Acoel Single-Cell Transcriptomics: Cell Type Analysis of a Deep Branching Bilaterian.

Bilaterian animals display a wide variety of cell types, organized into defined anatomical structures and organ systems, which are mostly absent in prebilaterian animals. Xenacoelomorpha are an early-branching bilaterian phylum displaying an apparently relatively simple anatomical organization that have greatly diverged from other bilaterian clades. In this study, we use whole-body single-cell transcriptomics on the acoel Isodiametra pulchra to identify and characterize different cell types. Our analysis identifies the existence of ten major cell type categories in acoels all contributing to main biological functions of the organism: metabolism, locomotion and movements, behavior, defense, and development. Interestingly, although most cell clusters express core fate markers shared with other animal clades, we also describe a surprisingly large number of clade-specific marker genes, suggesting the emergence of clade-specific common molecular machineries functioning in distinct cell types. Together, these results provide novel insight into the evolution of bilaterian cell types and open the door to a better understanding of the origins of the bilaterian body plan and their constitutive cell types.

Immunostaining and In Situ Hybridization of the Developing Acoel Nervous System.

The study of acoel morphologies has been recently stimulated by the knowledge that this group of animals represents an early offshoot of the Bilateria. Understanding how organ systems and tissues develop and the molecular underpinnings of the processes involved has become an area of new research. The microscopic anatomy of these organisms is best understood through the systematic use of immunochemistry and in situ hybridization procedures. These methods allow us to map, in precise detail, the expression patterns of genes and proteins, in space and time. With the additional use of genomic resources, they provide us with insights on how a group of "early" bilaterians have diversified over time. As these animals are new to the world of molecular studies, the protocols have involved a lot of new and specific adaptations to their specific anatomical-histological characteristics. Here we explain some of these protocols in detail, with the aim that should prove useful in our much-needed understanding of the origins of bilaterian animals. An anatomical sketch is provided at the beginning as a necessary guide for those not familiar with the Acoela.

Confirmation and distribution of tetrodotoxin for the first time in terrestrial invertebrates: two terrestrial flatworm species (Bipalium adventitium and Bipalium kewense).

The potent neurotoxin tetrodotoxin (TTX) is known from a diverse array of taxa, but is unknown in terrestrial invertebrates. Tetrodotoxin is a low molecular weight compound that acts by blocking voltage-gated sodium channels, inducing paralysis. However, the origins and ecological functions of TTX in most taxa remain mysterious. Here, we show that TTX is present in two species of terrestrial flatworm (Bipalium adventitium and Bipalium kewense) using a competitive inhibition enzymatic immunoassay to quantify the toxin and high phase liquid chromatography to confirm the presence. We also investigated the distribution of TTX throughout the bodies of the flatworms and provide evidence suggesting that TTX is used during predation to subdue large prey items. We also show that the egg capsules of B. adventitium have TTX, indicating a further role in defense. These data suggest a potential route for TTX bioaccumulation in terrestrial systems.

Some aspects of the immunolocalization of FMRFamide in the nervous system of turbellarians, Polycelis tenuis and Girardia tigrina. Short communication.

The details of the morphology of the nervous system has been investigated in two turbellarian species Polycelis tenuis and Girardia tigrina using confocal laser scanning microscopy and immunostaining to neuropeptide FMRFamide. Abundant FMRFamide immunoreactivity (FMRF-IR) has been observed in central and peripheral nervous systems of both species. Intensive staining has been found in the sensory elements: cells and fibres surrounded the mouth opening, in the fibres enclosed the photoreceptors, triangular auricles in the head region of G. tigrina. The possible function of FMRF-IR neurons in the realization of sensory function in turbellarians is discussed.

NF-Y and the transcriptional activation of CCAAT promoters.

The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the "innovative" NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y - also termed CBF and CP1 - is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; Hatamochi et al., 1988; Hooft van Huijsduijnen et al, 1987; Kim & Sheffery, 1990). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements (Suzuki et al., 2001, 2004; Elkon et al., 2003; Mariño-Ramírez et al., 2004; FitzGerald et al., 2004; Linhart et al., 2005; Zhu et al., 2005; Lee et al., 2007; Abnizova et al., 2007; Grskovic et al., 2007; Halperin et al., 2009; Häkkinen et al., 2011). The same consensus, as determined by mutagenesis and SELEX studies (Bi et al., 1997), was also retrieved in ChIP-on-chip analysis (Testa et al., 2005; Ceribelli et al., 2006; Ceribelli et al., 2008; Reed et al., 2008). Additional structural features of the CCAAT box - position, orientation, presence of multiple Transcriptional Start Sites - were previously reviewed (Dolfini et al., 2009) and will not be considered in detail here.

