The specific aims of this research is to test the hypothesis that the barring gene (B) and the dominant white gene (1) express themselves in Fowl by making the feather and choroid melanocytes more sensitive to a cytotoxic agent, possibly a compound produced by the melanocyte itself. Since these genes cause the feature melanocytes (and possibly the choroid melanocytes) in Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens to die prematurely as compared to the melanocytes of the wild type Jungle Fowl (JF), then the BPR and WL melanocytes may serve as useful models of vitiligo. The development of the pigmentation of the BPR choroid layer in the eye will be studied to determine if the reduced number of melanocytes in that layer is due to cell death or due to lack of migration of melanocyte stem cells to that region during the embryology of the eye. Thus an anatomical study would be initiated which would determine the fate of the choroid melanocytes during the early embryo stages (days 3 to 21) and, if necessary, through juvenile birds. If we find that the number of BPR melanocytes (compared to the JF choroid) is reduced due to premature cell death, then this finding would strengthen our argument for the use of the barring gene as a model for the cytotoxic hypothesis of vitiligo. We will also investigate the effect of the dominant white, gene 1 found in WL on the eye pigmentation in this breed. Tissue cultures of feather and choroid melanocytes for BPR, WL and JF will be tested for their sensitivity to a number of known cytotoxic agents, to melanocyte subfractions and to keratinocyte cytoplasm. Our hypothesis is that the WL melanocytes will be the most sensitive, followed by the BPR melanocytes. Since both genes exhibit a dosage effect, B/B, B/+, B/- 1/1 and 1/+ genotypes will be tested. Thin layer chromatography will be used to detect higher than normal levels of tyrosine and L-dopa in WL or BPR feathers as compared to JF features. Mitogens will be tested and added to the tissue culture media in an effort to increase the melanocyte populations. The ultrastructure of adjacent keratinocytes in the WL and BPR feather white areas will be examined for any possible degenerative changes. Enzymes associated with melanogenesis will be monitored in BPR, WL and JF to determine the effect of the B and 1 genes on the enzymes. Somatic cell fusion techniques will be used to determine whether or not the B and 1 genes exert their effect within the nucleus or in the cytoplasm. The goal of this grant, in addition to the scientific research, is to train MBRS students in research methodology.
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