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.     

Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.     

The nervous system of <Emphasis Type="Italic">Convolutriloba</Emphasis> (Acoela) and its patterning during regeneration after asexual reproduction

We describe the serotonergic and cholinergic nervous system of the asexually reproducing acoel Convolutriloba longifissura Bartolomaeus & Balzer, 1997 by means of immunohistochemistry, conventional histochemistry and transmission electron microscopy. Immunocytochemical staining for serotonin revealed neurons in the brain, in a pair of ventral main longitudinal cords, in two pairs of smaller dorsal longitudinal nerve cords, and in a submuscular nerve net. The brain comprises a ventral-anterior commissure and a less intensely stained dorsal commissure joined together by connectives into a three-ringed scaffold from which the longitudinal nerves extend. We followed the regeneration of the serotonergic part of the nervous system up to the second day after fission. Within this time period, the offspring reestablished bilateral symmetry in the nervous system and developed full motor control. The presence of aminergic cell bodies associated with the main lateral nerve cords of C. longifissura shows that the acoelan nervous system is more similar to that of other platyhelminths (triclads, rhabditophorans) than previously assumed. The presence of serotonergic cell bodies along the main nerve cord correlates with the capacity for asexual reproduction via fissioning. We also describe the single fission mode of C. hastifera Winsor 1990, which brings the modes of asexual reproduction employed by members of the Convolutrilobinae to three.     

Regeneration in <Emphasis Type="Italic">Macrostomum lignano</Emphasis> (Platyhelminthes): cellular dynamics in the neoblast stem cell system

Neoblasts are potentially totipotent stem cells and the only proliferating cells in adult Platyhelminthes. We have examined the cellular dynamics of neoblasts during the posterior regeneration of Macrostomum lignano. Double-labeling of neoblasts with bromodeoxyuridine and the anti-phospho histone H3 mitosis marker has revealed a complex cellular response in the first 48 h after amputation; this response is different from that known to occur during regeneration in triclad platyhelminths and in starvation/feeding experiments in M. lignano. Mitotic activity is reduced during the first 8 h of regeneration but, at 48 h after amputation, reaches almost twice the value of control animals. The total number of S-phase cells significantly increases after 1 day of regeneration. A subpopulation of fast-cycling neoblasts surprisingly shows the same dynamics during regeneration as those in control animals. Wound healing and regeneration are accompanied by the formation of a distinct blastema. These results present new insights, at the cellular level, into the early regeneration of rhabditophoran Platyhelminthes. This work was supported by FWF Grant (P16618; P.I. Rieger, Innsbruck).     

Fission in Convolutriloba longifissura: asexual reproduction in acoelous turbellarians revisited

Studies of the asexual reproduction of Convolutriloba longifissura (Acoela, Acoelomorpha) revealed that there is no longitudinal fission of the whole animal as has been described by Bartolomaeus and Balzer (1997) . Instead, the first step is a transverse fission. This results in the detachment of the caudal fourth of the mother animal. The detached part forms what we call the butterfly stage, which initially has no mouth and no eye fields. This stage gives rise to two new individuals by a longitudinal fission. Within 2–3 days the eye fields and a mouth develop in each of the two progenies formed in this way. In the meantime the mother individual grows and develops the three typical caudal lobes. The mother animal can repeat this process resulting in three individuals every fourth day. The finding of this new pattern of reproduction in the Acoela has prompted us to review the various ways by which asexual reproduction occurs in the group. The peculiar combination of few cases but high diversity of asexual reproduction in the Acoela is discussed from an evolutionary point of view.     

Stem cells in a basal bilaterian

In Platyhelminthes, totipotent stem cells (neoblasts) are supposed to be the only dividing cells. They are responsible for the renewal of all cell types during development, growth, and regeneration, a unique situation in the animal kingdom. In order to further characterize these cells, we have applied two immunocytochemical markers to detect neoblasts in different stages of the cell cycle in the acoel flatworm Convolutriloba longifissura: (1) the thymidine analog 5'-bromo-2'-deoxyuridine (BrdU) to identify cells in S-phase, and (2) an antibody to phosphorylated histone H3 to locate mitosis. BrdU pulse-chase experiments were carried out to follow differentiation of neoblasts. We demonstrate the differentation into four labeled, differentiated cell types. S-phase cells and mitotic cells showed a homogenous distribution pattern throughout the body of C. longifissura. Two different types of S-phase cells could be distinguished immunocytochemically by their pattern of incorporated BrdU in the nuclei. Transmission electron microscopy was used to study ultrastructural characters of neoblasts and revealed two different stages in maturation of neoblasts, each with a characteristic organization of heterochromatin. The stem-cell pool of C. longifissura is an important prerequisite for the extraordinary mode of asexual reproduction and the high capacity of regeneration. A comparison of the stem-cell pool in Acoela and higher platyhelminth species can provide evidence for the phylogenetic relationships of these taxa.     

To Be or Not to Be a Flatworm: The Acoel Controversy

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.     

Cell renewal and apoptosis in macrostomum sp. [Lignano

In platyhelminths, all cell renewal is accomplished by totipotent stem cells (neoblasts). Tissue maintenance is achieved in a balance between cell proliferation and apoptosis. It is known that in Macrostomum sp. the epidermis undergoes extensive cell renewal. Here we show that parenchymal cells also exhibit a high rate of cell turnover. We demonstrate cell renewal using continuous 5'bromo-2-deoxyuridine (BrdU) exposure. About one-third of all cells are replaced after 14 days. The high level of replacement requires an equivalent removal of cells by apoptosis. Cell death is characterized using a combination of three methods: (1). terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), (2). specific binding of phosphatidyl-serine to fluorescent-labelled annexin V and (3). identification of apoptotic stages by ultrastructure. The number of cells observed in apoptosis is insufficient to explain the homeostasis of tissues in Macrostomum. Apoptosis-independent mechanisms may play an additional role in tissue dynamics.