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Polycladida are the only free‐living flatworms with a planktonic larval stage in some species. Currently, it is not clear if a larval stage is ancestral in polyclads, and which type of larva that would be. Known polyclad larvae are Müller's larva, Kato's larva and Goette's larva, differing by body shape and the number of lobes and eyes. A valuable character for the comparison and characterisation of polyclad larval types is the ultrastructural composition of the apical organ. This organ is situated at the anterior pole of the larva and is associated with at least one ciliary tuft. The larval apical organ of Theama mediterranea features two multiciliated apical tuft sensory cells. Six unfurcated apical tuft gland cell necks are sandwiched between the apical tuft sensory cells and two anchor cells and have their cell bodies located lateral to the brain. Another type of apical gland cell necks is embedded in the anchor cells. Ventral to the apical tuft, ciliated sensory neurons are present, which are neighbouring the cell necks of two furcated apical tuft gland cells. Based on the ultrastructural organisation of the apical organ and other morphological features, like a laterally flattened wedge‐shaped body and three very small lobes, we recognise the larva of T. mediterranea as a new larval type, which we name Curini‐Galletti's larva after its first discoverer. The ultrastructural similarities of the apical organ in different polyclad larvae support their possible homology, that is, all polyclad larvae have likely evolved from a common larva. [ABSTRACT FROM AUTHOR]

The tiger flatworm Prostheceraeus crozieri (Polycladida) develops via an eight‐lobed, and three‐eyed planktonic Müller's larva. This larva has an apical organ, ultrastructural details of which remain elusive due to a scarcity of studies. The evolution and possible homology of the polyclad larva with other spiralian larvae is still controversial. Here, we provide ultrastructural data and three‐dimensional reconstructions of the apical organ of P. crozieri. The apical organ consists of an apical tuft complex and a dorso‐apical tuft complex. The apical tuft complex features a central tuft of five long cilia, which emerge from four or five individual cells that are themselves encircled by two anchor cells. The necks of six multibranched gland cells are sandwiched between ciliated tuft cell bodies and anchor cells. The proximal parts of the ciliated cell bodies are in contact with the lateral brain neuropil via gap junctions. Located dorsally of the apical tuft complex, the dorso‐apical tuft complex is characterized by several long cilia of sensory neurons, these emerge from an epidermal lumen and are closely associated with several gland cells that form a crescent apically around the dorsal anchor cell, and laterally touch the brain neuropil. Such ciliated sensory neurons emerging from a ciliated lumen are reminiscent of ampullary cells of mollusc and annelid larvae; a similar cell type can be found in the hoplonemertean decidula larva. We hypothesize that the ampullary‐like cells and the tuft‐forming sensory cells in the apical organs of these spiralian larvae could be homologous. [ABSTRACT FROM AUTHOR]

How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The transcriptional target of the Hippo pathway, the transcriptional coactivator YAP, inhibits the initiation of budding in Hydra and is regulated by Hydra LATS. In addition, we show functions of the Hippo pathway in regulation of actin organization and cell proliferation in Hydra. We hypothesize that the Hippo pathway served as a link between continuous cell division, cell density, and axis formation early in metazoan evolution. [ABSTRACT FROM AUTHOR]

Non-parasitic flatworms are known to temporarily attach to the substrate by secreting a multicomponent bioadhesive to counteract water movements. However, to date, only species of two higher-level flatworm taxa (Macrostomorpha and Proseriata) have been investigated for their adhesive proteins. Remarkably, the surface-binding protein is not conserved between flatworm taxa. In this study, we sequenced and assembled a draft genome, as well as a transcriptome, and generated a tail-specific positional RNA sequencing dataset of the polyclad Theama mediterranea. This led to the identification of 15 candidate genes potentially involved in temporary adhesion. Using in situ hybridisation and RNA interference, we determined their expression and function. Of these 15 genes, 4 are homologues of adhesion-related genes found in other flatworms. With this work, we provide two novel key components on the flatworm temporary adhesion system. First, we identified a Kringle-domain-containing protein (Tmed-krg1), which was expressed exclusively in the anchor cell. This in silico predicted membrane-bound Tmed-krg1 could potentially bind to the cohesive protein, and a knockdown led to a non-adhesive phenotype. Secondly, a secreted tyrosinase (Tmed-tyr1) was identified, which might crosslink the adhesive proteins. Overall, our findings will contribute to the future development of reversible synthetic glues with desirable properties for medical and industrial applications. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) ligands act as morphogens to direct patterning and proliferation during embryonic development. Protein kinase A (PKA) is a central negative regulator of Hh signalling, and in the absence of Hh ligands, PKA activity prevents inappropriate expression of Hh target genes. The orphan G-protein-coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA. Gpr161 acts as an A-kinase-anchoring protein, and is itself phosphorylated by PKA, but the functional significance of PKA phosphorylation of Gpr161 in the context of Hh signalling remains unknown. Here, we show that loss of Gpr161 in zebrafish leads to constitutive activation of medium and low, but not maximal, levels of Hh target gene expression. Furthermore, we find that PKA phosphorylation-deficient forms of Gpr161, which we show directly couple to Gαs, display an increased sensitivity to Shh, resulting in excess high-level Hh signalling. Our results suggest that PKA feedback-mediated phosphorylation of Gpr161 may provide a mechanism for fine-tuning Gpr161 ciliary localisation and PKA activity. [ABSTRACT FROM AUTHOR]

