The ultimate goal of this proposal is to identify, characterize, and study the function of genes involved in critical steps in early mammalian development. Initially, we will focus our attention on the Evx-1 gene, a mouse cognate of the Drosophila homeobox-containing even- skipped gene. Based on the observation that Evx-1 is expressed in a gradient in the primitive streak, we have hypothesized that Evx-1 functions in a dose-dependent fashion to provide dorso-ventral patterning of the newly formed mesoderm. To test this hypothesis we will determine the consequences of expressing Evx-1 RNA at high levels throughout the primitive streak, thereby ablating the gradient of Evx-1 RNA that is normally observed. We will also determine the consequences of ablating Evx-1 gene function, by creating embryos homozygous for a null allele of Evx-I. In addition, we will identify the genomic sequences that determine the tissue-specificity of Evx-1 expression in the developing embryo. A second major goal is to identify and isolate the gene encoding a molecule, VE-1, that is expressed in the visceral endoderm overlying the future anterior region of the embryo during the early post-implantation stages of development. Our long-term goal is to determine the function of this earliest known marker of A-P polarity in mouse embryogenesis. In the course of pursuing these experiments, we propose to test the efficacy of a new method (the cre/lox """"""""binary"""""""" transgenic mouse system) based on DNA recombination for manipulating gene expression in vivo. In these experiments the desired effect, e.g. ectopic gene expression, is achieved by mating mice of two different transgenic strains. One (the """"""""target"""""""" mouse) carries a target transgene that is innocuous but has the potential to cause the desired effect, and the other (the """"""""effector"""""""" mouse) carries a DNA recombinase capable of altering the target transgene so that it can produce the desired effect. Because the target transgene has no effect prior to recombination, and the effector (DNA recombinase) alone has no phenotype, both target and effector mice are normal. However, in offspring that inherit both transgenes the target transgene is altered by the effector, producing the desired phenotype. Specifically, the system we propose to test employs the cre gene of bacteriophage P1 as the effector. If this approach is successful, we propose further experiments aimed at increasing its versatility, by generating a standard target mouse strain that can be used with a variable effector strain to produce a new lineage map of the mouse, based on gene expression rather than anatomical position. We hope that in the long-term these experiments will not only lead to greater insights into the basic mechanisms controlling embryonic development, but will make a contribution towards the development of a new genre of transgenic mouse technology.
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