Stepwise emergence of the neuronal gene expression program in early animal evolution.

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.

The enigmatic Placozoa part 1: Exploring evolutionary controversies and poor ecological knowledge.

The placozoan Trichoplax adhaerens is a tiny hairy plate and more simply organized than any other living metazoan. After its original description by F.E. Schulze in 1883, it attracted attention as a potential model for the ancestral state of metazoan organization, the "Urmetazoon". Trichoplax lacks any kind of symmetry, organs, nerve cells, muscle cells, basal lamina, and extracellular matrix. Furthermore, the placozoan genome is the smallest (not secondarily reduced) genome of all metazoan genomes. It harbors a remarkably rich diversity of genes and has been considered the best living surrogate for a metazoan ancestor genome. The phylum Placozoa presently harbors three formally described species, while several dozen "cryptic" species are yet awaiting their description. The phylogenetic position of placozoans has recently become a contested arena for modern phylogenetic analyses and view-driven claims. Trichoplax offers unique prospects for understanding the minimal requirements of metazoan animal organization and their corresponding malfunctions.

The enigmatic Placozoa part 2: Exploring evolutionary controversies and promising questions on earth and in space.

The placozoan Trichoplax adhaerens has been bridging gaps between research disciplines like no other animal. As outlined in part 1, placozoans have been subject of hot evolutionary debates and placozoans have challenged some fundamental evolutionary concepts. Here in part 2 we discuss the exceptional genetics of the phylum Placozoa and point out some challenging model system applications for the best known species, Trichoplax adhaerens.

Innate immunity in the simplest animals - placozoans.

BACKGROUND: Innate immunity provides the core recognition system in animals for preventing infection, but also plays an important role in managing the relationship between an animal host and its symbiont. Most of our knowledge about innate immunity stems from a few animal model systems, but substantial variation between metazoan phyla has been revealed by comparative genomic studies. The exploration of more taxa is still needed to better understand the evolution of immunity related mechanisms. Placozoans are morphologically the simplest organized metazoans and the association between these enigmatic animals and their rickettsial endosymbionts has recently been elucidated. Our analyses of the novel placozoan nuclear genome of Trichoplax sp. H2 and its associated rickettsial endosymbiont genome clearly pointed to a mutualistic and co-evolutionary relationship. This discovery raises the question of how the placozoan holobiont manages symbiosis and, conversely, how it defends against harmful microorganisms. In this study, we examined the annotated genome of Trichoplax sp. H2 for the presence of genes involved in innate immune recognition and downstream signaling. RESULTS: A rich repertoire of genes belonging to the Toll-like and NOD-like receptor pathways, to scavenger receptors and to secreted fibrinogen-related domain genes was identified in the genome of Trichoplax sp. H2. Nevertheless, the innate immunity related pathways in placozoans deviate in several instances from well investigated vertebrates and invertebrates. While true Toll- and NOD-like receptors are absent, the presence of many genes of the downstream signaling cascade suggests at least primordial Toll-like receptor signaling in Placozoa. An abundance of scavenger receptors, fibrinogen-related domain genes and Apaf-1 genes clearly constitutes an expansion of the immunity related gene repertoire specific to Placozoa. CONCLUSIONS: The found wealth of immunity related genes present in Placozoa is surprising and quite striking in light of the extremely simple placozoan body plan and their sparse cell type makeup. Research is warranted to reveal how Placozoa utilize this immune repertoire to manage and maintain their associated microbiota as well as to fend-off pathogens.

The Trichoplax genome and the nature of placozoans.

As arguably the simplest free-living animals, placozoans may represent a primitive metazoan form, yet their biology is poorly understood. Here we report the sequencing and analysis of the approximately 98 million base pair nuclear genome of the placozoan Trichoplax adhaerens. Whole-genome phylogenetic analysis suggests that placozoans belong to a 'eumetazoan' clade that includes cnidarians and bilaterians, with sponges as the earliest diverging animals. The compact genome shows conserved gene content, gene structure and synteny in relation to the human and other complex eumetazoan genomes. Despite the apparent cellular and organismal simplicity of Trichoplax, its genome encodes a rich array of transcription factor and signalling pathway genes that are typically associated with diverse cell types and developmental processes in eumetazoans, motivating further searches for cryptic cellular complexity and/or as yet unobserved life history stages.

Studying Placozoa WBR in the Simplest Metazoan Animal, Trichoplax adhaerens.

Placozoans are a promising model system to study fundamental regeneration processes in a morphologically and genetically very simple animal. We here provide a brief introduction to the enigmatic Placozoa and summarize the state of the art of animal handling and experimental manipulation possibilities.

Mitochondrial Genome Evolution of Placozoans: Gene Rearrangements and Repeat Expansions.

