The long term objective of my research is to gain insight into complex cellular interactions underlying development of the mammalian embryo. Receptor-protein tyrosine kinases (R-PTKs) are involved in a number of developmental processes. To determine if they are involved in early mouse development, degenerate oligonucleotides were used to isolate members of this gene family which are expressed in 8.5 day mouse embryos. One such R- PTK, eck, exhibits a highly restricted pattern of expression in the post- implantation embryo. Transcripts first appear in ectodermal cells adjacent to the anterior region of primitive streak and later become restricted to the definitive node. The definitive node in the mouse embryo is analogous to the blastopore lip of Xenopus and Hensen's node in chick, all of which have been shown to induce secondary embryonic axes and thus possess """"""""organizer"""""""" activities. Cells in the region of the primitive streak where eck transcripts are localized also contribute to head process and notochordal cell populations. The expression of eck is thus consistent with a role in cell-cell interactions that leads to regionalization and specification of cell fate in node-derived structures during gastrulation. Since eck activity is rapidly down-regulated in cells leaving the node, the fate of cells specifically expressing this gene cannot be determined by conventional techniques, and thereby limits our understanding of how these gene products influence the generation of cell diversity. The major aim of this project is to develop a cell-autonomous marking system utilizing the FLP and Cre recombinase systems to determine the fate of cells which transiently express specific genes during development. This system will be used to determine the fate of cells expressing eck in early development. In particular, these experiments will address the hypothesis that eck plays a role in the specification of mesodermal cell fate in the mouse embryo.
The specific Aims are: I) To use homologous recombination to introduce a beta-galactosidase reporter gene into the eck locus using IRES sequences. II) To use an analogous technique to generate mouse lines with the recombinases expressed in an eck-specific pattern. III) To generate a second mouse line carrying a conditional beta-galactosidase reporter which can be activated by a recombinase-mediated recombination event. IV) To cross the two lines generated in II and III to generate embryos carrying both the recombinase and reporter loci and analyze them at successive stages of development to map the fate of cells expressing eck.
