TCDD, one of the most toxic halogenated dibenzodioxins, causes teratogenesis as well as epithelial metaplasia, tumor production and enzyme induction. The objectives of this research are to elucidate molecular mechanisms by which teratogens cause birth defects by exploring whether gene expression is altered. As a model system, the effects of TCDD on gene expression in the developing mouse palate is to be analyzed since it is known that TCDD (a) produces cleft palate in mice and (b) alters gene expression in other biological systems. The hypothesis to be tested is that TCDD quantitatively alters gene expression in the palate, the consequence of which prevents programmed cell death in the medial edge epithelium of the palate and cleft palate is produced. It should be emphasized that no published work in Teratology has as yet appeared which directly supports or refutes the concept that teratogenesis may be a consequence of altered gene expression.
The specific aims of this small grant application will be to show the feasibility of the project in the developing mouse palate by pursuing the following specific aims: (1) Quantitative analysis of specific mRNA sequences from TCCD and control palates using selected cDNA probes. After administration of teratogenic doses of TCDD to sensitive pregnant C57B1/6 mice, total or poly(A+)RNA will be prepared from dissected control and TCDD-palates. Employing radioactive cDNA probes containing message sequences to P1-450, to a number of cytokeratins, and to murine EGF receptor, message sequences will be quantitatively measured by blot hybridization of RNA. (2) Preparation of a cDNA library from palate in lambda gt-11. Message sequences from control and TCDD-palates will be separately made into cDNA libraries to allow screening of palatal poly (A+) RNA to detect differences from control and TCDD-treated embryos. (3) Preparation of a subtracted cDNA probe from control and TCDD poly (A+) RNA. Selected hybridization will be used to enrich for those sequences in a poly (A+) RNA population whose concentration is altered by TCDD (stimulated or inhibited) to allow their isolation and subsequent cloning. Control and TCDD-palatal RNA will be assayed with isolated cDNA probes from the lambda gt-11 library.
