This proposal is based upon the hypothesis that phase 1 enzymes are involved in an embryonic switch and that the default position for the switch at the time of fertilization is "OFF". This would occur in a subset of genes that have retinoic acid response elements (RAREs) in their regulatory DNA. The "OFF" position would be due to a function of retinoic acid receptors that has not received sufficient notice: in the absenc of retinoic acid, corepressor molecules bind to the RAR? on the RAREs, the corepresor prevents the RAR? from interacting with a coactivator and also brings in a histone deacetylase that condenses the chromatin. As development proceeds, retinoic acid is precisely parsed out through aldehyde dehydrogenases, and as it leaves its cellular source the retinoic acid can enter adjacent cells but is limited by boundaries of cells expressing specific cytochrome p450 activity (cyp26a1, cy26b1, cyp26c1) that oxidize retinoic acid to a nonfunctional ligand. All of these components are carefully regulated in the developing embryo and have direct and/or indirect feedback loops that are affected by retinoic acid. The allotment of retinoic acid then derepresses genes and affects the differentiation of the embryonic cells. So what does this mean and why is it important? Genes recognized as functional Phase I enzymes in the adult play a critical in the development of the embryo-drugs;hormones that affect these genes or designed to interact with them could then unknowingly play dramatic or subtle roles in the development of the embryo in general and specifically, the nervous system. We will be testing this hypothesis mechanistically with a toolbox of antisense morpholinos, indicator transgenic embryos, and additional reagents and approaches to identify the genes repressed and to see if experimental manipulation of these components can have transgenerational effects upon the germline. Specifically we will be using transgenic retinoic acid indicator zebrafish embryos (developed in this laboratory) to help us identify what genes might be repressed via this mechanism at the time of zygotic gene expression initiation. We will confirm this association by immunoprecipitating zebrafish smrt corepressor associated chromatin using anti-Smrt antisera. We will compare the identified family of genes that go through this double screen with genes others have found to turn on when embryonic stem cells differentiate and we will interrogate our 400+ Agilent microarray zebrafish developmental database to identify general developmental expression patterns and genes that appear to be coregulated in time. Through this work we hope to not only test the hypothesis but to begin to identify an important subset of genes "held-back" by this RAR-corepressor mechanism
The developmental expression of Phase I enzymes can play a critical role in developmental decisions in the embryos. In this proposal, the molecular nature and control of these events will be described and evaluated.