Terrestrial gastropods express metal‐selective metallothioneins (MTs) by which they handle metal ions such as Zn2+, Cd2+, and Cu+/Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu‐dependent MT and Hc pathways might be metabolically connected. In fact, the Cu‐specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interactions between the metal‐regulatory and detoxifying activity of MTs and the Cu demand of Hc isoforms was explored in the edible snail Cornu aspersum, one of the most common European helicid land snails. This species possesses CdMT and CuMT isoforms involved in metal‐selective physiological tasks. In addition, C. aspersum expresses three different Hc isoforms (CaH ɑD, CaH ɑN, CaH β). We have examined the effect of Cd2+ and Cu2+ exposure on metal accumulation in the midgut gland and mantle of C. aspersum, testing the impact of these metals on transcriptional upregulation of CdMT, CuMT, and the three Hc genes in the two organs. We found that the CuMT and CaH ɑD genes exhibit an organ‐specific transcriptional upregulation in the midgut gland of Cu‐exposed snails. These results are discussed in view of possible interrelationships between the metal‐selective activity of snail MT isoforms and the synthesis and metabolism of Hc isoforms. Highlights: Cadmium and copper are accumulated in the midgut gland and mantle of Cornu aspersum.Metallothionein (MT) genes show differential metal‐dependent upregulation patterns.Hemocyanin gene expression can be upregulated due to copper exposure. [ABSTRACT FROM AUTHOR]

Primordial germ cells (PGCs) are the precursors of germ cells, which migrate to the genital ridge during early development. Relatively little is known about PGCs after their migration. We studied this postmigratory stage using microscopy and sequencing techniques, and found that many PGC-specific genes, including genes known to induce PGC fate in the mouse, are only activated several days after migration. At this same time point, PGC nuclei become extremely gyrated, displaying general broad opening of chromatin and high levels of intergenic transcription. This is accompanied by changes in nuage morphology, expression of large loci (PGC-expressed noncoding RNA loci, PERLs) that are enriched for retro-transposons and piRNAs, and a rise in piRNA biogenesis signatures. Interestingly, no nuclear Piwi protein could be detected at any time point, indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post-migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation. [ABSTRACT FROM AUTHOR]

The graphene was doped by introducing atomized sulfur gas flow as sulfur source and acetylene as carbon source at 800 °C in chemical vapor deposition on copper foil. FE‐SEM, TGA, XPS and Raman tests were used to investigate the morphology, thermal behavior and surface properties of the synthesized graphene. A novel, facile and efficient methodology for the synthesis of 1,5–benzodiazepines by the reaction of chalcone with 1,2–phenylenediamine with the mediation of sulfur–doped graphene is described. This method is simple, efficient, environmentally benign, free of toxic catalysts, cost‐effective and has good yields for the formation of these seven‐membered heterocycles. [ABSTRACT FROM AUTHOR]

