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

The genome of the sea urchin Strongylocentrotus purpuratus

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes

Expression of Tgf-beta in the regenerative process of crinoid echinoderms

For many years, we focused our studies on the remarkable power of regeneration exhibited by crinoid echinoderms. Current investigations are exploring the aspects related to the 'putative' growth factors crucial for the regenerative process in crinoids. Due to its importance in wound healing phenomena of vertebrates and invertebrates, both in the embryo and in adults, the present study concentrates on the possible presence/expression and roles of TGF-\u3b2 in arm regeneration processe of the crinoid Antedon mediterranea. This problem has been explored by employing 1) a biochemical and immunocytochemical approach, which allowed us to detect the presence of TGF-\u3b21 and its putative changes of pattern distribution during the regeneration process; 2) a molecular approach, thanks to which we cloned a BMP2/4 homologue from crinoids (AnBMP2/4) confirming the possible presence of different genes of the TGF-\u3b2 superfamily. Expression studies indicate an important role for AnBMP2/4 during the more advanced stages of regeneration at a time when new tissues are being established and the brachial nerve is extending into the regenerate. This expression in an \u2018adult\u2019 regenerating system shows remarkable parallels with recent investigations in sea urchin embryos where a BMP2/4 homologue is involved in the regulation of the ectoderm/endoderm boundary and epidermal/non epidermal fate decisions. Our results suggest in particular that AnBMP2/4 plays an important role in crinoid skeletogenesis as well as in neurogenesis and support the idea of an evolutionary developmental programme where essential gene families are conserved throughout phylogeny both in terms of expression and function. The future employment of techniques such as RNA interference or bead implantation, in adult echinoderms, will be of fundamental importance to understand the function of AnBMP2/4 and other related genes

Cellular and molecular mechanisms of arm regeneration in crinoid echinoderms: the potential of arm explants.

Crinoid echinoderms can provide a valuable experimental model for studying all aspects of regenerative processes from molecular to macroscopic level. Recently we carried out a detailed study into the overall process of arm regeneration in the crinoid Antedon mediterranea and provided an interpretation of its basic mechanisms. However, the problem of the subsequent fate of the amputated arm segment (explant) once isolated from the animal body and of its possible regenerative potential have never been investigated before. The arm explant in fact represents a simplified and controlled regenerating system which may be very useful in regeneration experiments by providing a valuable test of our hypotheses in terms of mechanisms and processes. In the present study we carried out a comprehensive analysis of double-amputated arm explants (i.e. explants reamputated at their distal end immediately after the first proximal amputation) subjected to the same experimental conditions as the regenerating donor animals. Our results showed that the explants undergo similar regenerative processes but with some significant differences to those mechanisms described for normal regenerating arms. For example, whilst the proximal-distal axis of arm growth is maintained, there are differences in terms of the recruitment of cells which contribute to the regenerating tissue. As with normal regenerating arms, the present work focuses on (1) timing and modality of regeneration in the explant; (2) proliferation, migration and contribution of undifferentiated and/or dedifferentiated/transdifferentiated cells; (3) putative role of neural growth factors. These problems were addressed by employing a combination of conventional microscopy and immunocytochemistry. Comparison between arm explants and regenerating arms of normal donor adults indicates an extraordinary potential and regenerative autonomy of crinoid tissues and the cellular plasticity of the phenomenon

The peripheral nervous system of an ascidian, Botryllus schlosseri, as revealed by cholinesterase activity

In this study we present the first detailed description of the motor component of the peripheral nervous system of an ascidian showing its three-dimensional organization and the spatial relationships between nerves and contiguous organs. Nerves of the oozooid and blastozooid of the colonial ascidian Botryllus schlosseri were analyzed using a histochemical method for detecting cholinesterase activity in whole-mount preparations and in sections for light and electron microscopy. Except for the neural gland and gonads, all tissues are well innervated by cholinesterase-reactive neurites. Each blastozooid of the colony possesses an individual nerve plexus which is not in continuity with that of adjacent zooids. The innervation of the mantle, oral and cloacal siphons, branchial basket, heart, and gut are described. Most organs possess a complex network of nerves often with multiple origins from different pathways. A sophisticated pattern is described for the first time in the gut, which receives innervation from the endostyle, the roof of the branchial basket, and the posterior mantle. Dilated axonal regions but no obvious cell bodies were recognized in the peripheral nervous system revealed by cholinesterase activity. The localization of nerves is discussed together with a consideration of their physiological role