Expression of a neuropeptide similar to allatotropin in free living turbellaria (platyhelminthes).

Mechanisms coordinating cell-cell interaction have appeared early in evolution. Allatotropin (AT), a neuropeptide isolated based on its ability to stimulate the synthesis of juvenile hormones (JHs) in insects has also been found in other invertebrate phyla. Despite this function, AT has proved to be myotropic. In the present study we analyze its expression in two groups of Turbellaria (Catenulida, Macrostomida), and its probable relationship with muscle tissue. The results show the presence of an AT-like peptide in the free living turbellaria analyzed. The analysis of the expression of the peptide together with phalloidin, suggests a functional relationship between the peptide and muscle tissue, showing that it could be acting as a myoregulator. The finding of immunoreactive fibers associated with sensory organs like ciliated pits in Catenulida and eyes in Macrostomida makes probable that AT could play a role in the physiological mechanisms controlling circadian activities. Furthermore, the existence of AT in several phyla of Protostomata suggests that this peptide could be a synapomorphic feature of this group. Indeed, the presence in organisms that do not undergo metamorphosis, could be signaling that it was first involved in myotropic activities, being the stimulation of the synthesis of JHs a secondary function acquired by the phylum Arthropoda.

Molecular architecture of muscles in an acoel and its evolutionary implications.

We have characterized the homologs of an actin, a troponin I, and a tropomyosin gene in the acoel Symsagittifera roscoffensis. These genes are expressed in muscles and most likely coexpressed in at least a subset of them. In addition, and for the first time for Acoela, we have produced a species-specific muscular marker, an antibody against the tropomyosin protein. We have followed tropomyosin gene and protein expression during postembryonic development and during the posterior regeneration of amputated adults, showing that preexisting muscle fibers contribute to the wound closure. The three genes characterized in this study interact in the striated muscles of vertebrates and invertebrates, where troponin I and tropomyosin are key regulators of the contraction of the sarcomere. S. roscoffensis and all other acoels so far described have only smooth muscles, but the molecular architecture of these is the same as that of striated fibers of other bilaterians. Given the proposed basal position of acoels within the Bilateria, we suggest that sarcomeric muscles arose from a smooth muscle type, which had the molecular repertoire of striated musculature already in place. We discuss this model in a broad comparative perspective.

The female gonad in two species of Microplana (Platyhelminthes, Tricladida, Rhynchodemidae): ultrastructural and cytochemical investigations.

The female gonad of the land planarians Microplana scharffi and Microplana terrestris consists of two small germaria located ventrally in the anterior third of the body and of two ventro-lateral rows of oblong vitelline follicles distributed between the intestinal pouches. Both these structures are enveloped by a tunica composed of an outer extracellular lamina and an inner sheath of accessory cells. Oocyte maturation is characterized by the appearance of chromatoid bodies and the development of endoplasmic reticulum and Golgi complexes. These organelles appear to be correlated with the production of egg granules with a fenestrated/granular content of medium electron density, about 4-5 mum in diameter, which remain dispersed in the ooplasm of mature oocytes. On the basis of cytochemical tests showing their glycoprotein composition, and their localization in mature oocytes, these egg granules have been interpreted as yolk. In the vitelline follicles, vitellocytes show the typical features of secretory cells with well-developed rough endoplasmic reticulum and Golgi complexes involved in the production of eggshell globules and yolk. The eggshell globules, which appear to arise from repeated coalescences of two types of Golgi-derived vesicles, contain polyphenols and, when completely mature, they measure about 1-1,2 mum in diameter and show a meandering/concentric content pattern as is typical of the situation observed in most Proseriata and Tricladida. Mature vitellocytes also contain a large amount of glycogen and lipids as further reserve material. On the basis of the ultrastructural features of the female gonad and in relation to the current literature the two species of rhynchodemids investigated appear to be closely related to the freshwater planarians belonging to the family Dugesiidae.

Smed-Evi/Wntless is required for beta-catenin-dependent and -independent processes during planarian regeneration.

Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), we analyzed RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands. We show that, during regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-beta-catenin1 RNAi animals. In addition, we observe regeneration defects of the nervous system that are not found after Smed-beta-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, we identify Smed-WntP-1 and Smed-Wnt11-2 as the putative posterior organizers, and demonstrate that Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, our study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through beta-catenin-dependent and -independent pathways.

A naturally occurring omega current in a Kv3 family potassium channel from a platyhelminth.

BACKGROUND: Voltage-gated ion channels are membrane proteins containing a selective pore that allows permeable ions to transit the membrane in response to a change in the transmembrane voltage. The typical selectivity filter in potassium channels is formed by a tetrameric arrangement of the carbonyl groups of the conserved amino-acid sequence Gly-Tyr-Gly. This canonical pore is opened or closed by conformational changes that originate in the voltage sensor (S4), a transmembrane helix with a series of positively charged amino acids. This sensor moves through a gating pore formed by elements of the S1, S2 and S3 helices, across the plane of the membrane, without allowing ions to pass through the membrane at that site. Recently, synthetic mutagenesis studies in the Drosophila melanogaster Shaker channel and analysis of human disease-causing mutations in sodium channels have identified amino acid residues that are integral parts of the gating-pore; when these residues are mutated the proteins allow a non-specific cation current, known as the omega current, to pass through the gating-pore with relatively low selectivity. RESULTS: The N.at-Kv3.2 potassium channel has an unusual weak inward rectifier phenotype. Several mutations of two amino acids in the voltage sensing (S4) transmembrane helix change the phenotype to a typical delayed rectifier. The inward rectifier channels (wild-type and mutant) are sensitive to 4-aminopyridine (4-AP) but not tetra-ethyl ammonium (TEA), whereas the delayed rectifier mutants are sensitive to TEA but not 4-AP. The inward rectifier channels also manifest low cation selectivity. The relative selectivity for different cations is sensitive to specific mutations in the S4 helix, CONCLUSION: N.at-Kv3.2, a naturally occurring potassium channel of the Kv3 sequence family, mediates ion permeation through a modified gating pore, not the canonical, highly selective pore typical of potassium channels. This channel has evolved to yield qualitatively different ion permeability when compared to all other members of this gene family.

Amphiphilic copolymer for delivery of xenobiotics: in vivo studies in a freshwater invertebrate, a Mesostominae flatworm.

The synthesis of an amphiphilic polymethacrylate copolymer containing cholesterol hydrophobic moieties and rhodamine as a fluorescent probe, the formation of microspheres, and the uptake of these microspheres in an invertebrate are reported. The cholesterol-derived methacryloyl monomer, which was prepared via a one-step synthesis, was copolymerized with methacrylic acid and methacryloxyethyl thiocarbamoyl rhodamine B in the presence of AIBN as initiator. The obtained dye-labeled copolymer was characterized by (1)H NMR and UV-vis spectroscopy. Fluorescence and TEM microscopies studies show that this amphiphilic copolymer aggregates to give microspheres with diameters ranging from approximately 3 to 11 microm. The in vivo study in a freshwater invertebrate, a Mesostominae flatworm (Rhabdocoela, Thyphloplanidae), indicates that the microspheres enter the cells by endocytosis. The data collected demonstrate that the rhodamine B covalently attached to the amphiphilic copolymers is bioaccumulated without being translocated out of the cell by the multixenobiotic resistance (MXR) transporters. As the MXR system is similar to the multidrug resistance (MDR) first observed in tumor cell lines resistant to anticancer drugs, the present data confirm the significant role that amphiphilic copolymers can play in the ongoing development of drug delivery strategies to overcome multidrug resistance. These investigations illustrate a promising approach for the development of new medical and ecotoxicological tools that can deliver specific molecules within cells.

[Activities and properties of phosphorylases of turbellariae Phagocata sibirica and cestodes Bothriocephalus scorpii].

Activities and properties of phosphorylases of cytosol and mitochondrial fractions are studied in free-living turbellaria Phagocata sibirica and cestodes Bothriocephalus scorpii. The phosphorylase activities in P. sibirica and B. scorpii differ significantly both in form and the total activity of this enzyme. Dependence of the phosphorylase reaction rate on substrate concentration is studied. The high activity of phosphorylase as compared with that of hexokinase suggests glycogen to be the main energy source of the studied flatworms. Effects of various effectors on activities of cytosol and mitochondrial phosphorylases are studied.

FMRFamide- and neurotensin-immunoreactive elements in the intestine of some polyclad and triclad flatworms (Turbellaria).

