The early ANTP gene repertoire: insights from the placozoan genome

The evolution of ANTP genes in the Metazoa has been the subject of conflicting hypotheses derived from full or partial gene sequences and genomic organization in higher animals. Whole genome sequences have recently filled in some crucial gaps for the basal metazoan phyla Cnidaria and Porifera. Here we analyze the complete genome of Trichoplax adhaerens, representing the basal metazoan phylum Placozoa, for its set of ANTP class genes. The Trichoplax genome encodes representatives of Hox/ParaHox-like, NKL, and extended Hox genes. This repertoire possibly mirrors the condition of a hypothetical cnidarian-bilaterian ancestor. The evolution of the cnidarian and bilaterian ANTP gene repertoires can be deduced by a limited number of cis-duplications of NKL and "extended Hox" genes and the presence of a single ancestral "ProtoHox" gene.     

Comparative genomics of large mitochondria in placozoans

The first sequenced mitochondrial genome of a placozoan, Trichoplax adhaerens, challenged the conventional wisdom that a compact mitochondrial genome is a common feature among all animals. Three additional placozoan mitochondrial genomes representing highly divergent clades have been sequenced to determine whether the large Trichoplax mtDNA is a shared feature among members of the phylum Placozoa or a uniquely derived condition. All three mitochondrial genomes were found to be very large, 32- to 37-kb, circular molecules, having the typical 12 respiratory chain genes, 24 tRNAs, rnS, and rnL. They share with the Trichoplax mitochondrial genome the absence of atp8, atp9, and all ribosomal protein genes, the presence of several cox1 introns, and a large open reading frame containing an intron group I LAGLIDADG endonuclease domain. The differences in mtDNA size within Placozoa are due to variation in intergenic spacer regions and the presence or absence of long open reading frames of unknown function. Phylogenetic analyses of the 12 respiratory chain genes support the monophyly of Placozoa. The similarities in composition and structure between the three mitochondrial genomes reported here and that of Trichoplax's mtDNA suggest that their uncompacted state is a shared ancestral feature to other nonmetazoans while their gene content is a derived feature shared only among the Metazoa.     

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.     

Rapid evolution of the compact and unusual mitochondrial genome in the ctenophore, Pleurobrachia bachei

Ctenophores are one of the most basally branching lineages of metazoans with the largest mitochondrial organelles in the animal kingdom. We sequenced the mitochondrial (mtDNA) genome from the Pacific cidipid ctenophore, Pleurobrachia bachei. The circular mitochondrial genome is 11,016 nts, with only 12 genes, and one of the smallest metazoan mtDNA genomes recorded. The protein coding genes are intronless cox1-3, cob, nad1, 3, 4, 4L and 5. The nad2 and 6 genes are represented as short fragments whereas the atp6 gene was found in the nuclear genome. Only the large ribosomal RNA subunit and two tRNAs were present with possibly the small subunit unidentifiable due to extensive fragmentation. The observed unique features of this mitochondrial genome suggest that nuclear and mitochondrial genomes have evolved at very different rates. This reduced mtDNA genome sharply contrasts with the very large sizes of mtDNA found in other basal metazoans including Porifera (sponges), and Placozoa (Trichoplax).     

Concatenated Analysis Sheds Light on Early Metazoan Evolution and Fuels a Modern “Urmetazoon” Hypothesis

For more than a century, the origin of metazoan animals has been debated. One aspect of this debate has been centered on what the hypothetical “urmetazoon” bauplan might have been. The morphologically most simply organized metazoan animal, the placozoan Trichoplax adhaerens, resembles an intriguing model for one of several “urmetazoon” hypotheses: the placula hypothesis. Clear support for a basal position of Placozoa would aid in resolving several key issues of metazoan-specific inventions (including, for example, head–foot axis, symmetry, and coelom) and would determine a root for unraveling their evolution. Unfortunately, the phylogenetic relationships at the base of Metazoa have been controversial because of conflicting phylogenetic scenarios generated while addressing the question. Here, we analyze the sum of morphological evidence, the secondary structure of mitochondrial ribosomal genes, and molecular sequence data from mitochondrial and nuclear genes that amass over 9,400 phylogenetically informative characters from 24 to 73 taxa. Together with mitochondrial DNA genome structure and sequence analyses and Hox-like gene expression patterns, these data (1) provide evidence that Placozoa are basal relative to all other diploblast phyla and (2) spark a modernized “urmetazoon” hypothesis.     

