Application): The riddle of morphogenesis or how the organs and tissues of the body are formed has been ranked as one of the most important unanswered questions in developmental biology. Although morphogenesis is usually studied during embryogenesis, the regeneration of tissues and organs in adult organisms provide alternate morphogenetic models. Moreover, regeneration provides the advantage of studying morphogenesis of an organ in the absence of other concomitant developmental events as occurs during embryogenesis. The sea cucumber Holothuria glaberrima provides a new model system where the formation of the intestine can be studied during regeneration. These coelomate organisms, members of the phylum Echinodermata, are closely related to the chordates and exhibit an impressive ability to regenerate complex structures. Sea cucumbers subjected to unfavorable environmental conditions undergo a process of evisceration; ejecting most of their internal organs. Evisceration is followed by a period of regeneration during which the ejected organs are replaced. The proposed research focuses on the changes in extracellular matrix (ECM) molecules during the formation of the new intestine. The working hypothesis in the laboratory is that the ECM undergoes large remodeling processes during regeneration and that these are essential for the organization of the tissue layers during the formation of the intestine. Initially, major ECM components of the sea cucumber intestine will be identified and their temporal and spatial changes during the regeneration process will be documented. Central to the remodeling changes are metalloproteases that can actively degrade ECM components, thus it is necessary to detect and identify the metalloprotease activity that might be involved in the formation of the new intestine. Functional studies will be done by interfering with the activity of the metalloproteases and with the function of collagen in order to determine the role the molecules are playing during intestinal morphogenesis. Finally, studies will be done at the nucleic acid level to determine the gene expression of molecules associated with the ECM remodeling. The outcome of the experiments will provide new evidence for the importance of the ECM in morphogenetic events and will help elucidate the steps that take place in the assembly of tissues and organs that make an organism.
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