The membrane sphingolipid glucosylceramide (GlcCer) plays an important role in fungal fitness and adaptation to most diverse environments. Moreover, reported differences in the structure of GlcCer between fungi, plants and animals render this pathway a promising target for new generation therapeutics. Our knowledge about the GlcCer biosynthesis in fungi is mainly based on investigations of yeasts, whereas this pathway is less well characterized in molds. We therefore performed a detailed lipidomic profiling of GlcCer species present in Neurospora crassa and comprehensively show that the deletion of genes encoding enzymes involved in GlcCer biosynthesis affects growth, conidiation and stress response in this model fungus. Importantly, our study evidences that differences in the pathway intermediates and their functional role exist between N. crassa and other fungal species. We further investigated the role of GlcCer in the susceptibility of N. crassa toward two small cysteine-rich and cationic antimicrobial proteins (AMPs), PAF and PAFB, which originate from the filamentous ascomycete Penicillium chrysogenum. The interaction of these AMPs with the fungal plasma membrane is crucial for their antifungal toxicity. We found that GlcCer determines the susceptibility of N. crassa toward PAF, but not PAFB. A higher electrostatic affinity of PAFB than PAF to anionic membrane surfaces might explain the difference in their antifungal mode of action. [ABSTRACT FROM AUTHOR]

Combined exposure to engineered nanoparticles (ENPs) and anthropogenic contaminants can lead to changes in bioavailability, uptake and thus effects of both groups of contaminants. In this study we investigated effects of single and combined exposures of silver (Ag) nanoparticles (AgNPs) and the synthetic hormone 17α-ethinylestradiol (EE2) on tissue uptake of both contaminants in juvenile turbot ( Scophthalmus maximus ). Silver uptake and tissue distribution (gills, liver, kidney, stomach, muscle and bile) were analyzed following a 14-day, 2-h daily pulsed exposure to AgNPs (2 μg L −1 and 200 μg L −1 ), Ag + (50 μg L −1 ), EE2 (50 ng L −1 ) and AgNP + EE2 (2 or 200 μg L −1 +50 ng L −1 ). Effects of the exposures on plasma vitellogenin (Vtg) levels, EE2 and steroid hormone concentrations were investigated. The AgNP and AgNP + EE2 exposures resulted in similar Ag concentrations in the tissues, indicating that combined exposure did not influence Ag uptake in tissues. The highest Ag concentrations were found in gills. For the Ag + exposed fish, the highest Ag concentrations were measured in the liver. Our results show dissolution processes of AgNPs in seawater, indicating that the tissue concentrations of Ag may partly originate from ionic release. Plasma EE2 concentrations and Vtg induction were similar in fish exposed to the single contaminants and the mixed contaminants, indicating that the presence of AgNPs did not significantly alter EE2 uptake. Similarly, concentrations of most steroid hormones were not significantly altered due to exposures to the combined contaminants versus the single compound exposures. However, high concentrations of AgNPs in combination with EE2 caused a drop of estrone (E1) (female fish) and androstenedione (AN) (male and female fish) levels in plasma below quantification limits. Our results indicate that the interactive effects between AgNPs and EE2 are limited, with only high concentrations of AgNPs triggering synergistic effects on plasma steroid hormone concentrations in juvenile turbots. [ABSTRACT FROM AUTHOR]

Based on light and electron microscopy observations, oogenesis in the cotylean polyclad Prosthiostomum siphunculus was investigated for the first time. The numerous ovarian follicles are dispersed essentially in the dorsal parenchyma. In the follicles, a ventral germinative zone with undifferentiated germs cells of different sizes and a dorsal growth zone with larger growing and abortive oocytes are present. The oogenesis could be subdivided into four stages: (1) Oogonia with a dark nucleus and a dark, ribosome-rich cytoplasm. (2) Early oocyte stage, represented by relatively small cells (10 μm in diameter), a cytoplasm showing some mitochondria and some endoplasmic reticula. (3) Previtellogenic stage, with a decrease of the nucleo-cytoplasmic ratio due to the remarkable increase in ooplasm volume. Immature eggshell globules are observed. (4) Vitellogenic stage, including early vitellogenic ovarian stage, in which a second type of globule (inclusion globule) is formed, and a late vitellogenic uterine stage, in which the inclusion globules are not present anymore. The mature eggshell globules form a peripheral layer under the cell membrane. Eggshell and inclusion globules were analyzed with electron energy loss spectroscopy, electron spectroscopic imaging, protease treatment, and with periodic acid thiocarbohydracide silver proteinate to detect polysaccharides. Chromatoid bodies are present in all four stages. For the first time in a flatworm, we provide evidence that accessory cells, forming a tunica around the ovarian follicles, are epithelial or epithelium-like and likely contribute nutrients for the growth of the oocytes. [ABSTRACT FROM AUTHOR]

