Insights from diploblasts; the evolution of mesoderm and muscle

The origin of both mesoderm and muscle are central questions in metazoan evolution. The majority of metazoan phyla are triploblasts, possessing three discrete germ layers. Attention has therefore been focused on two outgroups to triploblasts, Cnidaria and Ctenophora. Modern texts describe these taxa as diploblasts, lacking a mesodermal germ layer. However, some members of Medusozoa, one of two subphyla within Cnidaria, possess tissue independent of either the ectoderm or endoderm referred to as the entocodon. Furthermore, members of both Cnidaria and Ctenophora have been described as possessing striated muscle, a mesodermal derivative. While it is widely accepted that the ancestor of Eumetazoa was diploblastic, homology of the entocodon and mesoderm as well as striated muscle within Eumetazoa has been suggested. This implies a potential triploblastic ancestor of Eumetazoa possessing striated muscle. In the following review, I examine the evidence for homology of both muscle and mesoderm. Current data support a diploblastic ancestor of cnidarians, ctenophores, and triploblasts lacking striated muscle.     

An analysis of partial 28S ribosomal RNA sequences suggests early radiations of sponges.

Sequences from the 5' end terminal part of 28S ribosomal RNA were obtained and compared for 22 animals belonging to all diploblastic phyla and for a large number of representatives of triploblastic Metazoa and protists. Phylogenetic analyses undertaken using different methods showed deep radiations of phyla such as Ctenophora, Cnidaria and Placozoa but also for groups of Porifera of low taxonomic rank. Short internodes between these radiations suggested an early rapid diversification of diploblasts. A long internal branch preceding the diversification of all triploblasts analyzed could be explained either by a long period with a single ancestor or by the extinction of the earliest triploblastic radiations. Finally some unexpected relationships were revealed among Porifera.     

Phylogenetic analysis of the Hsp70 sequences reveals the monophyly of Metazoa and specific phylogenetic relationships between animals and fungi

To understand the early evolution of the Metazoa, it is necessary to determine the correct phylogenetic status of diploblastic animals. Despite cladistic studies of morphological characters and recent molecular phylogenetic studies, it remains uncertain whether diploblasts are monophyletic or paraphyletic, and how the phyla of diploblasts are phylogenetically related. The heat shock protein 70 (Hsp70) sequences, because of their ubiquity and high degree of conservation, could provide a useful model for phylogenetic analysis. We have sequenced almost the entire nucleic acid sequence of cytoplasmic Hsp70 from eight diploblastic species. Our data support the monophyly of diploblastic animals. However, the phylogenetic relationships of the diploblast groups were not significantly resolved. Our phylogenetic trees also support the monophyly of Metazoa with high bootstrap values, indicating that animals form an extremely robust clade.      

Eukaryogenesis: a model derivated from ribosomal RNA molecular phylogenise]

We have undertaken the construction of a broad molecular phylogeny of protists through the comparison of 28S rRNA molecules. The sequences from several major protistan phyla were aligned and combined with a broad database of metazoans, metaphytes, fungi and bacteria and we have derived dendrograms from both distance matrix and parsimony methods. In agreement with classical systematics, a number of monophyletic groups separated by large evolutionary distances were observed (those of the ciliates, the chlorophytes, etc.). From this analysis, several inferences on the eukaryogenesis can be made among which the ancient origin of the cytoskeleton, the late occurrence of the chloroplastic endosymbiosis and the simultaneous emergence of the triploblastic and diploblastic metazoan patterns.     

Conservation of Brachyury, Mef2, and Snail in the myogenic lineage of jellyfish: a connection to the mesoderm of bilateria

One major difference between simple metazoans such as cnidarians and all the bilaterian animals is thought to involve the invention of mesoderm. The terms diploblasts and triploblasts are therefore, often used to group prebilaterian and bilaterian animals, respectively. However, jellyfish contain well developed striated and smooth muscle tissues that derive from the entocodon, a mesoderm-like tissue formed during medusa development. We investigated the hypothesis, that the entocodon could be homologous to the third germ layer of bilaterians by analyzing the structures and expression patterns of the homologues of Brachyury, Mef2, and Snail in the jellyfish Podocoryne carnea. These are regulatory genes from the T-box, MADS-box and zinc finger families known to play important roles in bilaterian mesoderm patterning and muscle differentiation. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. Based on these data we conclude that the common ancestor of the cnidarians and bilaterians not only shared genes that play a role in regulating myogenesis but already used them to develop and differentiate muscle systems similar to those of triploblasts.     