Placozoans, nonbilaterian animals with the simplest known metazoan bauplan, are currently classified into 20 haplotypes belonging to three genera, Polyplacotoma, Trichoplax, and Hoilungia. The latter two comprise two and five clades, respectively. In Trichoplax and Hoilungia, previous studies on six haplotypes belonging to four different clades have shown that their mtDNAs are circular chromosomes of 32-43 kb in size, which encode 12 protein-coding genes, 24 tRNAs, and two rRNAs. These mitochondrial genomes (mitogenomes) also show unique features rarely seen in other metazoans, including open reading frames (ORFs) of unknown function, and group I and II introns. Here, we report seven new mitogenomes, covering the five previously described haplotypes H2, H17, H19, H9, and H11, as well as two new haplotypes, H23 (clade III) and H24 (clade VII). The overall gene content is shared between all placozoan mitochondrial genomes, but genome sizes, gene orders, and several exon-intron boundaries vary among clades. Phylogenomic analyses strongly support a tree topology different from previous 16S rRNA analyses, with clade VI as the sister group to all other Hoilungia clades. We found small inverted repeats in all 13 mitochondrial genomes of the Trichoplax and Hoilungia genera and evaluated their distribution patterns among haplotypes. Because Polyplacotoma mediterranea (H0), the sister to the remaining haplotypes, has a small mitochondrial genome with few small inverted repeats and ORFs, we hypothesized that the proliferation of inverted repeats and ORFs substantially contributed to the observed increase in the size and GC content of the Trichoplax and Hoilungia mitochondrial genomes.

The early evolution of Hox genes: a battle of belief?

For more than a century the origin of metazoan animals and for less than three years the early evolution of Hox genes has been debated. Both discussions are intrinsically tied together. New data from whole genome sequencing and recent progress in phylogeny of basal metazoans allow to provide an answer. The evolution of diploblastic animals (Placozoa, Porifera, Ctenophora and Cnidaria) and Bilateria (all higher animals) went parallel. The early split of these two lineages led to the evolution of a Hox system in Bilateria and the presence of Hox-like genes in Cnidaria and Placozoa.

Axial patterning and diversification in the cnidaria predate the Hox system.

Across the animal kingdom, Hox genes are organized in clusters whose genomic organization reflects their central roles in patterning along the anterior/posterior (A/P) axis . While a cluster of Hox genes was present in the bilaterian common ancestor, the origins of this system remain unclear (cf. ). With new data for two representatives of the closest extant phylum to the Bilateria, the sea anemone Nematostella and the hydromedusa Eleutheria, we argue here that the Cnidaria predate the evolution of the Hox system. Although Hox-like genes are present in a range of cnidarians, many of these are paralogs and in neither Nematostella nor Eleutheria is an equivalent of the Hox cluster present. With the exception of independently duplicated genes, the cnidarian genes are unlinked and in several cases are flanked by non-Hox genes. Furthermore, the cnidarian genes are expressed in patterns that are inconsistent with the Hox paradigm. We conclude that the Cnidaria/Bilateria split occurred before a definitive Hox system developed. The spectacular variety in morphological and developmental characteristics shown by extant cnidarians demonstrates that there is no obligate link between the Hox system and morphological diversity in the animal kingdom and that a canonical Hox system is not mandatory for axial patterning.

Polyplacotoma mediterranea is a new ramified placozoan species.

The enigmatic phylum Placozoa is harboring an unknown number of cryptic species and has become a challenge for modern systematics. Only recently, a second species has been described [1], while the presence of more than a hundred additional species has been suggested [2]. The original placozoan species Trichoplax adhaerens[3], the second species Hoilungia hongkongensis[1] and all yet undescribed species are morphologically indistinguishable (i.e. no species diagnostic characters are available [4]). Here, we report on a new placozoan species, Polyplacotoma mediterranea gen. nov., spec. nov., which differs from other placozoans in its completely different morphological habitus, including long polytomous body branches and a maximum body length of more than 10 mm. Polyplacotoma mediterranea also necessitates a different view of placozoan mitochondrial genetics. P. mediterranea harbors a highly compact mitochondrial genome with overlapping mitochondrial tRNA and protein coding genes. Furthermore, the new species lacks typical placozoan features, including the cox1 micro exon and cox1 barcode intron. As phylogenetic analyses suggest a sister group relationship of P. mediterranea to all other placozoans, this new species may also be relevant for studies addressing the relationships at the base of the metazoan tree of life.

Genome analyses of a placozoan rickettsial endosymbiont show a combination of mutualistic and parasitic traits.

Symbiotic relationships between eukaryotic hosts and bacteria range from parasitism to mutualism and may deeply influence both partners' fitness. The presence of intracellular bacteria in the metazoan phylum Placozoa has been reported several times, but without any knowledge about the nature of this relationship and possible implications for the placozoan holobiont. This information may be of crucial significance since little is known about placozoan ecology and how different species adapt to different environmental conditions, despite being almost invariable at the morphological level. We here report on the novel genome of the rickettsial endosymbiont of Trichoplax sp. H2 (strain "Panama"). The combination of eliminated and retained metabolic pathways of the bacterium indicates a potential for a mutualistic as well as for a parasitic relationship, whose outcome could depend on the environmental context. In particular we show that the endosymbiont is dependent on the host for growth and reproduction and that the latter could benefit from a supply with essential amino acids and important cofactors. These findings call for further studies to clarify the actual benefit for the placozoan host and to investigate a possible role of the endosymbiont for ecological separation between placozoan species.

