The Hippo pathway regulates axis formation and morphogenesis in Hydra.

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.

Feedback control of the Gpr161-Gαs-PKA axis contributes to basal Hedgehog repression in zebrafish.

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.

Extensive nuclear gyration and pervasive non-genic transcription during primordial germ cell development in zebrafish.

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 post-migratory 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 non-coding 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.

The ultrastructure of the apical organ of Curini-Galletti's larva, a new polyclad larval type.

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.

Empagliflozin protects mice against diet-induced obesity, insulin resistance and hepatic steatosis.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter 2 (SGLT2) inhibitors are widely used in the treatment of type 2 diabetes, heart failure and chronic kidney disease. Their role in the prevention of diet-induced metabolic deteriorations, such as obesity, insulin resistance and fatty liver disease, has not been defined yet. In this study we set out to test whether empagliflozin prevents weight gain and metabolic dysfunction in a mouse model of diet-induced obesity and insulin resistance. METHODS: C57Bl/6 mice were fed a western-type diet supplemented with empagliflozin (WDE) or without empagliflozin (WD) for 10 weeks. A standard control diet (CD) without or with empagliflozin (CDE) was used to control for diet-specific effects. Metabolic phenotyping included assessment of body weight, food and water intake, body composition, hepatic energy metabolism, skeletal muscle mitochondria and measurement of insulin sensitivity using hyperinsulinaemic-euglycaemic clamps. RESULTS: Mice fed the WD were overweight, hyperglycaemic, hyperinsulinaemic and insulin resistant after 10 weeks. Supplementation of the WD with empagliflozin prevented these metabolic alterations. While water intake was significantly increased by empagliflozin supplementation, food intake was similar in WDE- and WD-fed mice. Adipose tissue depots measured by MRI were significantly smaller in WDE-fed mice than in WD-fed mice. Additionally, empagliflozin supplementation prevented significant steatosis found in WD-fed mice. Accordingly, hepatic insulin signalling was deteriorated in WD-fed mice but not in WDE-fed mice. Empagliflozin supplementation positively affected size and morphology of mitochondria in skeletal muscle in both CD- and WD-fed mice. CONCLUSIONS/INTERPRETATION: Empagliflozin protects mice from diet-induced weight gain, insulin resistance and hepatic steatosis in a preventative setting and improves muscle mitochondrial morphology independent of the type of diet.

The ultrastructure of the apical organ of the Müller's larva of the tiger flatworm Prostheceraeus crozieri.

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.

Sticking Together an Updated Model for Temporary Adhesion.

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.

A mechanism for temporary bioadhesion.

The flatworm Macrostomum lignano features a duo-gland adhesive system that allows it to repeatedly attach to and release from substrates in seawater within a minute. However, little is known about the molecules involved in this temporary adhesion. In this study, we show that the attachment of M. lignano relies on the secretion of two large adhesive proteins, M. lignano adhesion protein 1 (Mlig-ap1) and Mlig-ap2. We revealed that both proteins are expressed in the adhesive gland cells and that their distribution within the adhesive footprints was spatially restricted. RNA interference knockdown experiments demonstrated the essential function of these two proteins in flatworm adhesion. Negatively charged modified sugars in the surrounding water inhibited flatworm attachment, while positively charged molecules impeded detachment. In addition, we found that M. lignano could not adhere to strongly hydrated surfaces. We propose an attachment-release model where Mlig-ap2 attaches to the substrate and Mlig-ap1 exhibits a cohesive function. A small negatively charged molecule is secreted that interferes with Mlig-ap1, inducing detachment. These findings are of relevance for fundamental adhesion science and efforts to mitigate biofouling. Further, this model of flatworm temporary adhesion may serve as the starting point for the development of synthetic reversible adhesion systems for medicinal and industrial applications.

In Vitro Investigation of Thiolated Chitosan Derivatives as Mucoadhesive Coating Materials for Solid Lipid Nanoparticles.

In the present study, chitosan (CS) was thiolated by introducing l-cysteine via amide bond formation. Free thiol groups were protected with highly reactive 6-mercaptonicotinic acid (6-MNA) and less-reactive l-cysteine, respectively, via thiol/disulfide-exchange reactions. Unmodified CS, l-cysteine-modified thiolated CS (CS-Cys), 6-MNA-S-protected thiolated CS (CS-Cys-MNA), and l-cysteine-S-protected thiolated CS (CS-Cys-Cys) were applied as coating materials to solid lipid nanoparticles (SLN). The strength of mucus interaction followed the rank order plain < CS < CS-Cys-Cys < CS-Cys < CS-Cys-MNA, whereas mucus diffusion followed the rank order CS-Cys < CS-Cys-Cys < CS < CS-Cys-MNA < plain. In accordance with lower reactivity, CS-Cys-Cys-coated SLN were immobilized to a lower extent than CS-Cys-coated SLN, while CS-Cys-MNA-coated SLN dissociated from their coating material resulting in a similar diffusion behavior as plain SLN. Consequently, CS-Cys-Cys-coated SLN and CS-Cys-MNA-coated SLN showed the highest retention on porcine intestinal mucosa by enabling a synergism of efficient mucus diffusion and strong mucoadhesion.

