Adhesive organ regeneration in <i>Macrostomum lignano</i>

BackgroundFlatworms 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.ResultsWe 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.ConclusionOur 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

<i>Melav2</i>, an <i>elav</i>-like gene, is essential for spermatid differentiation in the flatworm <i>Macrostomum lignano</i>

BackgroundFailure of sperm differentiation is one of the major causes of male sterility. During spermiogenesis, spermatids undergo a complex metamorphosis, including chromatin condensation and cell elongation. Although the resulting sperm morphology and property can vary depending on the species, these processes are fundamental in many organisms. Studying genes involved in such processes can thus provide important information for a better understanding of spermatogenesis, which might be universally applied to many other organisms.ResultsIn a screen for genes that have gonad-specific expression we isolated an elav-like gene, melav2, from Macrostomum lignano, containing the three RNA recognition motifs characteristic of elav-like genes. We found that melav2 mRNA was expressed exclusively in the testis, as opposed to the known elav genes, which are expressed in the nervous system. The RNAi phenotype of melav2 was characterized by an aberrant spermatid morphology, where sperm elongation often failed, and an empty seminal vesicle. Melav2 RNAi treated worms were thus male-sterile. Further analysis revealed that in melav2 RNAi treated worms precocious chromatin condensation occurred during spermatid differentiation, resulting in an abnormally tightly condensed chromatin and large vacuoles in round spermatids. In addition, immunostaining using an early-spermatid specific antibody revealed that melav2 RNAi treated worms had a larger amount of signal positive cells, suggesting that many cells failed the transition from early spermatid stage.ConclusionWe characterize a new function for elav-like genes, showing that melav2 plays a crucial role during spermatid differentiation, especially in the regulation of chromatin condensation and/or cell elongation

Horizontal gene transfer contributed to the evolution of extracellular surface structures

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment

The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano

Background: Macrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts) in S-phase. Results: The tail plate accomodates the male genital apparatus and consists of about 3,100 cells, about half of which are epidermal cells. A distinct regeneration blastema, characterized by a local accumulation of rapidly proliferating neoblasts and consisting of about 420 cells (excluding epidermal cells), was formed 24 hours after amputation. Differentiated cells in the blastema were observed two days after amputation (with about 920 blastema cells), while the male genital apparatus required four to five days for full differentiation. At all time points, mitoses were found within the blastema. At the place of organ differentiation, neoblasts did not replicate or divide. After three days, the blastema was made of about 1420 cells and gradually transformed into organ primordia, while the proliferation rate decreased. The cell number of the tail plate, including about 960 epidermal cells, was restored to 75% at this time point. Conclusion: Regeneration after artificial amputation of the tail plate of adult specimens of Macrostomum lignano involves wound healing and the formation of a regeneration blastema. Neoblasts undergo extensive proliferation within the blastema. Proliferation patterns of S-phase neoblasts indicate that neoblasts are either determined to follow a specific cell fate not before, but after going through S-phase, or that they can be redetermined after S-phase. In pulse-chase experiments, dispersed distribution of label suggests that S-phase labeled progenitor cells of the male genital apparatus undergo further proliferation before differentiation, in contrast to progenitor cells of epidermal cells. Mitotic activity and proliferation within the blastema is a feature of M. lignano shared with many other regenerating animals

A new model organism among the lower Bilateria and the use of digital microscopy in taxonomy of meiobenthic Platyhelminthes Macrostomum lignano, n. sp. (Rhabditophora, Macrostomorpha)

Macrostomum lignano n. sp. is a member of the Macrostomorpha, the basal-most subtaxon of the PlatyhelminthesRhabditophora. This new species can be easily cultured in the laboratory and has been already the subject of several developmental/evolutionary studies. The small size, with only about 25 000 cells constituting the major bilaterian organ systems, makes this simultaneous hermaphrodite a possible candidate for a new model organism that is phylogenetically more basal than any of the model organisms currently used in such studies within the Bilateria. M. lignano belongs to the largest genus of the Macrostomorpha. Over 100 marine, fresh water and brackish water species are contained in the genus Macrostomum, some of them with worldwide distribution pattern. Within it, M. lignano is a member of the M. tuba-species group, which we have summarized here. In the species description, we have used a novel approach to document such small soft-bodied meiobenthic organisms: we provide extensive digital micrographical documentation, which are deposited as a CD together with the type material

New insights into the target site and mode of action of the antifungal protein of Aspergillus giganteus.

