Echinoderms comprise a group of animals with the capability of replacing complex internal organs following injury or autotomy. In holothurians or sea cucumbers, cellular processes of intestinal regeneration have been extensively studied, however, the molecular machinery behind this faculty remains to be understood. In this study, a de novo transcriptome was assembled and annotated using RNA-seq data consisting of regenerating and non-regenerating intestinal tissues from the sea cucumber Holothuria glaberrima. Comparisons of differential expression were made using the mesentery as a reference against 24 hours and 3 days regenerating intestine, revealing numerous differentially expressed transcripts. Gene ontology and pathway enrichment analysis showed evidence of increasing transcriptional activity. Further analysis of transcripts associated with transcription factors revealed diverse expression patterns with developmental and cancer-related mechanisms linked with regenerative responses. This study demonstrates the diverse gene expression profile during the early stages of regeneration using the mesentery as a focal point....

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.<br /> <br /> 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.<br /> <br /> 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....

Members of the phylum Echinodermata possess an impressive regenerative potential that could further develop the field of regenerative medicine. Holothurians have one of the most developed musculatures in terms of the myogenesis that leads to regeneration of muscle tissue. We aimed to develop an in vitro model for dissociated muscle tissue that could be used for regenerative studies. We focus on molecular candidates that have been previously associated with regeneration, to define their roles in the dedifferentiation of muscle cells. To do this, we optimized protocols for intestinal tissue cultures by changing parameters for ones that best suited our muscle tissue. Then, we applied pharmacological agents and analyzed the presence of SLS's and dedifferentiating fibers, structures that have been associated with the process of dedifferentiation, to elucidate which molecular pathways could induce the dedifferentiation of muscle cells. We found specific parameters such as changes in the enzymatic dissociation media, the adherence protocol and the number of cells cultured, that yielded healthy muscle fibers. Our cultures also showed a cellular component that was added to structures that indicated that dedifferentiation was occurring. For pharmacological assays, we found an increase in dedifferentiating fibers when we treated cultures with LiCl and an increase in SLS's when treated with DPI, EGTA and UK-383,367. Our results indicate that our in vitro model serves as an effective way to determine the effects that pharmacological agents have on dedifferentiation. We conclude that pathways involving Wnt/β- catenin and GSK-3, reactive oxygen species, Ca²⁺ and BMP-1 modulate in vitro dedifferentiation of muscle fibers....

Organogenesis is a process normally reserved for developing organisms, however, some adult organisms are capable of such a feat. Adult sea cucumbers, in the highly regenerative clade Echinodermata, can undergo intestinal organogenesis after a process of evisceration where the intestine is severed then expelled through the cloaca. Studying this phenomenon has implications in regenerative biology to help discover the cellular and molecular processes that allow some species to regenerate better than others. Also, the field of regenerative medicine can benefit by enhancing existing biomedical applications, particularly that of intestinal tissue engineering. Thus, the sea cucumber Holothuria glaberrima is being used in this study to investigate intestinal organogenesis. The cellular properties of its regeneration have largely been elucidated, but the molecular mechanisms are now just starting to be unveiled. In this study we use a bioinformatic approach to investigate molecular mechanisms by collecting RNA-seq data from various early- and late-stage intestinal regenerate timepoints. These new timepoints not only contain newly sequenced tissue with luminal epithelium, but also samples that are spatially separated, i.e., anterior and posterior regenerates. Analyses from differential gene expression, gene set enrichment analyses, and weighted gene co-expression revealed differences in gene expression and molecular pathways operating at distinct temporal and spatial timepoints. For example, various signaling components of the Wnt/β-catenin pathway appear to be upregulated during early-stage rudiment regeneration, while signaling components of the Wnt/Planar Cell Polarity pathway are upregulated during late-stage regeneration in tissue that contains luminal epithelium. Even distinct gene ontological terms are functionally enriched in anterior regenerates that are not in posterior regenerates such as chromosome organization and cilium assembly. Altogether, this study demonstrates the diversity of molecular mechanism that occur during a dynamic process like intestinal organogenesis and serves as a baseline to guide future hypothesis-driven molecular studies....

