Function and phylogeny support the independent evolution of an ASIC-like Deg/ENaC channel in the Placozoa.

ASIC channels are bilaterian proton-gated sodium channels belonging to the large and functionally-diverse Deg/ENaC family that also includes peptide- and mechanically-gated channels. Here, we report that the non-bilaterian invertebrate Trichoplax adhaerens possesses a proton-activated Deg/ENaC channel, TadNaC2, with a unique combination of biophysical features including tachyphylaxis like ASIC1a, reduced proton sensitivity like ASIC2a, biphasic macroscopic currents like ASIC3, as well as low sensitivity to the Deg/ENaC channel blocker amiloride and Ca2+ ions. Structural modeling and mutation analyses reveal that TadNaC2 proton gating is different from ASIC channels, lacking key molecular determinants, and involving unique residues within the palm and finger regions. Phylogenetic analysis reveals that a monophyletic clade of T. adhaerens Deg/ENaC channels, which includes TadNaC2, is phylogenetically distinct from ASIC channels, instead forming a clade with BASIC channels. Altogether, this work suggests that ASIC-like channels evolved independently in T. adhaerens and its phylum Placozoa. Our phylogenetic analysis also identifies several clades of uncharacterized metazoan Deg/ENaC channels, and provides phylogenetic evidence for the existence of Deg/ENaC channels outside of Metazoa, present in the gene data of select unicellular heterokont and filasterea-related species.

[Spatial Models of Piezo Proteins and Protein-Protein Interaction Networks in Trichoplax Animals (Placozoa)].

The marine free-living organism Trichoplax (phylum Placozoa) resembles a unicellular amoeba in shape and type of movement. Trichoplax diverged from the main evolutionary tree in the Neoproterozoic Era. Trichoplax provides one of the simplest models of multicellular animals and a strong example of how cells of an organism interact to form an ensemble during its development and movement. Two orthologs of the mouse Piezo1 protein (6B3R) were found in two Trichoplax haplotypes, H1 and H2, as a result of a search for similar sequences in the NCBI databases. Spatial models of the respective proteins XP_002112008.1 and RDD46920.1 were created via a structural alignment with 6KG7 (mouse Piezo2) template. Their domain structures were analyzed, and a limited graph of protein-protein interactions was constructed for the hypothetical mechanosensor XP_002112008.1. The possibility of signal transduction from the mechanoreceptor to membrane complexes, the cytoplasm, and the cell nucleus was shown. Trichoplax mechanoreceptors were assumed to play a role in perception of force stimuli from neighbor cells and the environment. Based on the results, the primitive Trichoplax organism was proposed as the simplest multicellular model of mechanical and morphogenetic movements.

Non-Bilaterians as Model Systems for Tissue Mechanics.

In animals, epithelial tissues are barriers against the external environment, providing protection against biological, chemical, and physical damage. Depending on the organism's physiology and behavior, these tissues encounter different types of mechanical forces and need to provide a suitable adaptive response to ensure success. Therefore, understanding tissue mechanics in different contexts is an important research area. Here, we review recent tissue mechanics discoveries in three early divergent non-bilaterian systems-Trichoplax adhaerens, Hydra vulgaris, and Aurelia aurita. We highlight each animal's simple body plan and biology and unique, rapid tissue remodeling phenomena that play a crucial role in its physiology. We also discuss the emergent large-scale mechanics in these systems that arise from small-scale phenomena. Finally, we emphasize the potential of these non-bilaterian animals to be model systems in a bottom-up approach for further investigation in tissue mechanics.

Growth produces coordination trade-offs in Trichoplax adhaerens, an animal lacking a central nervous system.

