Experimental taphonomy of giant sulphur bacteria: implications for the interpretation of the embryo-like Ediacaran Doushantuo fossils.

The Ediacaran Doushantuo biota has yielded fossils interpreted as eukaryotic organisms, either animal embryos or eukaryotes basal or distantly related to Metazoa. However, the fossils have been interpreted alternatively as giant sulphur bacteria similar to the extant Thiomargarita. To test this hypothesis, living and decayed Thiomargarita were compared with Doushantuo fossils and experimental taphonomic pathways were compared with modern embryos. In the fossils, as in eukaryotic cells, subcellular structures are distributed throughout cell volume; in Thiomargarita, a central vacuole encompasses approximately 98 per cent cell volume. Key features of the fossils, including putative lipid vesicles and nuclei, complex envelope ornament, and ornate outer vesicles are incompatible with living and decay morphologies observed in Thiomargarita. Microbial taphonomy of Thiomargarita also differed from that of embryos. Embryo tissues can be consumed and replaced by bacteria, forming a replica composed of a three-dimensional biofilm, a stable fabric for potential fossilization. Vacuolated Thiomargarita cells collapse easily and do not provide an internal substrate for bacteria. The findings do not support the hypothesis that giant sulphur bacteria are an appropriate interpretative model for the embryo-like Doushantuo fossils. However, sulphur bacteria may have mediated fossil mineralization and may provide a potential bacterial analogue for other macroscopic Precambrian remains.

Synthesis and storage of microtubule proteins by sea urchin embryos.

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-(3)H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 microg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified.

A sea urchin gene encodes a polypeptide homologous to epidermal growth factor.

A sea urchin DNA clone complementary to an embryonic messenger RNA whose protein product bears striking homology to the epidermal growth factor family of proteins has been identified and characterized. The structure of the protein is similar to that of previously identified regulatory genes in Drosophila and Caenorhabditis. RNA gel blot hybridization showed a unique temporal pattern of expression of this gene during embryogenesis and transcript enrichment in the embryonic ectoderm. These results suggest that this member of the epidermal growth factor gene family plays a role in early development decisions in sea urchin embryos.

Molecular phylogeny of the animal kingdom.

A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups: chordates, echinoderms, arthropods, and eucoelomate protostomes.

Protein-DNA interactions at putative regulatory regions of two coordinately expressed genes, msp130 and PM27, during skeletogenesis in sea urchin embryos.

Development of the primary mesenchyme cells (PMCs) of the sea urchin embryo, which give rise to the larval skeleton, involves the coordinate onset of expression of several structural genes. As part of an effort to identify cis-acting elements that might play a role in this regulatory event, co-regulated genes were examined by two approaches. First, they were compared for conserved sequence elements. Four conserved elements were found as a cluster in all three genes examined, suggesting a regulatory role. Second, as a test for potential function, the putative regulatory regions of two of these genes were examined for protein binding sites. DNase I protection and gel mobility shift assays were used to: 1) identify several nuclear protein binding sites in these regions, two of which correspond to conserved elements among the genes; 2) demonstrate that the developmental time of appearance of the proteins that interact with these sites corresponds to the time of activation of the genes; and 3) show that two of the conserved sequence elements shared by these genes compete for the same binding proteins. These data identify putative regulatory elements, whose specific roles in the coordinate regulation of PMC-expressed genes can now be addressed directly using appropriate transgenic expression constructs.

A sea urchin homologue of ceh-19, an unusual homeobox-containing gene from a nematode.

When screening for homeobox-containing genes from the sea urchin Heliocidaris erythrogramma (He), we isolated an exon of a gene which appears to be a homologue of the homeobox-containing gene, ceh-19, of Caenorhabditis elegans (Ce). The predicted translation of the sea urchin sequence shows 77% identity and 92% similarity to the first 53 amino acids of the homeodomain of ceh-19. The ceh-19 gene exhibits an intron in an unusual location in the 3' end of the homeobox; the He gene shares this feature.

Structure and evolution of CyI cytoplasmic actin-encoding genes in the indirect- and direct-developing sea urchins Heliocidaris tuberculata and Heliocidaris erythrogramma.

The CyI cytoplasmic actin-encoding genes of Heliocidaris erythrogramma (He), a direct-developing sea urchin, and H. tuberculata, an indirect developer, were isolated and compared to the homologous CyI gene of another indirect developer, Strongylocentrotus purpuratus. Comparisons show that despite the differences in development, the actin gene structures and sequences are highly similar. The coding and 3' untranslated regions are conserved. The 5' He regulatory region has an inserted repeat element, but is otherwise similar to its homologues in the arrangement of presumptive transcription control elements.

Larval homologies and radical evolutionary changes in early development.

Larval forms are highly conserved in evolution, and phylogeneticists have used shared larval features to link disparate phyla. Despite long-term conservation, early development has in some cases evolved radically. Analysis of evolutionary change depends on identification of homologues, and this concept of descent with modification applies to embryo cells and territories as well. Difficulties arise because evolutionary changes in development can obscure homologies. Even more difficult, threshold effects can yield changes in process whereby apparently homologous features can arise from new precursors or pathways. We have observed phenomena of this type in closely related sea urchins that differ in developmental mode. A species developing via a complex feeding larva and its congener, which develops directly, have different embryonic cell lineages and divergent patterns of early development, but converge on the adult sea urchin body plan. Despite differences in embryonic developmental pathways, conserved gene expression territories are evident, as are territories whose homologies are in doubt. The highly derived development of the direct developer evidently arises from an interplay of novel organization of the egg, loss of expression of regulatory gene involved in production of feeding larval features, and changes in site and timing of expression of a number of genes.