The microbiota, the set of microorganisms associated with a particular environment or host, has acquired a prominent role in the study of many physiological and developmental processes. Here I explore a possible role of the microbiota in regeneration, focusing primarily on the regeneration of the intestine in the sea cucumber Holothuria glaberrima, an echinoderm common in local waters. In CHAPTER 1, I review the involvement of the microbiota in regeneration-related cellular events, providing examples of regenerative models that extend from the repair of tissue layers to the regeneration of complete organs or animals. In addition, I highlight the role of the microbiota in the digestive tract, in echinoderms, primarily holothuroids, which are models for regeneration studies. As a first step in these studies, I characterize the microbiota of normal and regenerating animals (CHAPTER 2). In this chapter the microbial community of sea cucumbers undergoing intestinal regeneration was studied using 16S rRNA amplicon sequencing. For this, sea cucumbers were eviscerated and left to regenerate in seawater aquaria for up to 21 days. Among the findings reported are: (i) the existence of microbial compartmentalization in normal animals which changes in regenerating animals. (ii) a strong effect of the environment, in this case the closed aquarium system, on the normal and regenerating microbiota, (iii)) an increase in species richness and evenness in regenerating animals and (iv) differential abundance of specific bacterial taxa. To determine the role of the microbiota in the regeneration process an initial approach was executed in the following chapters. CHAPTER 3, shows the experiments where the effects of various antibiotics on intestinal regeneration of H. glaberrima were evaluated. For this, eviscerated holothurians were exposed to different antibiotic cocktails and left to regenerate for 10 days. Immunohistological and histochemical analyses were performed to analyze regenerative processes, including rudiment size, extracellular matrix (ECM) remodeling, cell proliferation, and muscle dedifferentiation. Reduction in muscle dedifferentiation, ECM remodeling, and the size of their regenerating rudiments were observed in antibiotic-treated animals in comparison to untreated ones. Our results demonstrate a negative effect of antibiotics on intestinal regeneration and strongly suggest that these effects are due to alterations in the microbial community. To discard the possibility that the antibiotics directly affect holothurian metabolic activity, their effect on the metabolism of H. glaberrima tissues was assayed. A second approach to test the effect of microbiota on intestinal regeneration is shown on CHAPTER 4. Here I utilized fecal microbial transplants (FMT) to validate the effectiveness of microbial transplants in enhancing intestinal regeneration. For this, eviscerated holothuroids, exposed to antibiotics prior to evisceration, were subjected to FMT from non-eviscerated donors. Control animals received no FMT or autoclaved FMT. Bacterial samples were cultured from each group to verify if antibiotics could induce changes in the animal microbiota. Histological analysis revealed that different combinations of antibiotics, along with FMT, have specific effects on various regeneration processes. These findings support the role of the microbiota in the sea cucumber's intestinal regeneration process, reveal factors contributing to organ regeneration, and provide insight into little studied functions of the gut microbiota....

Transcriptomic databases have become one of the main sources for protein discovery. In our studies of transcripts from normal echinoderms, we have identified several transcripts that have attracted our attention. One of these is a previously unidentified transcript (Orpin) that appeared to be upregulated during intestinal regeneration. In Chapter 2, using bioinformatics tools we: (1) identified a second Orpin sequence (2) describe their motifs and domains, and perform phylogenetic analyses that suggest that Orpins might comprise a novel subfamily of EF-hand containing proteins specific to the Ambulacraria clade. Semiquantitative RT-PCR analyses revealed that Orpin mRNAs are expressed in various tissues but no significant differential expression was found in regenerating tissues.<br /> <br /> In Chapter 3 we expressed and purified genetically modified versions of Orpin to further characterize this EF-hand protein. We developed two protocols:<br /> <br /> The first protocol consisted in the production of His-OBSm, which is His tagged with two additional genetic modifications: the deletion of the signal peptide encoding sequence and the addition of a 25 residues peptide from the pET200 plasmid vector. These modifications made possible the purification of a soluble recombinant version of Orpin B.<br /> <br /> The second protocol was developed to produce a soluble recombinant Orpin that best resembled the original protein. This protocol consisted of the expression of a GST-Orpin lacking the signal peptide region. For this protocol, we tested different parameters that affect protein expression, including, additives, host cells, growth media and supplementation, and protein extraction methods. The best parameters were identified and used to obtain a soluble Orpin form. We propose that the developed strategies can be used to increase the soluble expression and purification of other EF-hand proteins that are difficult to express by standard methods.<br /> <br /> In chapter 4 we produced antibodies against His-OBSm and used these to identify the cells expressing the protein in H. glaberrima tissues. Antibody specificity was tested through Western Blots.<br /> <br /> Finally, we investigated the effect of the Wnt signaling pathway on Orpin mRNA expression in regenerating gut explants and found an apparent decrease expression of an Orpin B isoform following Wnt/B-catenin pathway activation....

Bone morphogenetic proteins (BMPs) are widely known for their ability to induce bone formation in development and/or when it has been some damage in bone tissue. Nonetheless, these proteins are also involved on many other processes and exert their functions in other tissues. The BMPs are recently study in cancer, possible therapy for repairing cardio vascular tissues and in regeneration process. Our lab focus the study on understanding the dynamics of regeneration process once there is loss or damaged to a tissue, to bring an insight of which are the mechanisms that ensure a complete regeneration. The phylum of Echinoderms possess impressive regenerative capacities, which makes them a great example of model systems to study and comprehend the regeneration process. Our model system, the Caribbean Sea cucumber Holothuria glaberrima helps us understand how the regeneration is accomplished since this echinoderm is able to regenerate most of its organs once they are eviscerated. Here we explore the functions of BMPs under the intestinal regeneration since previous studies in our lab prove that these proteins were present during this process. We used pharmacological agents to see how the BMPs played a role in the process of regeneration. Our results demonstrates that these proteins compromised the muscle cell dedifferentiation, cell proliferation and affect the variability of the blastema size during the intestinal regeneration....