Methods for cell isolation and analysis of the highly regenerative tunicate Polycarpa mytiligera.

Background: Polycarpa mytiligera is the only molecularly characterized solitary ascidian capable of regenerating all organs and tissue types. The cellular basis for regeneration in P. mytiligera is largely unknown, and methods for isolating live cells from this species for functional analyses are unavailable. Results: Here, we developed a method for isolating live cells from P. mytiligera, overcoming major experimental challenges, including the dissociation of its thick body wall and native cellular autofluorescence. We demonstrated the applicability of our approach for tissue dissociation and cell analysis using three flow cytometry platforms, and by using broadly used non-species-specific cell labeling reagents. In addition to live cell isolation, proof-of-concept experiments showed that this approach was compatible with gene expression analysis of RNA extracted from the isolated cells, and with ex vivo analysis of phagocytosis. Conclusion: We presented efficient methods for cell purification from a highly regenerative ascidian, which could be transferable to diversity of non-model marine organisms. The ability to purify live cells will promote future studies of cell function in P. mytiligera regeneration.

Stemness Activity Underlying Whole Brain Regeneration in a Basal Chordate.

Understanding how neurons regenerate following injury remains a central challenge in regenerative medicine. Adult mammals have a very limited ability to regenerate new neurons in the central nervous system (CNS). In contrast, the basal chordate Polycarpa mytiligera can regenerate its entire CNS within seven days of complete removal. Transcriptome sequencing, cellular labeling, and proliferation in vivo essays revealed that CNS regeneration is mediated by a newly formed neural progeny and the activation of neurodevelopmental pathways that are associated with enhanced stem-cell activity. Analyzing the expression of 239 activated pathways enabled a quantitative understanding of gene-set enrichment patterns at key regeneration stages. The molecular and cellular mechanisms controlling the regenerative ability that this study reveals can be used to develop innovative approaches to enhancing neurogenesis in closely-related chordate species, including humans.

Molecular characterization of the immediate wound response of the solitary ascidian Polycarpa mytiligera.

BACKGROUND: Injury response is key to successful regeneration. Yet, transcriptome analyses of injury response were performed only on a handful of regenerative organisms. Here, we studied the injury response of the solitary ascidian Polycarpa mytiligera, an emerging model system, capable of regenerating any body part. We used the siphon as a model for studying transcriptional changes following injury, and identified genes that were activated in the initial 24 hours post amputation (hpa). RESULTS: Highly conserved genes, such as bone morphogenetic protein-1 (BMP1), growth hormone secretagogue receptor (GHSR) and IL-17, were upregulated by 12 hpa, yet their expression was sustained only in non-regenerating tissue fragments. We optimized fluorescent in situ hybridization, and found that the majority of BMP1+ cells were localized to the rigid tunic that covers the animal. This highlights the importance of this tissue, particularly during injury response. BMP1 was overexpressed following injuries to other body regions, suggesting that it was a part of a common injury-induced program. CONCLUSION: Our study suggests that, initially, specific injury-induced genes were upregulated in P. mytiligera organs, yet, later, a unique transcriptional profile was observed only in regenerating tissues. These findings highlight the importance of studying diverse regenerating and non-regenerating organisms for complete understanding of regeneration.

And Then There Were Three : Extreme Regeneration Ability of the Solitary Chordate Polycarpa mytiligera.

Extensive regenerative ability is a common trait of animals capable of asexual development. The current study reveals the extraordinary regeneration abilities of the solitary ascidian Polycarpa mytiligera. Dissection of a single individual into separate fragments along two body axes resulted in the complete regeneration of each fragment into an independent, functional individual. The ability of a solitary ascidian, incapable of asexual development, to achieve bidirectional regeneration and fully regenerate all body structures and organs is described here for the first time. Amputation initiated cell proliferation in proximity to the amputation line. Phylogenetic analysis demonstrated the close affinity of P. mytiligera to colonial species. This evolutionary proximity suggests the ability for regeneration as an exaptation feature for colonial lifestyle. P. mytiligera's exceptional regenerative abilities and phylogenetic position highlight its potential to serve as a new comparative system for studies seeking to uncover the evolution of regeneration and coloniality among the chordates.

