CRANIOFACIAL DEVELOPMENT IN A MURINE DOWN SYNDROME MODEL

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We propose to apply two recently developed techniques for gene discovery, differential display (DDPCR) and representational difference analysis (RDA), to an analysis of differential gene expression in the trisomy 16 (Ts 16) mouse model for human Down syndrome. These techniques allow for the direct comparison of differentially expressed mRNA species between tissue sources. Trisomic embryos will be identified in utero by visual examination and confirmed by Karyotyping. Embryos will be removed from dams on gestational days 10 through 14 (the period of craniofacial morphogenesis) and RNA prepared from tissues dissected from the midfacial region. Because midfacial hypoplasia is a hallmark of both human Down syndrome and mouse Ts 16, tissue from this region of the embryo is likely to reveal genes whose expression is important to the Down syndrome phenotype. RNA from tissues isolated from trisomic animals and their normal littermates will be compared by DDPCR and RDA. Genes whose expression is observed to be either increased or decreased as a result of the trisomy will be sobcloned into plasmid vectors and sequenced. Sequence analysis utilizing DNA sequence databases will determine the identity of the subcloned gene or indicate if it is a novel sequence. The subcloned bands will also be used as probes for northern blot and in situ hybridization analysis to determine the spatiotemporal expression pattern of these genes in Ts 16 and control embryos. The advantage of this approach is that it allows for the assessment of potential downstream effects of overexpressed chromosome 21 genes, which cannot be addressed by chromosomal mapping and identification of chromosome 21 genes. The proposed studies therefore represent a powerful and novel approache to the study of Down syndrome, and will lead to a greater understanding the etiology of this condition.