By means of immunohistochemistry with antisera to tetrapeptide FMRFamide and regulatory peptides neurotensin and calcitonin intestines of marine turbellarians Notoplana atomata, N. humilis (Polycladida) and Procerodes littoralis (Tricladida) were investigated. In all flatworms polymorphous cells and processes reacting with antibodies to FMRFamide and neurotensin but not with calcitonin were revealed. These cell elements are localized both in the epithelium and beneath it. FMRFamide-immunoreactive cells and processes of investigated turbellarians and neurotensin-immunoreactive elements in P. littoralis obviously belong to the nervous system, while intraepithelial neurotensin-immunoreactive cells of polyclads share some morphological features with endocrine-like cells.

Nutritional and respiratory pathways to parasitism exemplified in the Turbellaria.

Symbiosis is a dominant trait in the Platyhelminthes. The Neodermata (Aspidogastrea, Monogenea, Digenea, Udonellidea, Cestoda) are wholly parasitic and even the predominantly free-living Turbellaria have almost 200 species from 35 families living in permanent associations with other animals. In the simplest turbellarian symbioses, ectosymbiotes such as the Temnocephalida, some other Rhabdocoela and a few Tricladida live on the body surfaces or in the branchial chambers of their mainly arthropodan or chelonian hosts. They feed on the same types of prey as their free-living relatives but supplement their diet by opportunistic commensalism. Their digestive physiology and food reserves are the same as in free-living species. The entosymbiotic Umagillidae, Graffillidae, Pterastericolidae, Fecamplidae and Acholadidae live in internal body cavities or body wall derivatives of echinoderms, molluscs or arthropods and show increasing metabolic dependence on their hosts. Patterns of digestive physiology and food storage generally differ markedly from those of ectosymbiotic and free-living species. Some umagillids, in echinoids, feed as entozoic predators on co-symbiotic protozoa, supplemented by opportunistic ingestion of the hosts' ingesta, gut cells or coelomocytes. Others, in holothurians, feed mainly on gut cells, which also provide some digestive enzymes, and to a lesser extent on host ingesta and co-symbiotes. Graffillids, in molluscs, lack endogenous digestive enzymes and rely entirely on those taken in with host ingesta and gut tissues. Pterastericolids, in asteroids, similarly utilise gut tissues both as food and enzyme sources. The climax to metabolic dependence occurs in the Fecamplidae and Acholadidae. The former, in crustacean haemocoels and myzostomid tissues, lack conventional alimentary systems and absorb soluble nutrients through the epidermis. In the latter the only known species lives in the tube feet of its asteroid host, lacks a normal endodermal gut, but has a modified epidermis performing both digestive and absorptive functions. Most of these entosymbiotes show a shift from the lipid storage characteristic of free-living and ectosymbiotic species to the glycogen storage predominating in the Neodermata. In both groups this emphasis on carbohydrate metabolism is often independent of the PO2 of their environment. Both groups also show high fecundity and it is suggested that there is a direct relationship between this and glycogen storage. High fecundity, while clearly of adaptive value in entosymbiotes, is arguably primarily related to the assured food supply conferred by the entosymbiotic habit and thus can be viewed as a consequence of the latter rather than a prerequisite for it. Some entosymbiotic Turbellaria have evolved physiologically active haemoglobins, allowing them to abstract oxygen preferentially from host tissues; some have also evolved facultative glycolytic mechanisms comparable to those of the Cestoda. All these adaptations to ecto- and entosymbiotic life in the Turbellaria exemplify possible pathways to wholly parasitic lifestyles, with total metabolic dependence on the hosts, which may have been followed during the evolution of the Neodermata.

The nervous system of Tricladida. I. Neuroanatomy of Procerodes littoralis (Maricola, Procerodidae): an immunocytochemical study.

The organization of the nervous system of Procerodes littoralis (Tricladida, Maricola, Procerodidae) was studied by immunocytochemistry, using antibodies to authentic flatworm neuropeptide F (NPF) (Moniezia expansa). Compared to earlier investigations of the neuroanatomy of tricladid flatworms, the pattern of NPF immunoreactivity in Procerodes littoralis reveals differences in the following respects: 1. Shape and structure of the brain. 2. Number and composition of longitudinal nerve cords. 3. Shape of branches of, and transverse connections between, main ventral nerve cords. 4. Composition of the pharyngeal nervous system. The rich innervation by NPF immunoreactive (IR) fibres and cells of the subepithelial muscle layer, the pharynx musculature and the musculature of the male copulatory apparatus indicates a neurotransmitter or neuromodulatory influence on muscular activity.

