Maxime Willems

Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.

Characterization of the stem cell system of the acoel Isodiametra pulchra

BackgroundTissue plasticity and a substantial regeneration capacity based on stem cells are the hallmark of several invertebrate groups such as sponges, cnidarians and Platyhelminthes. Traditionally, Acoela were seen as an early branching clade within the Platyhelminthes, but became recently positioned at the base of the Bilateria. However, little is known on how the stem cell system in this new phylum is organized. In this study, we wanted to examine if Acoela possess a neoblast-like stem cell system that is responsible for development, growth, homeostasis and regeneration.ResultsWe established enduring laboratory cultures of the acoel Isodiametra pulchra (Acoela, Acoelomorpha) and implemented in situ hybridization and RNA interference (RNAi) for this species. We used BrdU labelling, morphology, ultrastructure and molecular tools to illuminate the morphology, distribution and plasticity of acoel stem cells under different developmental conditions. We demonstrate that neoblasts are the only proliferating cells which are solely mesodermally located within the organism. By means of in situ hybridisation and protein localisation we could demonstrate that the piwi-like gene ipiwi1 is expressed in testes, ovaries as well as in a subpopulation of somatic stem cells. In addition, we show that germ cell progenitors are present in freshly hatched worms, suggesting an embryonic formation of the germline. We identified a potent stem cell system that is responsible for development, homeostasis, regeneration and regrowth upon starvation.ConclusionsWe introduce the acoel Isodiametra pulchra as potential new model organism, suitable to address developmental questions in this understudied phylum. We show that neoblasts in I. pulchra are crucial for tissue homeostasis, development and regeneration. Notably, epidermal cells were found to be renewed exclusively from parenchymally located stem cells, a situation known only from rhabditophoran flatworms so far. For further comparison, it will be important to analyse the stem cell systems of other key-positioned understudied taxa.

The free-living flatworm Macrostomum lignano: A new model organism for ageing research

To study the several elements and causes of ageing, diverse model organisms and methodologies are required. The most frequently used models are Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and rodents. All have their advantages and disadvantages and allow studying particular aspects of the ageing process. During the last few years, several ageing studies focussed on stem cells and their role in tissue homeostasis. Here we present a new model organism which can study this relation where other model systems fail. The flatworm Macrostomum lignano possesses a dynamic population of likely totipotent somatic stem cells known as neoblasts. Several characteristics qualify M. lignano as a suitable model system for ageing studies in general and more specifically for gaining more insight in the causal relation between stem cells, ageing and rejuvenation. In this review, we will briefly describe the species and its life history, and discuss the role of its stem cells in ageing and rejuvenation. We also give an overview of the available experimental tools that allow a multidisciplinary approach for studying ageing in M. lignano.

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

BackgroundMacrostomum 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.ResultsThe 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.ConclusionRegeneration 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.

Lack of metabolic ageing in the long-lived flatworm Schmidtea polychroa

Freshwater planarians have a large totipotent stem cell population allowing high rates of cell renewal and morphological plasticity. It is often suggested that they are able to rejuvenate during fission, regeneration and starvation. These features, together with the rapidly expanding molecular toolset, make planarians such as Schmidtea polychroa and S. mediterranea interesting for ageing research. Yet, the basic demographic and physiological data are lacking or still based on fragmentary observations of one century ago. Here, we present the first longitudinal physiological study of the species S. polychroa. Survival, size and metabolic rate, measured by microcalorimetry, of a cohort of 28 individuals were followed over a period of three years. Sexual maturity was reached during the second month after which the worms continued growing up to 5 months. This initial growth phase was followed by alternating periods of synchronised growth and degrowth. Although mass-specific metabolic rates declined during the initial growth phase, no changes were found later in life. The absence of metabolic ageing may be explained by the very high rate of cell renewal during homeostasis and alternating phases of degrowth and growth during which tissues are renewed. Surprisingly, all deaths occurred in pairs of worms that were housed in the same culture recipient, suggesting that worms did not die from ageing. Taking into account the metabolic and demographic data, we suggest that S. polychroa shows negligible ageing. Detailed analyses of size and metabolic rate revealed a remarkable biphasic allometric scaling relation. During the initial growth phase (months 1-5) the allometric scaling exponent b was 0.86 while later in life, it increased to an unusually large value of 1.17, indicating that mass-specific metabolic rate increases with size in adult S. polychroa.

