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.

Plectus - a stepping stone in embryonic cell lineage evolution of nematodes.

BACKGROUND: Recent studies have challenged the widespread view that the pattern of embryogenesis found in Caenorhabditis elegans (clade 9) is characteristic of nematodes in general. To understand this still largely unexplored landscape of developmental events, we set out to examine more distantly related nematodes in detail for temporospatial differences in pattern formation and cell specification. Members of the genus Plectus (clade 6) seem to be suitable candidates to show variety, with certain idiosyncratic features during early development and the convenient availability of cultivatable species. METHODS: The study was conducted using 4-D lineage analysis, 3-D modeling of developing embryos and laser-induced ablation of individual blastomeres. RESULTS: Detailed cell lineage studies of several Plectus species reveal that pattern formation and cell fate assignment differ markedly from C. elegans. Descendants of the first somatic founder cell S1 (AB) - but not the progeny of other founder cells - demonstrate extremely variable spatial arrangements illustrating that here distinct early cell-cell interactions between invariant partners, as found in C. elegans, cannot take place. Different from C. elegans, in Plectus alternative positional variations among early S1 blastomeres resulting in a 'situs inversus' pattern, nevertheless give rise to adults with normal left-right asymmetries. In addition, laser ablations of early blastomeres uncover inductions between variable cell partners. CONCLUSIONS: Our results suggest that embryonic cell specification in Plectus is not correlated with cell lineage but with position. With this peculiarity, Plectus appears to occupy an intermediate position between basal nematodes displaying a variable early development and the C. elegans-like invariant pattern. We suggest that indeterminate pattern formation associated with late, position-dependent fate assignment represents a plesiomorphic character among nematodes predominant in certain basal clades but lost in derived clades. Thus, the behavior of S1 cells in Plectus can be considered an evolutionary relict in a transition phase between two different developmental strategies.

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.

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.

Refuge from predation, the benefit of living in an extreme acidic environment?

Organisms living in extreme habitats require costly adaptations to cope with these conditions. Among the suggested potential benefits that trade off these costs is refuge from predation. To study these interactions in extreme environments, samples were taken in the cave Cueva de Villa Luz, Tabasco, Mexico, where more than 32 subterranean springs, some H(2)S rich, rise from the floor. Hydrogen sulfide gas plus oxygen is absorbed by freshwater, and oxidation forms concentrated sulfuric acid. Snottites, whitish hollow mucous tubes, hang from the ceiling of the cave. Fluid drops from these snottites were recorded as having pH values of 0-3. We report the discovery of a new species of nematode that thrives in the highly acidic environment of the snottite. Micro CT scan of snottites reveals a complex interaction between the acidic snottite, nematodes, and abundant nematode-eating mites. The nematode adaptation to low pH probably protects them against mite predation, for which nematodes are most likely the most important source of carbon in this sulfur-driven ecosystem.

Embryonic origins of hull cells in the flatworm Macrostomum lignano through cell lineage analysis: developmental and phylogenetic implications.

The development of macrostomid flatworms is of interest for evolutionary developmental biology research because these taxa combine characteristics of the canonical spiral cleavage pattern with significant deviations from this pattern. One such deviation is the formation of hull cells, which surround the remaining embryonic primordium during early development. Using live observations with a 4D microscope system, histology, and 3D reconstructions, we analyzed the ontogeny of these hull cells in the macrostomid model organism Macrostomum lignano. Our cell lineage analysis allowed us to find the precursors of the hull cells in this species. We discuss the relation between macrostomid development and the development of other spiralians and the question of whether hull cells are homologous within rhabditophoran flatworms.

Analysis of the translationally controlled tumour protein in the nematodes Ostertagia ostertagi and Caenorhabditis elegans suggests a pivotal role in egg production.

The translationally controlled tumour protein (TCTP) is a conserved protein which has been described for a wide range of eukaryotic organisms including protozoa, yeasts, plants, nematodes and mammals. Several parasitic organisms have been shown to actively secrete TCTP during host infection as part of their immuno-evasive strategy. In this study, we have studied TCTP in Ostertagia ostertagi, a parasitic nematode of cattle, and in the free-living nematode Caenorhabditis elegans. An analysis of the transcription and expression patterns showed that TCTP was present in the eggs of both species. This localisation is consistent for some other Strongylida such as Teladorsagia circumcincta, Cooperia oncophora and Haemonchus contortus. TCTP was also detected at low levels in excretory-secretory material from adult O. ostertagi worms. The role of TCTP in nematode biology was also investigated by RNA interference in C. elegans. Knock-down of C. elegans tctp (tct-1) transcription reduced the numbers of eggs laid by the hermaphrodite in the F(0) and F(1) generations by 90% and 72%, respectively, indicating a pivotal role of TCTP in reproduction.