ABSTRACT Stem cells or undifferentiated cells can cope more easily with external stresses. To evaluate the impact of toxic compounds on stem cell dynamics in vivo, in relation to other biological responses, we use the carcinogenic element cadmium and the regenerating model organism Macrostomum lignano. Through both BrdU and anti-histone H3 immunostainings, cadmium-induced effects were investigated at different stages of the stem cell cycle. A 24-h exposure to 100 and 250 μM CdCl2 significantly decreased the number of stem cells (neoblasts) in mitosis, whereas the number of cells in the S phase remained unchanged. After this short-term exposure, the ultrastructure of the neoblasts was minimally affected in contrast to the epidermal tissues. These results were supported by gene expression data: transcripts of cdc2 and pig3 were significantly upregulated during all treatments. Both genes are involved in the cell cycle progression and are transcribed in the gonadal region, where stem cells are highly represented. Based on a substantial increase in gene expression of heat shock proteins (HSP) and their high activity in the gonadal region, we hypothesize that these proteins are key players in the protection of stem cells against external stresses. Apart from the strong HSP induction, other protective processes including cell division, apoptosis and anti-oxidative defence, were also activated. We, therefore, conclude that the protection of stem cells against external stressors may be based on the interplay between stem cell maintenance, i.e. repair and recovery through division, on one hand and apoptosis on the other hand. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1217-1228, 2016. [ABSTRACT FROM AUTHOR]

Background: Flatworms possess pluripotent stem cells that can give rise to all cell types, which allows them to restore lost body parts after injury or amputation. This makes flatworms excellent model systems for studying regeneration. In this study, we present the adhesive organs of a marine flatworm as a simple model system for organ regeneration. Macrostomum lignano has approximately 130 adhesive organs at the ventral side of its tail plate. One adhesive organ consists of three interacting cells: one adhesive gland cell, one releasing gland cell, and one modified epidermal cell, called an anchor cell. However, no specific markers for these cell types were available to study the regeneration of adhesive organs. Results: We tested 15 commercially available lectins for their ability to label adhesive organs and found one lectin (peanut agglutinin) to be specific to adhesive gland cells. We visualized the morphology of regenerating adhesive organs using lectin- and antibody staining as well as transmission electron microscopy. Our findings indicate that the two gland cells differentiate earlier than the connected anchor cells. Using EdU/lectin staining of partially amputated adhesive organs, we showed that their regeneration can proceed in two ways. First, adhesive gland cell bodies are able to survive partial amputation and reconnect with newly formed anchor cells. Second, adhesive gland cell bodies are cleared away, and the entire adhesive organ is build anew. Conclusion: Our results provide the first insights into adhesive organ regeneration and describe ten new markers for differentiated cells and tissues in M. lignano. The position of adhesive organ cells within the blastema and their chronological differentiation have been shown for the first time. M. lignano can regenerate adhesive organs de novo but also replace individual anchor cells in an injured organ. Our findings contribute to a better understanding of organogenesis in flatworms and enable further molecular investigations of cell-fate decisions during regeneration. [ABSTRACT FROM AUTHOR]

Apical-basal and planar cell polarities are hallmarks of metazoan epithelia required to separate internal and external environments and to regulate trans- and intracellular transport, cytoskeletal organization, and morphogenesis. Mechanisms of cell polarization have been intensively studied in bilaterian model organisms, particularly in early embryos and cultured cells, while cell polarity in pre-bilaterian tissues is poorly understood. Here, we have studied apical-basal and planar polarization in regenerating (aggregating) clusters of epitheliomuscular cells of Hydra , a simple representative of the ancestral, pre-bilaterian phylum Cnidaria. Immediately after dissociation, single epitheliomuscular cells do not exhibit cellular polarity, but they polarize de novo during aggregation. Reestablishment of the Hydra -specific epithelial bilayer is a result of short-range cell sorting. In the early phase of aggregation, apical-basal polarization starts with an enlargement of the epithelial apical-basal diameter and by the development of belt-like apical septate junctions. Specification of the basal pole of epithelial cells occurs shortly later and is linked to synthesis of mesoglea, development of hemidesmosome-like junctions, and formation of desmosome-like junctions connecting the basal myonemes of neighbouring cells. Planar polarization starts, while apical-basal polarization is already ongoing. It is executed gradually starting with cell-autonomous formation, parallelization, and condensation of myonemes at the basal end of each epithelial cell and continuing with a final planar alignment of epitheliomuscular cells at the tissue level. Our findings reveal that epithelial polarization in Hydra aggregates occurs in defined steps well accessible by histological and ultrastructural techniques and they will provide a basis for future molecular studies. [ABSTRACT FROM AUTHOR]

