomes are labeled. The nucleolar label is visualized at diplotene stages and onwards. The nuclear envelope differentiates by the appearance of numerous nuclear pore complexes with dense material in the annuli, and the chromosomes become markedly diffused. At vitellogenesis stage the nucleolus and chromatin become highly labeled after pulse incorporation of Uridine, indicating synthesis of ribosomal and chromosomal RNAs.This investigation was supported by grants No. A-5049, A-3624 and D-17 from National Research Council, Canada, grant No. DRB-9340-05 (U6) from Defense Research Board, Canada, and grant No. DRG-918 AT from Damon Runyon Memorial Fund for Cancer Research.     

Variability of sperm specific histones in sea urchins

The variability of sperm histones was compared in two species of sea urchin. Whole sperm specific histones (SpH), were isolated from Tetrapygus niger (Arbacoida) and Parechinus angulosus (Echinoida). Individual histones were purified by chromatography on BioGel P-60 followed by reverse high pressure liquid chromatography (HPLC). The heterogeneity of each major histone type from T. niger was established from their HPLC elution patterns and further confirmed by electrophoresis in polyacrylamide gels containing 6 mM Triton X-100 combined with a transverse urea gradient (0--8 M). In T. niger, as well as in P. angulosus, a single form of SpH1 and SpH2A were found. In contrast, SpH2B was found to be heterogeneous, but represented by one major form in both species. The relatedness between both sets of histones was determined by establishing their immunological cross-reactivity. In this context, polyclonal antibodies elicited against T. niger sperm histones were assayed against individual histones from P. angulosus. From the results obtained, it emerged that histone SpH2A was the more closely related protein between these two species, followed by histone SpH1. In contrast, histone SpH2B was found to be only moderately related. These results confirm that SpH2A did not co-evolve with SpH2B, as was predicted for most species.     

Evolutionary crossroads in developmental biology: sea urchins

Embryos of the echinoderms, especially those of sea urchins and sea stars, have been studied as model organisms for over 100 years. The simplicity of their early development, and the ease of experimentally perturbing this development, provides an excellent platform for mechanistic studies of cell specification and morphogenesis. As a result, echinoderms have contributed significantly to our understanding of many developmental mechanisms, including those that govern the structure and design of gene regulatory networks, those that direct cell lineage specification, and those that regulate the dynamic morphogenetic events that shape the early embryo.     

Muscles advance the teeth in sand dollars and other sea urchins

We demonstrate the action of the dental promoter muscles in advancing the continuously growing teeth of sand dollars and sea urchins. Teeth wear at the occlusal end, while new calcite is added to the opposite end. Dental ligaments rigidly hold teeth during chewing, but soften and reform during advancement. The source of forces that advance the teeth was unknown until our discovery of the dental promoter muscles. The muscles, which underly the tooth, attach centrally to the stereom of the pyramid of the Aristotle''s lantern (jaw) and peripherally to a membrane that covers the distal end of the tooth. The muscles shorten along an axis nearly parallel to the long axis of the tooth. We stimulated contraction by addition of acetylcholine, with increasing concentrations of acetylcholine generating higher forces. Forces exerted by this muscle are appropriate for its size and are 1000 times lower than forces exerted by interpyramidal muscles that generate chewing forces. In sand dollars, a single muscle contraction of the dental promoter muscle can account for half the mean daily advancement of the teeth.     

Antimicrobial activity of polyhydroxynaphthoquinones from sea urchins

Antimicrobial activity of polyhydroxynaphthoquinones from sea urchins was studied. 6-Ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone (equinochrome A) containing the beta-ethyl functional group was found to be the most active substance. Methylation of beta-hydroxyls markedly increased the inhibitory effect of the derivatives as compared to that of the initial quinones.     

Anti-inflammatory drugs promote polyspermic fertilization in sea urchins

Sea urchin eggs are known to release H₂O₂ during the cortical reaction at fertilization to help prevent polyspermy by inactivating excess sperm in the vicinity. This process resembles the peroxidatic killing of bacteria by phagocytic leukocytes during inflammation. Associated with these reactions in leukocytes, arachidonic acid is released from phospholipids and can be oxidized via the cyclooxygenase pathway to produce prostaglandins. Nonsteroidal anti-inflammatory drugs (NSAID) that are cyclooxygenase inhibitors in somatic cells were used to determine whether Arbacia punctulata and Strongylocentrotus purpuratus eggs use these processes to help prevent polyspermy. The potent cyclooxygenase inhibitor indomethacin causes a dose (10–100 μM) and sperm density dependent induction of polyspermy if added before the egg completes the cortical reaction. It does not retard elevation of the fertilization envelope and does not promote polyspermy by protecting sperm from peroxidatic inactivation by egg-derived H₂O₂. Other potent cyclooxygenase inhibitors, flufenamate and meclofenamate, also induce polyspermy at 10–60 μM. Aspirin, a weak cyclooxygenase inhibitor in somatic cells, does not cause polyspermy at 5 mM. These findings provide evidence that prostaglandins or other cyclooxygenase-derived metabolites may help assure monospermic fertilization in sea urchins.