The embryonic cell lineage of the nematode Rhabditophanes sp.

One of the unique features of the model organism Caenorhabditis elegans is its invariant development, where a stereotyped cell lineage generates a fixed number of cells with a fixed cell type. It remains unclear how embryonic development evolved within the nematodes to give rise to the complex, invariant cell lineage of C. elegans. Therefore, we determined the embryonic cell lineage of the nematode, Rhabditophanes sp. (family Alloionematidae) and made detailed cell-by-cell comparison with the known cell lineages of C. elegans, Pellioditis marina and Halicephalobus gingivalis. This gave us a unique data set of four embryonic cell lineages, which allowed a detailed comparison between these cell lineages at the level of each individual cell. This lineage comparison revealed a similar complex polyclonal fate distribution in all four nematode species (85% of the cells have the same fate). It is striking that there is a conservation of a 'C. elegans' like polyclonal cell lineage with strong left-right asymmetry. We propose that an early symmetry-breaking event in nematodes of clade IV-V is a major developmental constraint which shapes their asymmetric cell lineage.

Evolutionary loss of parasitism by nematodes? Discovery of a free-living filaroid nematode.

A cattle-drinking pool in nature reserve "Zwin" on the Belgian coast contained free-living third-stage infective filaroid juveniles. These juveniles clearly differ morphologically from all known nematodes. Morphological and molecular analyses indicate a position within the Filaroidea. The aberrant biology of this nematode, namely, a free-living stage in an aquatic environment, is unknown within this superfamily, and the evolution of the parasitic phenotype to a free-living state is generally thought to be unlikely. However, the obtained placement in the small subunit molecular phylogenetic tree suggests that this free-living stage is most likely a secondary adaptation. It is reasonable to assert that nematodes with complex life cycles still have the genetic potential for a reversion from parasitism to a (partial) free-living stage.

Different roads to form the same gut in nematodes.

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.

Unusual intestinal lamellae in the nematode Rhabditophanes sp. KR3021 (Nematoda: Alloinematidae).

The free-living nematode Rhabditophanes sp. has recently been placed in a clade of animal parasites and may be a unique example of a reversal to a nonparasitic lifestyle. Detailed morphological analysis of the intestine reveals the unusual and unique structure of splitting microlamellae forming a meshwork with cavities along the entire intestinal tract. Secretion vesicles were observed along the whole tract and along the length of the lamellae. It is suggested that these lamellae are adaptations to a different digestive strategy where low food availability and a low absorption surface are compensated for by maximizing the nutrient uptake efficiency along the entire length of the intestine. The likely reversal to a free-living life cycle may have caused drastic changes in diet, providing the necessary driving forces to such morphological changes.

The simplicity of metazoan cell lineages.

Developmental processes are thought to be highly complex, but there have been few attempts to measure and compare such complexity across different groups of organisms. Here we introduce a measure of biological complexity based on the similarity between developmental and computer programs. We define the algorithmic complexity of a cell lineage as the length of the shortest description of the lineage based on its constituent sublineages. We then use this measure to estimate the complexity of the embryonic lineages of four metazoan species from two different phyla. We find that these cell lineages are significantly simpler than would be expected by chance. Furthermore, evolutionary simulations show that the complexity of the embryonic lineages surveyed is near that of the simplest lineages evolvable, assuming strong developmental constraints on the spatial positions of cells and stabilizing selection on cell number. We propose that selection for decreased complexity has played a major role in moulding metazoan cell lineages.

Embryonic cell lineage of the marine nematode Pellioditis marina.

We describe the complete embryonic cell lineage of the marine nematode Pellioditis marina (Rhabditidae) up to somatic muscle contraction, resulting in the formation of 638 cells, of which 67 undergo programmed cell death. In comparison with Caenorhabditis elegans, the overall lineage homology is 95.5%; fate homology, however, is only 76.4%. The majority of the differences in fate homology concern nervous, epidermal, and pharyngeal tissues. Gut and, remarkably, somatic muscle is highly conserved in number and position. Partial lineage data from the slower developing Halicephalobus sp. (Panagrolaimidae) reveal a lineage largely, but not exclusively, built up of monoclonal sublineage blocs with identical fates, unlike the polyclonal fate distribution in C. elegans and P. marina. The fate distribution pattern in a cell lineage could be a compromise between minimizing the number of specification events by monoclonal specification and minimizing the need for migrations by forming the cells close at their final position. The latter could contribute to a faster embryonic development. These results reveal that there is more than one way to build a nematode.