Proposal of Thermoactinomyces mirandus sp. nov., a filamentous, anaerobic bacterium isolated from a biogas plant.

We isolated a filamentous, thermophilic, and first anaerobic representative of the genus Thermoactinomyces, designated strain AMNI-1T, from a biogas plant in Tyrol, Austria and report the results of a phenotypic, genetic, and phylogenetic investigation. Strain AMNI-1T was observed to form a white branching mycelium that aggregates into pellets when grown in liquid medium. Cells could primarily utilize lactose, glucose, and mannose as carbon and energy sources, with acetate accelerating and yeast extract being mandatory for growth. The optimum growth temperature and pH turned out to be 55 °C and pH 7.0, respectively, with an optimum NaCl concentration of 0-2% (w/v). 16S rRNA gene sequence comparison indicated that the genetic relatedness between strain AMNI-1T and Thermoactinomyces intermedius, Thermoactinomyces khenchelensis, and Thermoactinomyces vulgaris was less than 97%. The G + C content of the genomic DNA was 44.7 mol%. The data obtained suggest that the isolate represents a novel and first anaerobic species of the genus Thermoactinomyces, for which the name Thermoactinomyces mirandus is proposed. The type strain is AMNI-1T (= DSM 110094T = LMG 31503T). The description of the genus Thermoactinomyces is emended accordingly.

(Un)expected Similarity of the Temporary Adhesive Systems of Marine, Brackish, and Freshwater Flatworms.

Many free-living flatworms have evolved a temporary adhesion system, which allows them to quickly attach to and release from diverse substrates. In the marine Macrostomum lignano, the morphology of the adhesive system and the adhesion-related proteins have been characterised. However, little is known about how temporary adhesion is performed in other aquatic environments. Here, we performed a 3D reconstruction of the M. lignano adhesive organ and compared it to the morphology of five selected Macrostomum, representing two marine, one brackish, and two freshwater species. We compared the protein domains of the two adhesive proteins, as well as an anchor cell-specific intermediate filament. We analysed the gene expression of these proteins by in situ hybridisation and performed functional knockdowns with RNA interference. Remarkably, there are almost no differences in terms of morphology, protein regions, and gene expression based on marine, brackish, and freshwater habitats. This implies that glue components produced by macrostomids are conserved among species, and this set of two-component glue functions from low to high salinity. These findings could contribute to the development of novel reversible biomimetic glues that work in all wet environments and could have applications in drug delivery systems, tissue adhesives, or wound dressings.

Papillae revisited and the nature of the adhesive secreting collocytes.

Ascidian papillae (palps) constitute a transient sensory adhesive organ that assures larval settlement and the onset of metamorphosis to the filterfeeding adult. Despite the importance of papillae for the ascidian development, their cellular composition is only roughly described. For Ciona intestinalis/robusta, a clear definition of cell numbers and discriminative molecular markers for the different cell types is missing. While some attention was given to neural cell types and their connectivity little is known about the adhesive producing collocytes. We converge serial-section electron microscopy and confocal imaging with various marker combinations to document the 3D organization of the Ciona papillae. We show the papillar development with 4 axial columnar cells (ACCs), 4 lateral primary sensory neurons (PSNs) and 12 central collocytes (CCs). We propose molecular markers for each cell type including novel ones for collocytes. The subcellular characteristics are suggestive of their role in papillar function: the ACCs featuring apical protrusions and microvilli, also contain neuroactive and endocytic vesicles indicative of a chemosensory role. They are clearly distinct from the ciliated glutamatergic PSNs. CCs encircle the ACCs and contain microvilli, small endocytic vesicles and notably a large numbers of adhesive granules that, according to element analysis and histochemistry, contain glycoproteins. Interestingly, we detect two different types of collocyte granules, one of them containing fibrous material and larger quantities of polysaccharides. Consistently, carbohydrate specific lectins label the papillar apex, the granules within CCs and the adhesive plaques upon larval attachment. We further propose CCs to derive from an evolutionary ancient neurosecretory cell type. Our findings contribute to understanding the development of the anterior ('new head') region of the Ciona larva and notably the adhesive secreting cells which has implications for developmental biology, cell differentiation and evolution, but also bioadhesion.