The antifungal protein (AFP) secreted by Aspergillus giganteus exerts growth inhibitory effects on various filamentous fungi. In order to obtain more information on the mode of action of AFP, we used transmission electron microscopy in this study to compare the cellular ultrastructure of the AFP-sensitive Aspergillus niger and of the AFP-resistant Penicillium chrysogenum upon AFP treatment. Furthermore, AFP was localized by immunogold staining in both fungi. Severe membrane alterations in A. niger were observed, whereas the membrane of P. chrysogenum was not affected after treatment with AFP. The protein localized predominantly to a cell wall attached outer layer which is probably composed of glycoproteins, as well as to the cell wall of A. niger. It was found to accumulate within defined areas of the cell wall, pointing towards a specific interaction of AFP with cell wall components. In contrast, very little protein was bound to the outer layer and cell wall of P. chrysogenum. For future applications of AFP as an antimycotic drug, the mode of action of the protein was further characterized. The protein was found to act in a dose-dependent manner: it was fungistatic when applied at concentrations below the minimal inhibitory concentration, but fungicidal at higher concentrations. Using an in vivo model system, we were able to finally show that AFP indeed prevented the infection of tomato roots (Lycopersicon esculentum) by the plant-pathogenic fungus Fusarium oxysporum f. sp. lycopersici

Impacts of climate warming on Alpine lake biota over the past decade

Alpine temperatures have risen at twice the rate compared to the northern-hemispheric average during the past century. This can be expected to affect Alpine lake ecosystems via, for example, intensified thermal stratification, shorter ice cover periods, and altered catchment processes. Our study assesses changes in some main constituents of the plank tic and benthic communities of five mid-Alpine lakes in the Niedere Tauern region in Austria in relation to climatic warming, by comparing community and environmental data from 1998-1999 to data from 2010-2011. Although lake chemistry remained relatively stable between the study periods, we observed an increase in lake water temperatures and a decrease in ice cover durations. Several of the dominant diatom species and chrysophyte cyst types show relatively clear changes; the responses of the whole communities, however, are less evident. Yet, in particular, diatoms show distinct assemblage changes along the climatic gradients in the two lakes with the largest decrease in ice-cover duration. Chironomid communities appear to be less sensitive compared to diatoms and chrysophyte cysts, which are known for reacting quickly to changes in their environment. Finally, Alpine lakes, which are moderately nutrient-enriched because of human activities in the catchment area, are likely to experience increases in their productivity with climate warming.Peer reviewe

The stem cell system of the basal flatworm Macrostomum lignano

The scope of this review is to introduce the free-living flatworm Macrostomum lignano as an excellent model organism to address questions of platyhelminth stem cell biology. First, we sketch the historical origin of flatworm stem cell research. Second, we introduce M. lignano, and summarize the main advantages that we think it has over the classical planarian model. Third, we give a short summary of the simple culture techniques. Fourth, we give a detailed overview over its morphology and embryology as far as it is relevant for stem cell biology. Fifth, we summarize our main findings on stem cell biology, with respect to the identification of neoblasts, their distribution and number. We describe the ultrastructure of neoblasts, their dynamics and gene expression. Sixth, we outline ways to study sex allocation by means of stem cell labeling and manipulation. Last, we highlight the regeneration capacity of this species and link it to the stem cell system. We conclude that M. lignano is a highly suitable model organism to gain knowledge about flatworm stem cells and to provide insight into stem cell systems of higher organisms, including humans