How collectives remain coordinated as they grow in size is a fundamental challenge affecting systems ranging from biofilms to governments. This challenge is particularly apparent in multicellular organisms, where coordination among a vast number of cells is vital for coherent animal behavior. However, the earliest multicellular organisms were decentralized, with indeterminate sizes and morphologies, as exemplified by Trichoplax adhaerens, arguably the earliest-diverged and simplest motile animal. We investigated coordination among cells in T. adhaerens by observing the degree of collective order in locomotion across animals of differing sizes and found that larger individuals exhibit increasingly disordered locomotion. We reproduced this effect of size on order through a simulation model of active elastic cellular sheets and demonstrate that this relationship is best recapitulated across all body sizes when the simulation parameters are tuned to a critical point in the parameter space. We quantify the trade-off between increasing size and coordination in a multicellular animal with a decentralized anatomy that shows evidence of criticality and hypothesize as to the implications of this on the evolution hierarchical structures such as nervous systems in larger organisms.

Review: The evolution of peptidergic signaling in Cnidaria and Placozoa, including a comparison with Bilateria.

Bilateria have bilateral symmetry and are subdivided into Deuterostomia (animals like vertebrates) and Protostomia (animals like insects and mollusks). Neuropeptides occur in both Proto- and Deuterostomia and they are frequently structurally related across these two lineages. For example, peptides belonging to the oxytocin/vasopressin family exist in both clades. The same is true for the G protein-coupled receptors (GPCRs) of these peptides. These observations suggest that these neuropeptides and their GPCRs were already present in the common ancestor of Proto- and Deuterostomia, which lived about 700 million years ago (MYA). Furthermore, neuropeptides and their GPCRs occur in two early-branching phyla that diverged before the emergence of Bilateria: Cnidaria (animals like corals and sea anemones), and Placozoa (small disk-like animals, feeding on algae). The sequences of these neuropeptides and their GPCRs, however, are not closely related to those from Bilateria. In addition, cnidarian neuropeptides and their receptors are not closely related to those from Placozoa. We propose that the divergence times between Cnidaria, Placozoa, and Bilateria might be too long for recognizing sequence identities. Leucine-rich repeats-containing GPCRs (LGRs) are a special class of GPCRs that are characterized by a long N-terminus containing 10-20 leucine-rich domains, which are used for ligand binding. Among the ligands for LGRs are dimeric glycoprotein hormones, and insulin-like peptides, such as relaxin. LGRs have been found not only in Proto- and Deuterostomia, but also in early emerging phyla, such as Cnidaria and Placozoa. Humans have eight LGRs. In our current review, we have revisited the annotations of LGRs from the sea anemone Nematostella vectensis and the placozoan Trichoplax adhaerens. We identified 13 sea anemone LGRs and no less than 46 LGRs from T. adhaerens. All eight human LGRs appear to have orthologues in sea anemones and placozoans. LGRs and their ligands, therefore, have a long evolutionary history, going back to the common ancestor of Cnidaria and Placozoa.

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.

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria.

CaV1 and CaV2 voltage-gated calcium channels evolved from an ancestral CaV1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian CaV1 channels are uniquely subject to pronounced, buffer-resistant Ca2+/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca2+ rises. Early diverging, nonbilaterian invertebrates also possess CaV1 and CaV2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both CaV1 and CaV2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of CaV1 and CaV2 channels. Here, we build upon previous characterization of Trichoplax CaV channels by determining the cellular expression and ion-conducting properties of the CaV1 channel orthologue, TCaV1. We show that TCaV1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage-activated Ca2+ currents with kinetics resembling those of rat CaV1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCaV1, but not TCaV2, exhibits buffer-resistant Ca2+/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca2+ signaling within various cellular contexts.

Placozoan fiber cells: mediators of innate immunity and participants in wound healing.

