During the earliest stages of development, molecular signals initiate hierarchies of gene expression confined to specific regions of the embryo. This spatial restriction is ultimately translated into cell and tissue- specific patterns of gene expression within defined cell lineages. Erythroid cells provide a useful model system to analyze vertebrate lineage determination at the biochemical level, because primary cells expressing lineage-and stage-specific markers are obtained easily. The erythroid-specific transcription factor Eryfl appears to be the major regulatory protein for genes of the erythroid lineage. The proposed study is designed to determine whether Eryfl is the primary determinant for establishment of the erythroid lineage, and to investigate what molecular events regulate its expression. Three specific questions will be addressed: 1. What are the initial trans-signals and cis-regulatory elements required during the earliest stages of development (mesoderm induction) for the lineage-restricted expression of the Eryfl gene. 2. Is Eryfl involved in determining stage-specific expression among different established erythroid lineages? 3. How does Eryfl activate erythroid- specific genes in the terminally differentiated cell? No single system is optimal for biochemical analysis of mechanisms operating both early and late in development. The frog provides a tractable approach to analyze molecules involved in early development. Therefore, using the pluripotent zygote of Xenopus laevis the signals that lead to the initial activation of Eryfl will be investigated. However, primary erythroid cells of later defined development stages are obtained most easily from the developing chick embryo. Therefore, using the committed cells of the chick erythroid system, the regulatory events at the other end of the cascade will be analyzed. It is anticipated that by working in both directions, the order can be determined of a complex series of molecular events, which ultimately establish and the maintain a unique pattern of tissue-specific gene expression. Defining the order of events will help us understand how different kinds of cells are formed, and why the process sometimes fails.
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