With this proposal we expect to create new knowledge on the biological action of vitamin K in fish skeletal development and deformities, with a multidisciplinary team that can deliver state-of- the-art knowledge on the biological, nutritional and genetic regulation of skeletogenesis in Senegalese sole and zebrafish. Nutritional imbalances affecting bone development have been  implicated in the incidence of skeletal deformities, which are acknowledged to constitute one of the major constraints in the production of high quality fish. In aquaculture production, deformities in the skeleton affect growth, morphology, fish survival and increase production costs and decrease market value of the final products. Several studies have focused on the identification of causes of deformities and associated them to factors such as rearing environment, nutrition and genetics (Boglione et al, 2003; Cahu et al, 2003). In particular the most active form of vitamin A, retinoids, have been proven to cause marked effects over the skeleton, with treatments in larvae causing a high teratogenicity even at very low concentrations. Vitamin K is another liposoluble vitamin classically involved in blood coagulation, and until recently largely disregarded in aquaculture nutrition. Vitamin K is known to play an important role in bone metabolism by acting over gamma carboxylation of matrix proteins as Osteocalcin (Oc) and Matrix gla protein (Mgp) and regulating Msx gene expression, through the recently identified vitamin K binding Pregnane X receptor (PXR). The mechanisms underlying the association of vitamin K with the changes of skeletal microanatomy are still not fully understood. It has been recently shown by Roy and Lall (2007) that supplementation of feed pellets with menadione sodium bisulphite (Vitamin K3) have positive effects over the incidence of deformities during juvenile growth in haddock (Melanogrammus aeglefinus). Conversely, dietary Vitamin K nutritional deficiency caused a decrease in bone mineralization and bone mass, related to an increasing susceptibility to bone deformity. Similar results were also observed by Udagawa (2001) using mummichog (Fundulos heteroclitus) fish fed vitamin K rich diets showed better skeletal quality than vitamin K deprived ones and mature fish fed vitamin K rich diets resulted in better skeletal quality in progeny. There are also clear evidences that supplemented phylloquinone is accumulated in tissues at a higher levels than supplemented menaquinone and also that the use of phylloquinone was more effective than menadione (Udagawa, 2001, 2007). However there are no studies supplementing Vitamin K in live prey and no information on the effectiveness of this system to deliver this nutrient to fish larvae. In a preliminary experiment we obtained some indications of the positive effects of Vitamin K1 supplementation by prey enrichment, on the skeletal development of early stages of S. senegalensis, with a reduction on the incidence of deformities and in the number of deformities per individual. We could also observe that there is differential protein expression in supplemented fish. These results were recently presented in a international meeting (Annex file Richard et al- ISFNF2008.pdf). Given the interesting results obtained with our preliminary experiment and the described for other species, we aim with this project to improve the scarce knowledge in this field and propose to investigate the effects of dietary vitamin K supplementation on skeletal deformities and on the expression of genes and proteins involved in skeletal development. For this we will use two fish models with different bone types: the Senegalese sole (Solea senegalensis), a species with commercial interest that as an acellular bone devoid of osteocytes, and zebrafish (Danio rerio), a known model for studying osteocyte-containing cellular bone, with the advantages of the large array of existing tools and genomic databases for both species. The analysis of the proteome and the use of available gene micro arrays will be used to identify differentially expressed genes and proteins induced by vitamin K. By cloning the genes for vitamin K receptor (PXR) and its obvious target Msx, and studying the expression levels and patterns along with other known skeletal gene markers, we intend to gain new knowledge on the molecular mechanisms that mediate vitamin K action on skeletogenesis in fish. In addition we will verify the influence of bone type and look for any differences in the chosen models. Finally we intend to conduct several trials with larval and juvenile stages using new formulated diets and enrichment medias that meet nutritional vitamin K needs in all life stages of Senegalese sole. These results altogether can bring new knowledge important for the improvement of aquaculture industry by reducing deformity incidence in produced fish and can have a high potential for industrial production of new diets.