Placozoa is a phylum of non-bilaterian marine animals. These small, flat organisms adhere to the substrate via their densely ciliated ventral epithelium, which mediates mucociliary locomotion and nutrient uptake. They have only six morphological cell types, including one, fiber cells, for which functional data is lacking. Fiber cells are non-epithelial cells with multiple processes. We used electron and light microscopic approaches to unravel the roles of fiber cells in Trichoplax adhaerens, a representative member of the phylum. Three-dimensional reconstructions of serial sections of Trichoplax showed that each fiber cell is in contact with several other cells. Examination of fiber cells in thin sections and observations of live dissociated fiber cells demonstrated that they phagocytose cell debris and bacteria. In situ hybridization confirmed that fiber cells express genes involved in phagocytic activity. Fiber cells also are involved in wound healing as evidenced from microsurgery experiments. Based on these observations we conclude that fiber cells are multi-purpose macrophage-like cells. Macrophage-like cells have been described in Porifera, Ctenophora, and Cnidaria and are widespread among Bilateria, but our study is the first to show that Placozoa possesses this cell type. The phylogenetic distribution of macrophage-like cells suggests that they appeared early in metazoan evolution.

Upregulation of DNA repair genes and cell extrusion underpin the remarkable radiation resistance of Trichoplax adhaerens.

Trichoplax adhaerens is the simplest multicellular animal with tissue differentiation and somatic cell turnover. Like all other multicellular organisms, it should be vulnerable to cancer, yet there have been no reports of cancer in T. adhaerens or any other placozoan. We investigated the cancer resistance of T. adhaerens, discovering that they are able to tolerate high levels of radiation damage (218.6 Gy). To investigate how T. adhaerens survive levels of radiation that are lethal to other animals, we examined gene expression after the X-ray exposure, finding overexpression of genes involved in DNA repair and apoptosis including the MDM2 gene. We also discovered that T. adhaerens extrudes clusters of inviable cells after X-ray exposure. T. adhaerens is a valuable model organism for studying the molecular, genetic, and tissue-level mechanisms underlying cancer suppression.

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.

Motility disorders and disintegration into separate cells of Trichoplax sp. H2 in the presence of Zn2+ ions and L-cysteine molecules: A systems approach.

Placozoa remain an ancient multicellular system with a dynamic body structure where calcium ions carry out a primary role in maintaining the integrity of the entire animal. Zinc ions can compete with calcium ions adsorption. We studied the effect of zinc ions and l-cysteine molecules on the interaction of Trichoplax sp. H2 cells. The regularity of formless motion was diminished in the presence of 20-25 µM of Zn2+ ions leading to the formation of branching animal forms. Locomotor ciliated cells moved chaotically and independently of each other leaving the Trichoplax body and opening a network of fiber cells. Application of 100 µM cysteine resulted in dissociation of the plate into separate cells. The combined chemical treatment shifted the effect in a random sample of animals toward disintegration, i.e. initially leading to disorder of collective cell movement and then to total body fragmentation. Two dissociation patterns of Trichoplax plate as "expanding ring" and "bicycle wheel" were revealed. Analysis of the interaction of Ca2+ and Zn2+ ions with cadherin showed that more than half (54%) of the amino acid residues with which Ca2+ and Zn2+ ions bind are common. The contact interaction of cells covered by the cadherin molecules is important for the coordinated movements of Trichoplax organism, while zinc ions are capable to break junctions between the cells. The involvement of other players, for example, l-cysteine in the regulation of Ca2+-dependent adhesion may be critical leading to the typical dissociation of Trichoplax body like in a calcium-free environment. A hypothesis about the essential role of calcium ions in the emergence of Metazoa ancestor is proposed.

Hidden cell diversity in Placozoa: ultrastructural insights from Hoilungia hongkongensis.

From a morphological point of view, placozoans are among the most simple free-living animals. This enigmatic phylum is critical for our understanding of the evolution of animals and their cell types. Their millimeter-sized, disc-like bodies consist of only three cell layers that are shaped by roughly seven major cell types. Placozoans lack muscle cells and neurons but are able to move using their ciliated lower surface and take up food in a highly coordinated manner. Intriguingly, the genome of Trichoplax adhaerens, the founding member of the enigmatic phylum, has disclosed a surprising level of genetic complexity. Moreover, recent molecular and functional investigations have uncovered a much larger, so-far hidden cell-type diversity. Here, we have extended the microanatomical characterization of a recently described placozoan species-Hoilungia hongkongensis. In H. hongkongensis, we recognized the established canonical three-layered placozoan body plan but also came across several morphologically distinct and potentially novel cell types, among them novel gland cells and "shiny spheres"-bearing cells at the upper epithelium. Thus, the diversity of cell types in placozoans is indeed higher than anticipated.