The nervous system of Tricladida. II. Neuroanatomy of Dugesia tigrina (Paludicola, Dugesiidae): an immunocytochemical study.

The nervous system (NS) of Dugesia tigrina has been studied by immunocytochemical double-staining, using the authentic flatworm neuropeptide, neuropeptide F (NPF), and serotonin (5-HT) on cryosections. This technique provides a precise morphological (descriptive) account of the NS. The results show that the central nervous system is shaped like a horseshoe. The brain is composed of two lateral lobes connected by three commissures, one antero-dorsal in front of the cerebral eyes and two, more ventral, behind the eyes. The pair of main nerve cords extend from the lateral lobes of the brain to the tail end of the worm. Cross sections reveal a very close contact between lateral branches from the main cords and the submuscular plexus. Thin cord-like lateral nerves are formed by longitudinal plexal fibres. No dorsal cords were observed. The patterns of immunoreactivity to NPF and 5-HT differ from each other in several respects. In the walls of gut diverticula only NPF immunoreactive (IR) cells and fibres were observed. Only NPF-immunoreactive cells occur in the parenchyma along dorso-ventral nerve fibres connecting the dorsal and ventral parts of the submuscular plexus. The number of 5-HT-immunoreactive cells associated with the main nerve cords (MCs) is greater than that of the NPF-immunoreactive cells, and the spongy structure of the MCs is more apparent following immunostaining for 5-HT. Thin 5-HT-immunoreactive fibres were observed in the subepithelial plexus, penetrating the basal lamina and innervating a rhabdite-free ventro-lateral sensory area along the body periphery. The correspondence between MCs in the lower flatworms (Catenulida and Macrostomida) and the Seriata (Tricladida and Proseriata) confirms the status of the MCs in flatworms as the most important and stable neuronal characteristic, and constitutes support for the hypothesized common origin of the MCs in flatworms.

Eggshell formation in Bdelloura candida, an ectoparasitic turbellarian of the horseshoe crab Limulus polyphemus.

The marine triclad Bdelloura candida (Turbellaria) episodically deposits stalked eggshells onto the gill lamellae of the horseshoe crab Limulus polyphemus. Ultrastructural and biochemical analyses indicate that the eggshells consist of primary inner and secondary outer layers. Protein of the primary layer is rich in glycine, aspartate/asparagine, serine, and 3,4-dihydroxyphenylalanine (Dopa). In this regard it resembles the eggshell compositions of other members of the phylum Platyhelminthes. The primary layer appears to be derived from precursors produced in the vitelline cells of the flatworm. Each egg-laying episode consumes all of the precursor-containing vitelline cells. A Dopa-containing protein, vitelline protein 1 (Vp1), has been isolated from B. candida and resembles the primary eggshell layer in its composition. Vp1 has an apparent molecular weight of 34.4 and an acidic pI. Dopa-containing proteins are presumed to be directly involved in the cross-linking reactions that accompany quinone-tanning. The secondary outer layer of the eggshell may serve to adhere the eggshell to the gill. Its amino acid composition is unlike that of the primary eggshell and nothing is known about its precursors.

Demonstration by immunocytochemical staining of a somatostatin-28-(1-14)-like peptide in planarians (Plathyhelminthes Turbellaria Tricladida).

A specific polyclonal antiserum directed against the somatostatin-28(1-14) of vertebrates was applied to sections of the planarians Dugesia lugubris and Dendrocoelum lacteum. This made it possible to reveal nerve cells and processes specifically both in cerebral ganglia and in ventral nerve cords. The phylogenetic importance of this demonstration is pointed out.

Photoperiodic modulation of cephalic melatonin in planarians.

Endogenous melatonin was detected by high-performance liquid chromatography (HPLC) with electrochemical detection (EC) in the head of the planarian Dugesia dorotocephala. The identity of this elution peak was further confirmed by radioimmunoassay. In groups of planarians adapted to either normal or reversed photoperiods, the melatonin levels were always higher in those heads collected in the dark period than in those collected in the light period. This indicates that primitive animals such as planarians have already evolved a melatonin-metabolizing system that is photically driven in a manner suggestive of the way melatonin synthesis is influenced by light and dark cycles in vertebrates.