Microtubule protein pools in early development.

Microtubule protein pools have been demonstrated to exist in unfertilized eggs and the early embryonic stages of several organisms. The microtubule pool of the sea urchin embryo is constant in size (about 0.4% of the total embryo protein) throughout early development. Protein withdrawn from this pool for organelle assembly is replaced by new synthesis. Eggs and embryos of Drosophila similarly contain a pool of microtubule proteins (larger than or equal to 0.4% of the total embryo protein, congruent to 3% of the soluble protein), which is constant in size throughout early development. The Drosophila egg microtubule proteins are easily purified by self-assembly in vitro of microtubules, and are similar to microtubule proteins from other organisms in molecular weight and other properties. Synthesis of microtubule proteins in sea urchin embryos is supported by oogenetic mRNA. This appears also to be the case in molluscan (Ilyanassa) embryos. It is not known whether Drosophila embryos synthesize microtubule proteins during the early stages of development.

The active evolutionary lives of echinoderm larvae.

Echinoderms represent a researchable subset of a dynamic larval evolutionary cosmos. Evolution of echinoderm larvae has taken place over widely varying time scales from the origins of larvae of living classes in the early Palaeozoic, approximately 500 million years ago, to recent, rapid and large-scale changes that have occurred within living genera within a span of less than a million years to a few million years. It is these recent evolutionary events that offer a window into processes of larval evolution operating at a micro-evolutionary level of evolution of discrete developmental mechanisms. We review the evolution of the diverse larval forms of living echinoderms to outline the origins of echinoderm larval forms, their diversity among living echinoderms, molecular clocks and rates of larval evolution, and finally current studies on the roles of developmental regulatory mechanisms in the rapid and radical evolutionary changes observed between closely related congeneric species.

Evo-devo: the evolution of a new discipline.

The history of life documented in the fossil record shows that the evolution of complex organisms such as animals and plants has involved marked changes in morphology, and the appearance of new features. However, evolutionary change occurs not by the direct transformation of adult ancestors into adult descendants but rather when developmental processes produce the features of each generation in an evolving lineage. Therefore, evolution cannot be understood without understanding the evolution of development, and how the process of development itself blases or constrains evolution. A revolutionary synthesis of developmental biology and evolution is in progress.

Constraint, flexibility, and phylogenetic history in the evolution of direct development in sea urchins.

Development in sea urchins typically involves the production of an elaborate feeding larva, the pluteus, within which the juvenile sea urchin grows. However, a significant fraction of sea urchins have completely or partially eliminated the pluteus, and instead undergo direct development from a large egg. Direct development is achieved primarily by heterochrony, that is, by the abbreviation or elimination of larval developmental processes and the acceleration of processes involved in development of adult features. Direct development has evolved independently several times, and in several ways. These radically altered ontogenies offer remarkable opportunities for the study of the mechanisms by which early development undergoes evolutionary modification. The recent availability of monoclonal antibody and cDNA probes that recognize homologous cells in embryos of closely related typical and direct developing species makes possible an experimental analysis of the cellular and molecular bases for heterochronic changes in development.

Evolution of developmental decisions and morphogenesis: the view from two camps.

Modern developmental biology largely ignores evolution and instead focuses on use of standard model organisms to reveal general mechanisms of development. Evolutionary biologists more widely hold developmental biology to be of major consequence in providing potential insights into evolution. Evolutionary insights can enlighten our views of developmental mechanisms as much as developmental data offer clearer views of mechanisms which underlie evolutionary change. However, insights have been limited by the long-term disengagement of the two fields dating to the rise of experimental embryology in the 1890s. Molecular genetics now provides a powerful tool to probe both gene function and evolutionary relationships, and a greater connection has become possible. The expansion of experimental organisms beyond the standard model animals used in most studies of development allows us to ask deeper questions about the interaction of development and evolution. This paper presents an analysis of the complementary uses of the resulting data in the two fields as they grope for accommodation. Analysis of the radical changes in early development seen in closely related sea urchins with alternate modes of development illustrate the complementarity of developmental and evolutionary data. These studies show that what have been thought to be constrained mechanisms of axial determination, cell lineage patterning, and gastrulation in fact evolve readily and provide the means for the rapid evolution of development.

Direct-developing sea urchins and the evolutionary reorganization of early development.

The evolution of development can be made accessible to study by exploiting closely related species that exhibit distinct ontogenies. The direct-developing sea urchin Heliocidaris erythrogramma is closely related to indirect-developing sea urchins that develop via a feeding larval stage. Superficial consideration would suggest that simple heterochronies resulting in loss of larval features and acceleration of adult features could explain the substitution of direct for indirect development. However, our experiments show that early development has in fact been extensively remodeled, with modified localization of maternal determinants coupled with dissociation of cell cleavage from axis formation resulting in novel patterns of cell lineage differentiation and fate map. Gene expression has undergone concomitant changes.