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.

Regeneration in Macrostomum lignano (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.

Stem cell dynamics during growth, feeding, and starvation in the basal flatworm Macrostomum sp. (Platyhelminthes).

Development, growth, and regeneration in Macrostomum are based--as in all Platyhelminthes--on likely totipotent stem cells (neoblasts), basic for all Bilaterians. We demonstrate dynamics and migration of neoblasts during postembryonic development, starvation, and feeding of Macrostomum sp. Double labeling of S-phase and mitotic cells revealed a fast cell turnover. Conflicting with recent results from planarians, we have some indication of slow cycling neoblasts. As in planarians, starvation dramatically reduced mitotic activity and a very basic level was maintained after 30 days of starvation. Afterward, feeding induced a dramatic immediate proliferative response probably caused by G2-arrested neoblasts. The following 12 hr showed a significant mitotic decline, caused by the depletion of the G2 neoblast pool. Neoblasts that pass through S-phase led to a maximum of mitoses after 48 hr. Our results allow deeper insight into cellular dynamics of an ancestral bilaterian stem cell system of a basal Platyhelminth.

Development of the swimbladder in the European eel ( Anguilla anguilla).

The swimbladder of the adult eel, Anguilla anguilla, with its bipolar countercurrent system, the rete mirabile, is a widely used model for swimbladder function, but very little is known about the development of this swimbladder. Our histological studies on the developing swimbladder revealed that during metamorphosis the swimbladder becomes present as a dorsal outgrowth of the esophagus. It is filled with surfactant, and gas was not detected in the swimbladder. In the young glass-eel, the epithelial (gas gland) cells of the swimbladder are columnar, but do not yet have the typical basolateral labyrinth established in adult animals. Few blood vessels are found in the swimbladder tissue, and the submucosa is present as a thick layer of connective tissue, giving a large diffusion distance between blood vessel and swimbladder lumen. Within the next 2 or 3 months of development, gas gland cells develop their typical basolateral labyrinth, and the thickness of the submucosa is significantly reduced, resulting in a short diffusion distance between blood vessels and the swimbladder lumen. The first filling of the swimbladder with gas is observed while the gas gland cells are still in a poorly differentiated status and it appears unlikely that these cells can accomplish their typical role in gas deposition. The presence of small gas bubbles in the swimbladder as well as in the ductus pneumaticus at the time of initial swimbladder inflation suggests that the swimbladder is filled by air gulping or possibly by taking up gas bubbles from the water.