Choanoflagellates, choanocytes, and animal multicellularity

Abstract. It is widely accepted that multicellular animals (metazoans) constitute a monophyletic unit, deriving from ancestral choanoflagellate‐like protists that gave rise to simple choanocyte‐bearing metazoans. However, a re‐assessment of molecular and histological evidence on choanoflagellates, sponge choanocytes, and other metazoan cells reveals that the status of choanocytes as a fundamental cell type in metazoan evolution is unrealistic. Rather, choanocytes are specialized cells that develop from non‐collared ciliated cells during sponge embryogenesis. Although choanocytes of adult sponges have no obvious homologue among metazoans, larval cells transdifferentiating into choanocytes at metamorphosis do have such homologues. The evidence reviewed here also indicates that sponge larvae are architecturally closer than adult sponges to the remaining metazoans. This may mean that the basic multicellular organismal architecture from which diploblasts evolved, that is, the putative planktonic archimetazoan, was more similar to a modern poriferan larva lacking choanocytes than to an adult sponge. Alternatively, it may mean that other metazoans evolved from a neotenous larva of ancient sponges. Indeed, the Porifera possess some features of intriguing evolutionary significance: (1) widespread occurrence of internal fertilization and a notable diversity of gastrulation modes, (2) dispersal through architecturally complex lecithotrophic larvae, in which an ephemeral archenteron (in dispherula larvae) and multiciliated and syncytial cells (in trichimella larvae) occur, (3) acquisition of direct development by some groups, and (4) replacement of choanocyte‐based filter‐feeding by carnivory in some sponges. Together, these features strongly suggest that the Porifera may have a longer and more complicated evolutionary history than traditionally assumed, and also that the simple anatomy of modern adult sponges may have resulted from a secondary simplification. This makes the idea of a neotenous evolution less likely than that of a larva‐like choanocyte‐lacking archimetazoan. From this perspective, the view that choanoflagellates may be simplified sponge‐derived metazoans, rather than protists, emerges as a viable alternative hypothesis. This idea neither conflicts with the available evidence nor can be disproved by it, and must be specifically re‐examined by further approaches combining morphological and molecular information. Interestingly, several microbial lin°Cages lacking choanocyte‐like morphology, such as Corallochytrea, Cristidiscoidea, Ministeriida, and Mesomycetozoea, have recently been placed at the boundary between fungi and animals, becoming a promising source of information in addition to the choanoflagellates in the search for the unicellular origin of animal multicellularity.     

Current problems with the zootype and the early evolution of Hox genes

"Hox cluster type" genes have sparked intriguing attempts to unite all metazoan animals by a shared pattern of expression and genomic organization of a specific set of regulatory genes. The basic idea, the zootype concept, claims the conservation of a specific set of "Hox cluster type genes" in all metazoan animals, i.e., in the basal diploblasts as well as in the derived triploblastic animals. Depending on the data used and the type of analysis performed, different opposing views have been taken on this idea. We review here the sum of data currently available in a total evidence analysis, which includes morphological and the most recent molecular data. This analysis highlights several problems with the idea of a simple "Hox cluster type" synapomorphy between the diploblastic and triploblastic animals and suggests that the "zootype differentiation" of the Hox cluster most likely is an invention of the triploblasts. The view presented is compatible with the idea that early Hox gene evolution started with a single proto-Hox (possibly a paraHox) gene. J. Exp. Zool. (Mol. Dev. Evol.) 291:169-174, 2001.     

Animal evolution. The end of the intermediate taxa?

Contrary to general belief, there has not been a reliable, global phylogeny of animals at hand within the past few decades. Recent progress in molecular phylogeny is rapidly changing the situation and has provided trees that constitute a reference frame for discussing the still controversial evolution of body plans. These trees, once purged of their possible artefacts, have already yielded confirmation of traditional, anatomically based, phylogenies as well as several new and quite significant results. Of these, one of the most striking is the disappearance of two superphyla (acoelomates such as flatworms, pseudocoelomates such as nematodes) previously thought to represent grades of intermediate complexity between diploblasts (organisms with two germ layers) and triploblasts (organisms with three germ layers). The overall image now emerging is of a fairly simple global tree of metazoans, comprising only a small number of major branches. The topology nicely accounts for the striking conservation of developmental genes in all bilaterians and suggests a new interpretation of the 'Cambrian explosion' of animal diversity.     