Integrative Transcriptome and Proteome Analysis of the Tube Foot and Adhesive Secretions of the Sea Urchin Paracentrotus lividus.

Echinoderms, such as the rock-boring sea urchin Paracentrotus lividus, attach temporarily to surfaces during locomotion using their tube feet. They can attach firmly to any substrate and release from it within seconds through the secretion of unknown molecules. The composition of the adhesive, as well as the releasing secretion, remains largely unknown. This study re-analyzed a differential proteome dataset from Lebesgue et al. by mapping mass spectrometry-derived peptides to a P. lividus de novo transcriptome generated in this study. This resulted in a drastic increase in mapped proteins in comparison to the previous publication. The data were subsequently combined with a differential RNAseq approach to identify potential adhesion candidate genes. A gene expression analysis of 59 transcripts using whole mount in situ hybridization led to the identification of 16 transcripts potentially involved in bioadhesion. In the future these data could be useful for the production of synthetic reversible adhesives for industrial and medical purposes.

Organ specific gene expression in the regenerating tail of Macrostomum lignano.

Temporal and spatial characterization of gene expression is a prerequisite for the understanding of cell-, tissue-, and organ-differentiation. In a multifaceted approach to investigate gene expression in the tail plate of the free-living marine flatworm Macrostomum lignano, we performed a posterior-region-specific in situ hybridization screen, RNA sequencing (RNA-seq) of regenerating animals, and functional analyses of selected tail-specific genes. The in situ screen revealed transcripts expressed in the antrum, cement glands, adhesive organs, prostate glands, rhabdite glands, and other tissues. Next we used RNA-seq to characterize temporal expression in the regenerating tail plate revealing a time restricted onset of both adhesive organs and copulatory apparatus regeneration. In addition, we identified three novel previously unannotated genes solely expressed in the regenerating stylet. RNA interference showed that these genes are required for the formation of not only the stylet but the whole male copulatory apparatus. RNAi treated animals lacked the stylet, vesicula granulorum, seminal vesicle, false seminal vesicle, and prostate glands, while the other tissues of the tail plate, such as adhesive organs regenerated normally. In summary, our findings provide a large resource of expression data during homeostasis and regeneration of the morphologically complex tail regeneration and pave the way for a better understanding of organogenesis in M. lignano.

Sequential development of apical-basal and planar polarities in aggregating epitheliomuscular cells of Hydra.

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.

Pronephric tubule morphogenesis in zebrafish depends on Mnx mediated repression of irx1b within the intermediate mesoderm.

Mutations in the homeobox transcription factor MNX1 are the major cause of dominantly inherited sacral agenesis. Studies in model organisms revealed conserved mnx gene requirements in neuronal and pancreatic development while Mnx activities that could explain the caudal mesoderm specific agenesis phenotype remain elusive. Here we use the zebrafish pronephros as a simple yet genetically conserved model for kidney formation to uncover a novel role of Mnx factors in nephron morphogenesis. Pronephros formation can formally be divided in four stages, the specification of nephric mesoderm from the intermediate mesoderm (IM), growth and epithelialisation, segmentation and formation of the glomerular capillary tuft. Two of the three mnx genes in zebrafish are dynamically transcribed in caudal IM in a time window that proceeds segmentation. We show that expression of one mnx gene, mnx2b, is restricted to the pronephric lineage and that mnx2b knock-down causes proximal pronephric tubule dilation and impaired pronephric excretion. Using expression profiling of embryos transgenic for conditional activation and repression of Mnx regulated genes, we further identified irx1b as a direct target of Mnx factors. Consistent with a repression of irx1b by Mnx factors, the transcripts of irx1b and mnx genes are found in mutual exclusive regions in the IM, and blocking of Mnx functions results in a caudal expansion of the IM-specific irx1b expression. Finally, we find that knock-down of irx1b is sufficient to rescue proximal pronephric tubule dilation and impaired nephron function in mnx-morpholino injected embryos. Our data revealed a first caudal mesoderm specific requirement of Mnx factors in a non-human system and they demonstrate that Mnx-dependent restriction of IM-specific irx1b activation is required for the morphogenesis and function of the zebrafish pronephros.

Adhesive organ regeneration in Macrostomum lignano.

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.

Ultrastructure of the ovary and oogenesis in the flatworm Prosthiostomum siphunculus (Polycladida, Cotylea).

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.

Ultrastructure of spermatogenesis and mature spermatozoa in the flatworm Prosthiostomum siphunculus (Polycladida, Cotylea).

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.

Toxic effects of cadmium on flatworm stem cell dynamics: A transcriptomic and ultrastructural elucidation of underlying mechanisms.

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.