Toxicity profiles and solvent–toxicant interference in the planarian Schmidtea mediterranea after dimethylsulfoxide (DMSO) exposure

To investigate hydrophobic test compounds in toxicological studies, solvents like dimethylsulfoxide (DMSO) are inevitable. However, using these solvents, the interpretation of test compound‐induced responses can be biased. DMSO concentration guidelines are available, but are mostly based on acute exposures involving one specific toxicity endpoint. Hence, to avoid solvent–toxicant interference, we use multiple chronic test endpoints for additional interpretation of DMSO concentrations and propose a statistical model to assess possible synergistic, antagonistic or additive effects of test compounds and their solvents. In this study, the effects of both short‐ (1 day) and long‐term (2 weeks) exposures to low DMSO concentrations (up to 1000 µl l−1) were studied in the planarian Schmidtea mediterranea. We measured different biological levels in both fully developed and developing animals. In a long‐term exposure set‐up, a concentration of 500 µl l−1 DMSO interfered with processes on different biological levels, e.g. behaviour, stem cell proliferation and gene expression profiles. After short exposure times, 500 µl l−1 DMSO only affected motility, whereas the most significant changes on different parameters were observed at a concentration of 1000 µl l−1 DMSO. As small sensitivity differences exist between biological levels and developmental stages, we advise the use of this solvent in concentrations below 500 µl l−1 in this organism. In the second part of our study, we propose a statistical approach to account for solvent–toxicant interactions and discuss full‐scale solvent toxicity studies. In conclusion, we reassessed DMSO concentration limits for different experimental endpoints in the planarian S. mediterranea. Copyright

Stem Cells Propagate Their DNA by Random Segregation in the Flatworm Macrostomum lignano

Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts) are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized ‘Label-Retaining Cells’ (LRCs). Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells.We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU− double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.

Ontogeny of the complex sperm in the macrostomid flatworm Macrostomum lignano (Macrostomorpha, Rhabditophora)

Spermiogenesis in Macrostomum lignano (Macrostomorpha, Rhabditophora) is described using light‐ and electron microscopy of the successive stages in sperm development. Ovoid spermatids develop to highly complex, elongated sperm possessing an undulating distal (anterior) process (or “feeler”), bristles, and a proximal (posterior) brush. In particular, we present a detailed account of the morphology and ontogeny of the bristles, describing for the first time the formation of a highly specialized bristle complex consisting of several parts. This complex is ultimately reduced when sperm are mature. The implications of the development of this bristle complex on both sperm maturation and the evolution and function of the bristles are discussed. The assumed homology between bristles and flagellae questioned. J. Morphol., 2009.

Different roads to form the same gut in nematodes

SUMMARY The morphogenesis of a gut from the endoderm has been well studied among the animal kingdom and is also well described in the nematode Caenorhabditis elegans. But are there other ways to build a nematode intestine? Sulston et al. (1983) described a different intestinal cell lineage in the species Panagrellus redivivus and Turbatrix aceti that includes two programmed cell deaths. However, no details are known about the three‐dimensional (3D) configuration and the role of the cell deaths. Here, we describe the intestinal morphogenesis of P. redivivus and five other nematode species by means of four‐dimensional microscopy, which gives us a 3D representation of gut formation at the cellular level. The morphological pathway of gut formation is highly conserved among these distantly related species. However, we found the P. redivivus pattern in another related species Halicephalobus gingivalis. In this pattern, the intestinal precursors migrate inward in concert with the mesoderm precursors. Based on the observations, we propose a hypothesis that could explain the differences. The positions of the mesoderm precursors create a possible spatial constraint, by which the establishment of bilateral symmetry in the intestine is delayed. This symmetry is corrected by cell migrations; other cells are eliminated and compensated by supplementary cell divisions. This pattern leads to the same result as in the other nematodes: a bilateral symmetrical intestine with nine rings. This illustrates how conserved body plans can be achieved by different developmental mechanisms.

Measurement of S-phase duration of adult stem cells in the flatworm Macrostomum lignano by double replication labelling and quantitative colocalization analysis

Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo. These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated Ts (S‐phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano. A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity‐based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object‐based colocalization approach was devised to score the relative number of double‐positive cells. Using this approach, Ts (S‐phase duration) in the main population of neoblasts was ∼13 h. During early regeneration, no significant change in Ts was observed.