This is the first study investigating spermatogenesis and spermatozoan ultrastructure in the polyclad flatworm Prosthiostomum siphunculus. The testes are numerous and scattered as follicles ventrally between the digestive ramifications. Each follicle contains the different stages of sperm differentiation. Spermatocytes and spermatids derive from a spermatogonium and the spermatids remain connected by intercellular bridges. Chromatoid bodies are present in the cytoplasm of spermatogonia up to spermatids. During early spermiogenesis, a differentiation zone appears in the distal part of spermatids. A ring of microtubules extends along the entire sperm shaft just beneath the cell membrane. An intercentriolar body is present and gives rise to two axonemes, each with a 9 + '1' micro-tubular pattern. Development of the spermatid leads to cell elongation and formation of a filiform, mature spermatozoon with two free flagella and with cortical microtubules along the sperm shaft. The flagella exit the sperm shaft at different levels, a finding common for acotyleans, but so far unique for cotylean polyclads. The Golgi complex produces numerous electron-dense bodies of two types and of different sizes. These bodies are located around a perinuclear row of mitochondria. The elongated nucleus extends almost along the entire sperm body. The nucleus is wide in the proximal part and becomes narrow going towards the distal end. Thread-like chromatin mixed with electron-dense intranuclear spindle-shaped bodies are present throughout nucleus. The general sperm ultrastructure, the presence of intranuclear bodies and a second type of cytoplasmic electron-dense bodies may provide characters useful for phylogenetic analysis. [ABSTRACT FROM AUTHOR]

Background Free-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process. Results In this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horseshoe- shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin. Conclusion Our findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of the animals to adhere. The RNAi mediated changes of the anchor cell morphology are comparable to situations observed in human gut epithelia. Therefore, our current findings and future investigations using this powerful flatworm model system might contribute to a better understanding of the function of intermediate filaments and their associated human diseases. [ABSTRACT FROM AUTHOR]

Introduction Regeneration is a widespread phenomenon in the animal kingdom, but the capacity to restore damaged or missing tissue varies greatly between different phyla and even within the same phylum. However, the distantly related Acoelomorpha and Platyhelminthes share a strikingly similar stem-cell system and regenerative capacity. Therefore, comparing the underlying mechanisms in these two phyla paves the way for an increased understanding of the evolution of this developmental process. To date, Isodiametra pulchra is the most promising candidate as a model for the Acoelomorpha, as it reproduces steadily under laboratory conditions and is amenable to various techniques, including the silencing of gene expression by RNAi. In order to provide an essential framework for future studies, we report the succession of regeneration events via the use of cytochemical, histological and microscopy techniques, and specify the total number of cells in adult individuals. Results Isodiametra pulchra is not capable of regenerating a new head, but completely restores all posterior structures within 10 days. Following amputation, the wound closes via the contraction of local muscle fibres and an extension of the dorsal epidermis. Subsequently, stem cells invade the wound area and form a loosely delimited blastema. After two days, the posterior end is re-patterned with the male (and occasionally the female) genital primordium being apparent. Successively, these primordia differentiate into complete copulatory organs. The size of the body and also of the male and female copulatory organs, as well as the distance between the copulatory organs, progressively increase and by nine days copulation is possible. Adult individuals with an average length of 670 μm consist of approximately 8100 cells. Conclusion Isodiametra pulchra regenerates through a combination of morphallactic and epimorphic processes. Existing structures are "re-modelled" and provide a framework onto which newly differentiating cells are added. Growth proceeds through the intercalary addition of structures, mirroring the embryonic and post-embryonic development of various organ systems. The suitability of Isodiametra pulchra for laboratory techniques, the fact that its transcriptome and genome data will soon be available, as well as its small size and low number of cells, make it a prime candidate subject for research into the cellular mechanisms that underlie regeneration in acoelomorphs. [ABSTRACT FROM AUTHOR]