Analysis and characterization of genes associated with intestinal regeneration in the sea cucumber (Echinodermata: Holothuroidea)

Regeneration is an attribute of all living organisms to replace a lost or damaged body part. Despite being a very common feature throughout the evolutionary scale the mechanisms of regeneration appear to be widely different among taxa. Differences in the ability of animals and their mechanism to regenerate have been directly related with the evolutionary complexity of the organisms. The most simple and primitive ones tend to have increased cellular plasticity that allows regeneration while in more complex organisms such plasticity is limited to few animals or structures. In mammals for example, as in other evolutionary recent organisms, regeneration is limited to some developmental stages or to some tissues. Echinoderms are exceptional organisms to study regeneration for several reasons: (1) they are philogenetically close to vertebrates (2) they exhibit astonishing regeneration abilities and (3) they show high variability in the regeneration processes.;At the molecular level, we are still distant from understanding the molecular mechanisms that control organ regeneration. However, in recent years, this distance is being curtailed as new genes and gene functions are known in regeneration events.;The present work is focused at the molecular level to identify those genes (known and novel) that might direct the regenerative processes and their evolution. Here we use as a model of study the intestinal regeneration of the sea cucumber H. glaberrima. Initially we construct three cDNA libraries, two of them from early regeneration stages that in total count 7209 ESTs, in which significant cellular events have been previously characterized. Inferential statistics was performed to determine differential expression due to EST abundance. This predictive test results in 17 genes that were 77% validated by microarrays and 87% by RT-PCR. Gene-expression microarrays were done to find and profile the genes associated with the intestinal regeneration process. From the approximately 2500 genes tested, more than 57% show significant expression changes during at least one stage of regeneration with significance p<0.05. The expression profile obtained by microarrays was validated using semiquantitative-PCR and further gene characterization was performed for a selected set of 26 genes. Molecular strategies were complemented with the use of bioinformatics, inferential statistics and phylogenetic analysis in order to characterize putative identities, location, function and evolution. Here are presented two sub-sets of genes, the known genes and the potentially novel genes (unknowns), that are associated with the process of intestinal regeneration. The first group includes genes whose function has been characterized in development, cytoskeleton organization and extra cellular matrix in other organisms and might represent pivotal roles in the cellular events that take place in the intestinal regeneration. The unknown genes in contrast, represent potential novel genes that might have specific functions in the molecular control of regeneration. In particular, further characterization and experimentation with one of the unknown genes confirmed that it is indeed a novel gene from the hundreds involved in the regeneration of the intestine.;In summary, this work demonstrates that the intestinal regeneration in the sea cucumber is not only a great model to study regeneration at molecular level but a complex process in which a large number of genes are involved. Moreover, it opens several doors that lead to further examination of candidate genes associated with the molecular control of organ regeneration.