Setting-up an in vitro model to study the signaling mechanisms associated with intestine regeneration in the sea cucumber Holothuria glaberrima
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Advisor
García-Arrarás, José E.Type
DissertationDegree Level
Ph.D.Date
2019-12Metadata
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The sea cucumber Holothuria glaberrima is a mighty model to study organ regeneration. Our group has described the cellular mechanisms underlying intestine re-growth in H. glaberrima and is exploring the signaling mechanisms involved in this process. One of the limitations to our studies has been the lack of suitable cell culture methodologies required to advance the regeneration studies. I have now established in vitro systems where individual cells or explants can be kept and studied. Cultured cells and explants were analyzed using various techniques that include light, fluorescence, and electron microscopy. Cells isolated by enzymatic dissociation from regenerating guts of H. glaberrima could be maintained in the right conditions for up to two weeks in vitro. Different phenotypes were identified using cellular markers established previously in our lab. However, no mature muscle cells or neurons were found, which is not suitable to perform studies about cell dedifferentiation/redifferentiation. Additionally, isolated cells showed deficient proliferative activity. For that reason, I characterized gut explants as an alternative. Remarkably, the histological characteristics, including the presence of mature muscle cells and the proliferative rate, in cultured explants resembled more accurately the in vivo conditions compared to the dissociated cultures. Thus, I decided to use gut explants to gain new insights about the signaling mechanisms associated with the intestine regeneration in H. glaberrima.
Initial in vivo studies using small molecules that are putative disruptors of the Wnt pathway supported the involvement of the Wnt pathway on intestine regeneration. These studies showed that iCRT14, a Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a Wnt pathway activator, increased its size. The possible cellular mechanisms by which the signaling disruptors affected the gut rudiment size were further studied in vitro using additional pharmacological agents. Among them, those that inhibited the GSK-3 enzyme (a component of the Wnt pathway) were found to increase muscle cell dedifferentiation. However, these agents also induced a reduction in cell proliferation, suggesting that cell dedifferentiation can be decoupled from cell proliferation during intestinal regeneration.
Interestingly, the drop of cell proliferation in explants was also caused by small molecules that block the Wnt pathway in other points different to GSK-3. For that reason, I propose that GSK-3 is the mediator of the cellular dedifferentiation response and that it takes place by a signaling pathway that is independent of Wnt. Differently, cellular proliferation appears to be controlled by the canonical Wnt pathway during intestine regeneration. My results open the door for future studies where the signaling pathways involved at each regeneration stage can be determined during intestine regeneration.
Initial in vivo studies using small molecules that are putative disruptors of the Wnt pathway supported the involvement of the Wnt pathway on intestine regeneration. These studies showed that iCRT14, a Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a Wnt pathway activator, increased its size. The possible cellular mechanisms by which the signaling disruptors affected the gut rudiment size were further studied in vitro using additional pharmacological agents. Among them, those that inhibited the GSK-3 enzyme (a component of the Wnt pathway) were found to increase muscle cell dedifferentiation. However, these agents also induced a reduction in cell proliferation, suggesting that cell dedifferentiation can be decoupled from cell proliferation during intestinal regeneration.
Interestingly, the drop of cell proliferation in explants was also caused by small molecules that block the Wnt pathway in other points different to GSK-3. For that reason, I propose that GSK-3 is the mediator of the cellular dedifferentiation response and that it takes place by a signaling pathway that is independent of Wnt. Differently, cellular proliferation appears to be controlled by the canonical Wnt pathway during intestine regeneration. My results open the door for future studies where the signaling pathways involved at each regeneration stage can be determined during intestine regeneration.
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