Concatenated Analysis Sheds Light on Early Metazoan Evolution and Fuels a Modern “Urmetazoon” Hypothesis

For more than a century, the origin of metazoan animals has been debated. One aspect of this debate has been centered on what the hypothetical “urmetazoon” bauplan might have been. The morphologically most simply organized metazoan animal, the placozoan Trichoplax adhaerens, resembles an intriguing model for one of several “urmetazoon” hypotheses: the placula hypothesis. Clear support for a basal position of Placozoa would aid in resolving several key issues of metazoan-specific inventions (including, for example, head–foot axis, symmetry, and coelom) and would determine a root for unraveling their evolution. Unfortunately, the phylogenetic relationships at the base of Metazoa have been controversial because of conflicting phylogenetic scenarios generated while addressing the question. Here, we analyze the sum of morphological evidence, the secondary structure of mitochondrial ribosomal genes, and molecular sequence data from mitochondrial and nuclear genes that amass over 9,400 phylogenetically informative characters from 24 to 73 taxa. Together with mitochondrial DNA genome structure and sequence analyses and Hox-like gene expression patterns, these data (1) provide evidence that Placozoa are basal relative to all other diploblast phyla and (2) spark a modernized “urmetazoon” hypothesis.     

Inhibitors of the p53-Mdm2 interaction increase programmed cell death and produce abnormal phenotypes in the placozoon Trichoplax adhaerens (F.E. Schulze)

Recent identification of genes homologous to human p53 and Mdm2 in the basal phylum Placozoa raised the question whether the network undertakes the same functions in the most primitive metazoan organism as it does in more derived animals. Here, we describe inhibition experiments on p53/Mdm2 interaction in Trichoplax adhaerens by applying the inhibitors nutlin-3 and roscovitine. Both inhibitors had a strong impact on the animals’ survival by significantly increasing programmed cell death (cf. apoptosis, measured via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay). Treatment with roscovitine decreased cell proliferation (visualized by means of bromodeoxyuridine incorporation), which is likely reducible to its function as cyclin-dependent kinase inhibitor. Obvious phenotypic abnormalities have been observed during long-term application of both inhibitors, and either treatment is highly lethal in T. adhaerens. The findings of this study suggest a conserved role of the p53/Mdm2 network for programmed cell death since the origin of the Metazoa and advocate the deployment of Placozoa as a model for p53, apoptosis, and possibly cancer research.     

Animal evolution: the enigmatic phylum placozoa revisited

A recent report of high levels of genetic variation between strains of Trichoplax adhaerens challenges the traditional view that the phylum Placozoa comprises only one species. At the morphological level, placozoans are amongst the simplest extant animals, but molecular evidence suggests that they may have more complex origins.     

The backbone of the post-synaptic density originated in a unicellular ancestor of choanoflagellates and metazoans

Background  

Comparative genomics of the early diverging metazoan lineages and of their unicellular sister-groups opens new window to reconstructing the genetic changes which preceded or accompanied the evolution of multicellular body plans. A recent analysis found that the genome of the nerve-less sponges encodes the homologues of most vertebrate post-synaptic proteins. In vertebrate excitatory synapses, these proteins assemble to form the post-synaptic density, a complex molecular platform linking membrane receptors, components of their signalling pathways, and the cytoskeleton. Newly available genomes from Monosiga brevicollis (a member of Choanoflagellata, the closest unicellular relatives of animals) and Trichoplax adhaerens (a member of Placozoa: besides sponges, the only nerve-less metazoans) offer an opportunity to refine our understanding of post-synaptic protein evolution.     

