The regeneration capacity of the flatworm Macrostomum lignano--on repeated regeneration, rejuvenation, and the minimal size needed for regeneration.

The lion's share of studies on regeneration in Plathelminthes (flatworms) has been so far carried out on a derived taxon of rhabditophorans, the freshwater planarians (Tricladida), and has shown this group's outstanding regeneration capabilities in detail. Sharing a likely totipotent stem cell system, many other flatworm taxa are capable of regeneration as well. In this paper, we present the regeneration capacity of Macrostomum lignano, a representative of the Macrostomorpha, the basal-most taxon of rhabditophoran flatworms and one of the most basal extant bilaterian protostomes. Amputated or incised transversally, obliquely, and longitudinally at various cutting levels, M. lignano is able to regenerate the anterior-most body part (the rostrum) and any part posterior of the pharynx, but cannot regenerate a head. Repeated regeneration was observed for 29 successive amputations over a period of almost 12 months. Besides adults, also first-day hatchlings and older juveniles were shown to regenerate after transversal cutting. The minimum number of cells required for regeneration in adults (with a total of 25,000 cells) is 4,000, including 160 neoblasts. In hatchlings only 1,500 cells, including 50 neoblasts, are needed for regeneration. The life span of untreated M. lignano was determined to be about 10 months.

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.

Stem cells in a basal bilaterian. S-phase and mitotic cells in Convolutriloba longifissura (Acoela, Platyhelminthes).

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.