Homology of Hox Genes and the Zootype Concept in Early Metazoan Evolution

The correct identification of homologous Hox genes within and between diplo- and triploblastic animals is of crucial importance for recent hypotheses on the anagenetic evolution of animal bauplans. While the homology discussion in general has reached new heights, we apply traditional homology criteria to assign homology to Hox genes from diploblastic animals. Comparison of theTrox-2gene from the presumably most basal metazoan animal, the placozoanTrichoplax adhaerens,to other Hox genes suggests the presence of unambiguous homologs in Hydrozoa and Scyphozoa and the absence of any specific homolog in triploblasts. Furthermore, the comparisons provide support for the idea that Hox genes—at least in diploblastic animals—are composed of functional subunits (modules), which to some degree have undergone independent evolution. The findings are not readily compatible with the existence of the “zootype” in diploblastic animals.     

The Trichoplax PaxB gene: a putative Proto-PaxA/B/C gene predating the origin of nerve and sensory cells

Pax genes play key regulatory roles in embryonic and sensory organ development in metazoans but their evolution and ancestral functions remain widely unresolved. We have isolated a Pax gene from Placozoa, beside Porifera the only metazoan phylum that completely lacks nerve and sensory cells or organs. These simplest known metazoans also lack any kind of symmetry, organs, extracellular matrix, basal lamina, muscle cells, and main body axis. The isolated Pax gene from Trichoplax adhaerens harbors a paired domain, an octapeptide, and a full-length homeodomain. It displays structural features not only of PaxB and Pax2/5/8-like genes but also of PaxC and Pax6 genes. Conserved splice sites between Placozoa, Cnidaria, and triploblasts, mark the ancient origin of intron structures. Phylogenetic analyses demonstrate that the Trichoplax PaxB gene, TriPaxB, is basal not only to all other known PaxB genes but also to PaxA and PaxC genes and their relatives in triploblasts (namely Pax2/5/8, Pax4/6, and Poxneuro). TriPaxB is expressed in distinct cell patches near the outer edge of the animal body, where undifferentiated and possibly multipotent cells are found. This expression pattern indicates a developmental role in cell-type specification and/or differentiation, probably in specifying-determining fiber cells, which are regarded as proto-neural/muscle cells in Trichoplax. While PaxB, Pax2/5/8, and Pax6 genes have been linked to nerve cell and sensory system/organ development in virtually all animals investigated so far, our study suggests that Pax genes predate the origin of nerve and sensory cells.     

An evolutionary history of the FGF superfamily

Fibroblast growth factors (FGF) are associated with multiple developmental and metabolic processes in triploblasts, and perhaps also in diploblasts. The evolution of the FGF superfamily has accompanied the major morphological and functional innovations of metazoan species. The study of FGFs throughout species shows that the FGF superfamily can be subdivided in eight families in present-day organisms and has evolved through phases of gene duplications and gene losses. At least two major expansions of the superfamily can be recognized: a first expansion increased the number of FGFs from one or few archeo-FGFs to eight proto-FGFs, prototypic of the eight families. A second expansion, which took place during euchordate evolution, is associated with genome duplications. It increased the number of members in the families. Subsequent losses reduced that number to the present-day figures.     

Phylogenetic analysis of the formin homology 2 domain

Formin proteins are key regulators of eukaryotic actin filament assembly and elongation, and many species possess multiple formin isoforms. A nomenclature system based on fundamental features would be desirable, to aid the rapid identification and characterization of novel formins. In this article, we attempt to systematize the formin family by performing phylogenetic analyses of the formin homology 2 (FH2) domain, an independently folding region common to all formins, which alone can influence actin dynamics. Through database searches, we identify 101 FH2 domains from 26 eukaryotic species, including 15 in mice. Sequence alignments reveal a highly conserved yeast-specific insert in the "knob loop" region of the FH2 domain, with unknown functional consequences. Phylogenetic analysis using minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML) algorithms strongly supports the existence of seven metazoan groups. Yeast FH2 domains segregate from all other eukaryotes, including metazoans, other fungi, plants, and protists. Sequence comparisons of non-FH2 regions support relationships between three metazoan groups (Dia, DAAM, and FRL) and examine previously identified coiled-coil and Diaphanous auto-regulatory domain sequences. This analysis allows for a formin nomenclature system based on sequence relationships, as well as suggesting strategies for the determination of biochemical and cellular activities of these proteins.     

