Mineralized skeletons, whether they are bones, teeth, mollusc shells or crustacean exoskeletons, are all comprised of two structural components: an organic matrix and the minerals that impregnate it. The organic matrix is a complex mixture of proteins and carbohydrates; sometimes the proteins have carbohydrates attached to them and are referred to as glycoproteins, proteoglycans or mucins. The minerals may be crystalline forms of calcium phosphate, like the hydroxyapatite of bones and teeth, or calcium carbonate, like the calcite of crab exoskeletons. One of the central and most basic questions in the field of skeleton formation (biomineralization) is which of the complex of matrix molecules are the ones that actually control the initiation of skeletal hardening and control the location and form of the mineral. This project utilizes the blue crab as a model for the control of biomineralization. Because crabs molt in order to grow, they provide an ideal system in which to study the biological control of mineralization and the interaction between the organic and mineral components of the skeleton. The outer two layers (epi- and exocuticle) of the new exoskeleton of the carapace of the crab are deposited beneath the old exoskeleton in preparation for the molt (premolt). They must remain unmineralized until after the crab emerges and expands. Subsequently (postmolt), the inner and thickest layer of the exoskeleton (endocuticle) is deposited and mineralized. The same temporal sequence occurs in the exoskeleton covering the joints (arthrodial membrane), but it never mineralizes in order to remain flexible. A number of biochemical changes that occur in the epi- and exocuticle of the carapace exoskeleton that coincide with their postmolt mineralization were previously catalogued by our laboratory. To determine if these changes are really important to this process, the arthrodial membrane will be similarly analyzed during the same time period. The same biochemical changes should not occur in the arthrodial membrane if, in fact, they are associated with the initiation of mineralization. A new approach to be taken in this grant is to compare the proteins that are manufactured by the tissue that makes the mineralized cuticle to those proteins that are manufactured by the non-mineralizing arthrodial membrane. In this way, it can definitively be determined which proteins are the ones that are involved in mineralization. With the modern molecular biological tools now available, the most efficient way to see what proteins are being made by a tissue is to extract the genetic blueprint for the proteins in the form of the messenger RNAs (mRNA). The plan is to extract the mRNA from the tissues of premolt crabs that are making proteins of the new epi- and exocuticle. This will be done for the tissues underlying both the mineralizing carapace exoskeleton and the non-mineralizing arthrodial membrane. The mRNA will be extracted from the same tissues of postmolt crabs when they are synthesizing the endocuticle. The mRNAs will be compared using a technique termed differential display of expression. In essence, this technique allows the identification of those pieces of mRNA that are found in the carapace tissue but not in the arthrodial membrane tissue. The ones that are identified from premolt tissue are the likely candidates for the messages for the proteins that are intimately involved in the mineralization process of epi- and exocuticle. Those that are identified from postmolt tissue will similarly be involved in endocuticle mineralization. A large part, if not the entire sequence, of the message can be reconstructed and these candidate proteins can be synthesized. Antibodies to these proteins will be made, which, when labeled, will allow the microscopic localization of these proteins spatially and temporally within the exoskeleton and the comparison of their presence to the sites of initial mineralization. To date, no one has firmly identified all of the components of an organic matrix that are actually responsible for initiating and controlling mineralization. The crustacean exoskeleton affords a unique system in which this can be accomplished and will provide basic information that can be applied to other mineralizing tissues.