Sexual and asexual development: two distinct programs producing the same tunicate.

Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri, we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways.

Spawning induction, development and culturing of the solitary ascidian Polycarpa mytiligera, an emerging model for regeneration studies.

BACKGROUND: Ascidians (phylum Chordata, class Ascidiacea) represent the closest living invertebrate relatives of the vertebrates and constitute an important model for studying the evolution of chordate development. The solitary ascidian Polycarpa mytiligera exhibits a robust regeneration ability, unique among solitary chordates, thus offering a promising new model for regeneration studies. Understanding its reproductive development and establishing land-based culturing methods is pivotal for utilizing this species for experimental studies. Its reproduction cycle, spawning behavior, and developmental processes were therefore studied in both the field and the lab, and methods were developed for its culture in both open and closed water systems. RESULTS: Field surveys revealed that P. mytiligera's natural recruitment period starts in summer (June) and ends in winter (December) when seawater temperature decreases. Laboratory experiments revealed that low temperature (21 °C) has a negative effect on its fertilization and development. Although spontaneous spawning events occur only between June and December, we were able to induce spawning under controlled conditions year-round by means of gradual changes in the environmental conditions. Spawning events, followed by larval development and metamorphosis, took place in ascidians maintained in either artificial or natural seawater facilities. P. mytiligera's fast developmental process indicated its resemblance to other oviparous species, with the larvae initiating settlement and metamorphosis at about 12 h post-hatching, and reaching the juvenile stage 3 days later. CONCLUSIONS: Polycarpa mytiligera can be induced to spawn in captivity year-round, independent of the natural reproduction season. The significant advantages of P. mytiligera as a model system for regenerative studies, combined with the detailed developmental data and culturing methods presented here, will contribute to future research addressing developmental and evolutionary questions, and promote the use of this species as an applicable model system for experimental studies.

Solitary Ascidians as Model Organisms in Regenerative Biology Studies.

Regeneration, the process of replacing lost or damaged body parts, has long captured human imagination and is a key feature among all animal phyla. Due to their close phylogenetic relationship to vertebrates and their high regenerative abilities, ascidians (Chordata, Ascidiacea) are often used as models to shed light on the cellular and genetic process involved in tissue regeneration. Surprisingly, ascidian regeneration studies are based on only a few model species. In this chapter, we point out the important potential of solitary ascidians in regenerative and stem cell studies. We review recent studies of regeneration among solitary ascidians and discuss the cellular mechanism of tissue regeneration and the possible involvement of circulating cells in these processes. New data regarding the relationship between age and regeneration abilities of the solitary ascidian Polycarpa mytiligera (Stolidobranchia, Styelidae) are presented. The unique regeneration abilities found in P. mytiligera following evisceration of its digestive system and following amputation of its neural complex and siphon-associated structures and nerves imply on its potential to serve as a novel model system for understanding tissue regeneration.

Gut-spilling in chordates: evisceration in the tropical ascidian Polycarpa mytiligera.

The ejection of internal organs, i.e., evisceration, is a well-known phenomenon in sea-cucumbers. We report the ability of a member of the Chordate phyla, the tropical ascidian Polycarpa mytiligera, to eviscerate and regenerate its gut within 12 days, and to rebuild its branchial sac within 19 days. Evisceration occurred within 4-43 seconds of gentle mechanical pressure exerted on the tunic in 47% of the tested P. mytiligera. Individuals were able to discard up to 3/4 of their digestive tract via the incurrent siphon by rupture of the branchial sac in this area. Although chemical analysis revealed no significant levels of toxic compounds, the eviscerated guts were unpalatable to the triggerfish and pufferfish on which they were tested, suggesting evisceration as a defense mechanism. Given the close affinity of ascidians to vertebrates, the regeneration pathway of the viscera and branchial sac of ascidians suggests its potential beneficial application in soft tissue regeneration research.