The complete mitochondrial genome sequence of Whitmania pigra (Annelida, Hirudinea): the first representative from the class Hirudinea

The mitochondrial genome is a significant tool for investigating the evolutionary history of metazoan animals. The currently available mitochondrial genome data in GenBank is limited to understand the detail evolutionary relationship among the metazoan animals, especially in the phylum Annelida. Here we present the mitochondrial gene organization, gene order and codon usage of the leech Whitmania pigra (Annelida), which is the first representative from the class Hirudinea. It is a circular molecule of 14,426bp, and encodes 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. All 37 genes of W. pigra mitochondrial genome are transcribed from the same strand, which is identical to studied annelids, two echiurans, two sipunculans and many other lophotrochozoans. Five conserved gene clusters can be found in mitochondrial genomes of nine studied annelids, including (1) cox1-N-cox2; (2) cox3-Q-nad6-cob-W-atp6; (3) H-nad5-F-E-P-T-nad4L-nad4; (4) srRNA-V-lrRNA; and (5) nad3-S(1)-nad2. Compared with that of other studied annelids, translocations of transfer RNAs were found in the gene arrangement of W. pigra mitochondrial genome. Phylogenetic analysis strongly support that the species from Hirudinina and Oligochaeta form a monophyletic group Clitellata (BPM=100, BPP=100), which is consistent with previous research based on morphological and other molecular data. Both gene order data and amino acid sequences reveal that echiurans are derived annelids and sipunculans should be clustered with annelids and echiurans.     

Isolation of Hox genes from the scyphozoan Cassiopeia xamachana: implications for the early evolution of Hox genes.

The isolation of Hox genes from two cnidarian groups, the Hydrozoa and Anthozoa, has sparked hypotheses on the early evolution of Hox genes and a conserved role for these genes for defining a main body axis in all metazoan animals. We have isolated the first five Hox genes, Scox-1 to Scox-5, from the third cnidarian class, the Scyphozoa. For all but one gene, we report full-length homeobox plus flanking sequences. Four of the five genes show close relationship to previously reported Cnox-1 genes from Hydrozoa and Anthozoa. One gene, Scox-2, is an unambiguous homologue of Cnox-2 genes known from Hydrozoa, Anthozoa, and also Placozoa. Based on sequence similarity and phylogenetic analyses of the homeobox and homeodomain sequences of known Hox genes from cnidarians, we suggest the presence of at least five distinct Hox gene families in this phylum, and conclude that the last common ancestor of the Recent cnidarian classes likely possessed a set of Hox genes representing three different families, the Cnox-1, Cnox-2, and Cnox-5 families. The data presented are consistent with the idea that multiple duplication events of genes have occurred within one family at the expense of conservation of the original set of genes, which represent the three ancestral Hox gene families.     

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.     

Trichoplax adhaerens (Phylum Placozoa) has Cells that React with Antibodies Against the Neuropeptide RFamide

Abstract The phylum Placozoa contains the most simply organized multicellular animals: they have a thick lower epithelium and a thin upper epithelium with some single cells sandwiched between these layers. Placozoans are generally considered to have no nerve cells or specialized sensory cells. However, the results of this investigation demonstrate the existence of a specific set of cells which react with antibodies against the neuropeptide family RFamide. The RFamide-positive cells of Trichoplax adhaerens are positioned at some distance along the margin of the animal.     

Evidence for a common pattern of peptidergic innervation of cnidocytes

Tentacles from representatives of all four classes of the phylum Cnidaria were examined using antibodies against the neuropeptides FMRFamide and RFamide to reveal the organization of neurons and nerve nets associated with cnidocytes. The tentacles of all species examined contained FMRFamide- or RFamide-immunoreactive neurons, in varying densities. In representatives from the Scyphozoa, Hydrozoa, and Cubozoa, the FMRFamide-immunoreactive neurons formed plexuses at the base of the cnidocyte assemblages; in anthozoans, the absence of discrete assemblies of cnidocytes precluded visual co-localization of cnidocytes and immunoreactive neurons. In all four classes, immunoreactive sensory cells connected these peptidergic nerve nets to the surface of the tentacle. These findings suggest that members of all four cnidarian classes share a common organizational pattern, and it is proposed that this peptidergic innervation may be involved in the chemosensory regulation of cnidocyte discharge.     

The mitochondrial genome of the sea anemone Metridium senile (Cnidaria): introns,a paucity of tRNA genes,and a near-standard genetic code.