Paracellular and Transcellular Leukocytes Diapedesis Are Divergent but Interconnected Evolutionary Events.

Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.

Neural versus alternative integrative systems: molecular insights into origins of neurotransmitters.

Transmitter signalling is the universal chemical language of any nervous system, but little is known about its early evolution. Here, we summarize data about the distribution and functions of neurotransmitter systems in basal metazoans as well as outline hypotheses of their origins. We explore the scenario that neurons arose from genetically different populations of secretory cells capable of volume chemical transmission and integration of behaviours without canonical synapses. The closest representation of this primordial organization is currently found in Placozoa, disk-like animals with the simplest known cell composition but complex behaviours. We propose that injury-related signalling was the evolutionary predecessor for integrative functions of early transmitters such as nitric oxide, ATP, protons, glutamate and small peptides. By contrast, acetylcholine, dopamine, noradrenaline, octopamine, serotonin and histamine were recruited as canonical neurotransmitters relatively later in animal evolution, only in bilaterians. Ligand-gated ion channels often preceded the establishment of novel neurotransmitter systems. Moreover, lineage-specific diversification of neurotransmitter receptors occurred in parallel within Cnidaria and several bilaterian lineages, including acoels. In summary, ancestral diversification of secretory signal molecules provides unique chemical microenvironments for behaviour-driven innovations that pave the way to complex brain functions and elementary cognition. This article is part of the theme issue 'Basal cognition: multicellularity, neurons and the cognitive lens'.

Reafference and the origin of the self in early nervous system evolution.

Discussions of the function of early nervous systems usually focus on a causal flow from sensors to effectors, by which an animal coordinates its actions with exogenous changes in its environment. We propose, instead, that much early sensing was reafferent; it was responsive to the consequences of the animal's own actions. We distinguish two general categories of reafference-translocational and deformational-and use these to survey the distribution of several often-neglected forms of sensing, including gravity sensing, flow sensing and proprioception. We discuss sensing of these kinds in sponges, ctenophores, placozoans, cnidarians and bilaterians. Reafference is ubiquitous, as ongoing action, especially whole-body motility, will almost inevitably influence the senses. Corollary discharge-a pathway or circuit by which an animal tracks its own actions and their reafferent consequences-is not a necessary feature of reafferent sensing but a later-evolving mechanism. We also argue for the importance of reafferent sensing to the evolution of the body-self, a form of organization that enables an animal to sense and act as a single unit. This article is part of the theme issue 'Basal cognition: multicellularity, neurons and the cognitive lens'.

Microscopy Studies of Placozoans.

Trichoplax adhaerens is an enigmatic animal with an extraordinarily simple morphology and a cellular organization, which are the focus of current research. Protocols outlined here provide detailed descriptions of advanced techniques for light and electron microscopic studies of Trichoplax. Studies using these techniques have enhanced our understanding of cell type diversity and function in placozoans and have provided insight into the evolution, development, and physiology of this little understood group.

Functional Studies of Trichoplax adhaerens Voltage-Gated Calcium Channel Activity.

Trichoplax adhaerens is a member of the phylum Placozoa, an enigmatic group of benthic animals with remarkably simple morphology. While initial work on these organisms has primarily focused on their morphology and the development of genomic resources, Trichoplax has received increased attention as a model for studying the evolution of nervous and sensory systems. This work is motivated by the fact that Trichoplax features distinct behaviours and responses to environmental stimuli. Therefore, much progress has been made in recent years on the molecular, cellular, and behavioral understanding of this organism. Methods outlined here provide hands-on approaches to cutting edge molecular and cellular techniques to record cellular activities in Trichoplax.

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.