Trichoplax genomes reveal profound admixture and suggest stable wild populations without bisexual reproduction.

The phylum Placozoa officially consists of only a single described species, Trichoplax adhaerens, although several lineages can be separated by molecular markers, geographical distributions and environmental demands. The placozoan 16S haplotype H2 (Trichoplax sp. H2) is the most robust and cosmopolitan lineage of placozoans found to date. In this study, its genome was found to be distinct but highly related to the Trichoplax adhaerens reference genome, for remarkably unique reasons. The pattern of variation and allele distribution between the two lineages suggests that both originate from a single interbreeding event in the wild, dating back at least several decades ago, and both seem not to have engaged in sexual reproduction since. We conclude that populations of certain placozoan haplotypes remain stable for long periods without bisexual reproduction. Furthermore, allelic variation within and between the two Trichoplax lineages indicates that successful bisexual reproduction between related placozoan lineages might serve to either counter accumulated negative somatic mutations or to cope with changing environmental conditions. On the other hand, enrichment of neutral or beneficial somatic mutations by vegetative reproduction, combined with rare sexual reproduction, could instantaneously boost genetic variation, generating novel ecotypes and eventually species.

High Cell Diversity and Complex Peptidergic Signaling Underlie Placozoan Behavior.

Placozoans, together with sponges, are the only animals devoid of a nervous system and muscles, yet both respond to sensory stimulation in a coordinated manner. How behavioral control in these free-living animals is achieved in the absence of neurons and, more fundamentally, how the first neurons evolved from more primitive cells for communication during the rise of animals are not yet understood [1-5]. The placozoan Trichoplax adhaerens is a millimeter-wide, flat, free-living marine animal composed of six morphologically identified cell types distributed across a simple body plan [6-9]: a thin upper epithelium and a columnar lower epithelium interspersed with a loose layer of fiber cells in between. Its genome contains genes encoding several neuropeptide-precursor-like proteins and orthologs of proteins involved in neurosecretion in animals with a nervous system [10-12]. Here we investigate peptidergic signaling in T. adhaerens. We found specific expression of several neuropeptide-like molecules in non-overlapping cell populations distributed over the three cell layers, revealing an unsuspected cell-type diversity of T. adhaerens. Using live imaging, we discovered that treatments with 11 different peptides elicited striking and consistent effects on the animals' shape, patterns of movement, and velocity that we categorized under three main types: (1) crinkling, (2) turning, and (3) flattening and churning. Together, the data demonstrate a crucial role for peptidergic signaling in nerveless placozoans and suggest that peptidergic volume signaling may have pre-dated synaptic signaling in the evolution of nervous systems.

GC Content of Early Metazoan Genes and Its Impact on Gene Expression Levels in Mammalian Cell Lines.

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure-function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines.

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.

Ancient linkage of a POU class 6 and an anterior Hox-like gene in cnidaria: implications for the evolution of homeobox genes.

Linkage analyses in metazoan genomes suggest two ancestral arrays for the majority of homeobox genes. The related homeobox genes and chromosomal regions that are dispersed in extant species derived possibly from only two single common ancestor regions. One proposed ancestral array, designated as ANTP mega-array, contains most of the ANTP class homeobox genes; the second, named the contraHox super-paralogon, would consist of the classes PRD, POU, LIM, CUT, prospero, TALE and SIX. Here, we report the tight linkage of a POU class 6 gene to an anterior Hox-like gene in the hydrozoan Eleutheria dichotoma and discuss its possible significance for the evolution of homeobox genes. POU class 6 genes also seem to be ancestrally linked to the HoxC and A clusters in vertebrates, despite POU homeobox genes belonging to the contraHox paralogon. Hence, the much tighter linkage of a POU class 6 gene to an anterior Hox-like gene in a cnidarian is possibly the evolutionary echo of an ancestral genomic region from which most metazoan homeobox classes emerged.

Ancient complexity of the non-Hox ANTP gene complement in the anthozoan Nematostella vectensis: implications for the evolution of the ANTP superclass.

The origin and evolution of ANTP superclass genes has raised controversial discussions. While recent evidence suggests that a true Hox cluster emerged after the cnidarian bilaterian split, the origin of the ANTP superclass as a whole remains unclear. Based on analyses of bilaterian genomes, it seems very likely that clustering has once been a characteristic of all ANTP homeobox genes and that their ancestors have emerged through several series of cis-duplications from the same genomic region. Since the diploblastic Cnidaria possess orthologs of some non-Hox ANTP genes, at least some steps of the expansion of this hypothetical homeobox gene array must have occurred in the last common ancestor of both lineages--but it is unknown to what extent. By screening the unassembled Nematostella genome, we have identified unambiguous orthologs to almost all non-Hox ANTP genes which are present in Bilateria--with the exception of En, Tlx and (possibly) Vax. Furthermore, Nematostella possesses ANTP genes that are missing in some bilaterian lineages, like the rough gene or NK7. In addition, several ANTP homeobox gene families have been independently duplicated in Nematostella. We conclude that the last cnidarian/bilaterian ancestor already harboured the almost full complement of non-Hox ANTP genes before the Hox system evolved.