The circular, 17,443 nucleotide-pair mitochondrial (mt) DNA molecule of the sea anemone, Metridium senile (class Anthozoa, phylum Cnidaria) is presented. This molecule contains genes for 13 energy pathway proteins and two ribosomal (r) RNAs but, relative to other metazoan mtDNAs, has two unique features: only two transfer RNAs (tRNA(f-Met) and tRNA(Trp)) are encoded, and the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes each include a group I intron. The COI intron encodes a putative homing endonuclease, and the ND5 intron contains the molecule''s ND1 and ND3 genes. Most of the unusual characteristics of other metazoan mtDNAs are not found in M. senile mtDNA: unorthodox translation initiation codons and partial translation termination codons are absent, the use of TGA to specify tryptophan is the only genetic code modification, and both encoded tRNAs have primary and secondary structures closely resembling those of standard tRNAs. Also, with regard to size and secondary structure potential, the mt-s-rRNA and mt-1-rRNA have the least deviation from Escherichia coli 16S and 23S rRNAs of all known metazoan mt-rRNAs. These observations indicate that most of the genetic variations previously reported in metazoan mtDNAs developed after Cnidaria diverged from the common ancestral line of all other Metazoa.     

Mitochondrial genome sequence of <Emphasis Type="Italic">Unionicola parkeri</Emphasis> (Acari: Trombidiformes: Unionicolidae): molecular synapomorphies between closely-related <Emphasis Type="Italic">Unionicola</Emphasis> gill mites

The mitochondrial genome of Unionicola parkeri is a 14,734 bp circular DNA molecule. The sequence and annotation revealed a unique gene order, related to but distinct from the gene order in the closely related species U. foili. Mitochondrial tRNA sequences annotated in this genome predict non-canonical secondary structures for these molecules. The continuing patterns of unique gene orders and unusual tRNA structures in the Trombidiformes in general and Unionicola in particular support the use of phylogenetic approaches that use these types of molecular features as shared, derived character states. Further progress in using these molecular character states to reconstruct phylogeny will depend on careful annotation, especially of tRNA genes.     

Phylogenetic analysis of the Cnidaria

The recent members of the phylum Cnidaria were analyzed with phylogenetic methodology and the help of the PAUP Computer program. The Cnidaria are established as a monophylum by their cnidocysts, planula larva, and a polyp stage. The Ctenophora were seen as the most probable sister group of the Cnidaria. Arguments for the monophyly of the cnidarian classes Anthozoa, Scyphozoa, Cubozoa, and Hydrozoa were providea. For the ground plan of the Cnidaria the following characters were postulated: triphasic life cycle consisting of a planula larva, a benthic polyp stage, and a sexually propagating medusa like stage. For the polyp a radial symmetry, lack of septae, and hollow tentacles were assumed. The original medusa probably was tetraradial and developed from the polyp stage by a total metamorphosis. Twelve polarized characters were used to generate cladograms. The most parsimonious one showed the Anthozoa as the first offshoot of the tree with the united Scyphozoa, Cubozoa and Hydrozoa forming its sister group. Within this sister group the Scyphozoa and Cubozoa were seen as sistergroups to each other. Both groups united are then the sistergroup of the Hydrozoa. A bootstrap analysis yielded the same tree with high probabilities for the internal nodes. Despite assuming a planktonic origin of the Cnidaria in this investigation, the resulting cladogram is also compatible with an evolution of the medusa stage within the Cnidaria after the splitting-off of the Anthozoa. The possible loss of the medusa stage in the Anthozoa is discussed.     

Evolution of striated muscle: jellyfish and the origin of triploblasty

The larval and polyp stages of extant Cnidaria are bi-layered with an absence of mesoderm and its differentiation products. This anatomy originally prompted the diploblast classification of the cnidarian phylum. The medusa stage, or jellyfish, however, has a more complex anatomy characterized by a swimming bell with a well-developed striated muscle layer. Based on developmental histology of the hydrozoan medusa this muscle derives from the entocodon, a mesoderm-like third cell layer established at the onset of medusa formation. According to recent molecular studies cnidarian homologs to bilaterian mesoderm and myogenic regulators are expressed in the larval and polyp stages as well as in the entocodon and derived striated muscle. Moreover striated and smooth muscle cells may have evolved directly and independently from non-muscle cells as indicated by phylogenetic analysis of myosin heavy chain genes (MHC class II). To accommodate all evidences we propose that striated muscle-based locomotion coevolved with the nervous and digestive systems in a basic metazoan Bauplan from which the ancestors of the Ctenophora (comb jellyfish), Cnidaria (jellyfish and polyps), as well as the Bilateria are derived. We argue for a motile tri-layered cnidarian ancestor and a monophyletic descent of striated muscle in Cnidaria and Bilateria. As a consequence, diploblasty evolved secondarily in cnidarian larvae and polyps.