Nucleotide sequence of the histone gene cluster in the coral acropora formosa (cnidaria; scleractinia): Features of histone gene structure and organization are common to diploblastic and triploblastic metazoans

We report the nucleotide sequence of the core histone gene cluster from the Cnidarian Acropora formosa. This is the first histone gene cluster to be sequenced from a diploblastic organism and the predicted amino acid sequences most resemble those of sea urchin equivalents. Each of the Cnidarian histone genes has two conserved regions 3 of the coding sequences and these closely resemble those of the metazoan a-class histone genes. In A. formosa the core histone genes are arranged as opposed (H3/H4 and H2A/H2B) pairs, a pattern common to the nondeuterostome metazoa, and tandem repetition is the predominant pattern of organization in the Cnidarian. With the recent identification of several classes of homeobox genes in Cnidarians these features clearly align the Cnidaria with triploblastic metazoans, supporting a monophyletic origin of the metazoa.     

EvoD/Vo: the origins of BMP signalling in the neuroectoderm

The genetic systems controlling body axis formation trace back as far as the ancestor of diploblasts (corals, hydra, and jellyfish) and triploblasts (bilaterians). Comparative molecular studies, often referred to as evo-devo, provide powerful tools for elucidating the origins of mechanisms for establishing the dorsal-ventral and anterior-posterior axes in bilaterians and reveal differences in the evolutionary pressures acting upon tissue patterning. In this Review, we focus on the origins of nervous system patterning and discuss recent comparative genetic studies; these indicate the existence of an ancient molecular mechanism underlying nervous system organization that was probably already present in the bilaterian ancestor.     

Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.     

Placozoa and the evolution of Metazoa and intrasomatic cell differentiation

The multicellular Metazoa evolved from single-celled organisms (Protozoa) and usually – but not necessarily – consist of more cells than Protozoa. In all cases, and thus by definition, Metazoa possess more than one somatic cell type, i.e. they show-in sharp contrast to protists–intrasomatic differentiation. Placozoa have the lowest degree of intrasomatic variation; the number of somatic cell types according to text books is four (but see also Jakob W, Sagasser S, Dellaporta S, Holland P, Kuhn K, and Schierwater B. The Trox-2 Hox/ParaHox gene of Trichoplax (Placozoa) marks an epithelial boundary. Dev Genes Evol 2004;214:170–5). For this and several other reasons Placozoa have been regarded by many as the most basal metazoan phylum. Thus, the morphologically most simply organized metazoan animal, the placozoan Trichoplax adhaerens, resembles a unique model system for cell differentiation studies and also an intriguing model for a prominent “urmetazoon” hypotheses—the placula hypothesis. A basal position of Placozoa would provide answers to several key issues of metazoan-specific inventions (including for example different lines of somatic cell differentiation leading to organ development and axis formation) and would determine a root for unraveling their evolution. However, the phylogenetic relationships at the base of Metazoa are controversial and a basal position of Placozoa is not generally accepted (e.g. Schierwater B, DeSalle R. Can we ever identify the Urmetazoan? Integr Comp Biol 2007;47:670–76; DeSalle R, Schierwater B. An even “newer” animal phylogeny. Bioessays 2008;30:1043–47). Here we review and discuss (i) long-standing morphological evidence for the simple placozoan bauplan resembling an ancestral metazoan stage, (ii) some rapidly changing alternative hypotheses derived from molecular analyses, (iii) the surprising idea that triploblasts (Bilateria) and diploblasts may be sister groups, and (iv) the presence of genes involved in cell differentiation and signaling pathways in the placozoan genome.     

Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.     

Characterization of protistan assemblages in the Ross Sea, Antarctica, by denaturing gradient gel electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.