Evidence of interactions among apoptosis, cell proliferation, and dedifferentiation in the rudiment during whole-organ intestinal regeneration in the sea cucumber.

Sea cucumbers have an extraordinary regenerative capability. Under stressful conditions, Holothuria glaberrima can eviscerate their internal organs, including the digestive tract. From the mesentery, a rudiment grows and gives rise to a new intestine within a few weeks. In the last decades, the cellular events that occur during intestinal regeneration have been characterized, including apoptosis, cell proliferation, and muscle cell dedifferentiation. Nevertheless, their contribution to the formation and early growth of the rudiment is still unknown. Furthermore, these cellular events' relationship and potential interdependence remain a mystery. Using modulators to inhibit apoptosis and cell proliferation, we tested whether rudiment growth or other regenerative cellular events like muscle cell dedifferentiation were affected. We found that inhibition of apoptosis by zVAD and cell proliferation by aphidicolin and mitomycin did not affect the overall size of the rudiment seven days post-evisceration (7-dpe). Interestingly, animals treated with aphidicolin showed higher levels of muscle cell dedifferentiation in the distal mesentery, which could act as a compensatory mechanism. On the other hand, inhibition of apoptosis led to a decrease in cell proliferation in the rudiment and a delay in the spatiotemporal progression of muscle cell dedifferentiation throughout the rudiment-mesentery structure. Our findings suggest that neither apoptosis nor cell proliferation significantly contributes to early rudiment growth during intestinal regeneration in the sea cucumber. Nevertheless, apoptosis may play an essential role in modulating cell proliferation in the rudiment (a process known as apoptosis-induced proliferation) and the timing for the progression of muscle cell dedifferentiation. These findings provide new insights into the role and relationship of cellular events during intestinal regeneration in an emerging regeneration model.

Insights into intestinal regeneration signaling mechanisms.

The cellular mechanisms underlying the amazing ability of sea cucumbers to regenerate their autotomized intestines have been widely described by us and others. However, the signaling pathways that control these mechanisms are unknown. Previous studies have shown that Wnt homologs are upregulated during early intestinal regenerative stages, suggesting that the Wnt/ß-catenin pathway is active during this process. Here, we used small molecules, putative disruptors of the Wnt pathway, to determine the potential role of the canonical Wnt pathway on intestine regeneration in the sea cucumber Holothuria glaberrima. We evaluated their effects in vivo by using histological analyses for cell dedifferentiation, cell proliferation and apoptosis. We found that iCRT14, an alleged Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a presumed Wnt pathway activator, increased its size. The possible cellular mechanisms by which signaling pathway disruptors affect the gut rudiment size were further studied in vitro, using cultures of tissue explants and additional pharmacological agents. Among the tested signaling activators, those that act through GSK-3 inhibition, LiCl, 1-Azakenpaullone, and CHIR99021 were found to increase muscle cell dedifferentiation, while the inhibitor iCRT14 blocked cell dedifferentiation. Differently, cell proliferation was reduced by all GSK-3 inhibitors, as well as by iCRT14 and C59, which interferes with Wnt ligand secretion. The in vivo temporal and spatial pattern of ß-catenin activity was determined using an antibody against phosphorylated ß-catenin and shown to correlate with cell proliferative activity. In vitro treatment using C59 decreased the number of cells immunostained for nuclear phosphorylated ß-catenin. Our results showed that the cell dedifferentiation observed during intestinal regeneration can be decoupled from the cell proliferation event and that these cellular processes can be modulated by particular signaling pathway inhibitors and activators. These results open the door for future studies where the cellular signaling pathways involved at each regeneration stage can be determined.