In 1991 we isolated and characterized a human gene and cDNA that codes for a protein with a domain structure similar to hepatocyte growth factor (HGF). We have called this protein HGF-like protein or HGFL. Since then HGFL has been found to be identical to macrophage stimulating protein (MSP). The cell surface receptor for HGFL has been found to be the Ron tyrosine kinase receptor; a member of the c-Met proto-oncogene family, c-Met is the receptor for HGF. Although c-Met and HGF have been implicated in many biological responses, little is known about the responses initiated by Ron and HGFL in vivo. In order to determine the physiological roles of HGFL and Ron, we generated mice deficient in each of these proteins. Mice lacking HGFL grow to adulthood with few phenotypic abnormalities. However, mice null for Ron possess a strikingly different phenotype. Ron-/- embryos are viable through the blastocyst stage of development but fail to survive past the peri-implantation period. In situ hybridization studies demonstrated Ron expression in the trophoblast cells surrounding the inner cell mass. These trophectoderm cells give rise to the invading trophoblast cells and the fetal portion of the placenta. Studies by others have demonstrated that overexpression of Ron and the presence of constitutively active forms of Ron promote an invasive phenotype. Therefore, the Specific Aims of this proposal will address whether Ron expression is a necessary factor for embryo implantation and sustained viability during early embryonic development and the paradoxical findings of the phenotypes of HGFL- and Ron-deficiency in mice.
In Specific Aim I we will test the hypothesis that Ron plays an active role during embryo implantation in the mouse by characterizing the temporal and spatial distribution of Ron and HGFL during early blastocyst development and implantation in mice, and determining the phenotype resulting from the loss of Ron alleles during mouse development.
In Specific Aim 2 we will test the hypothesis that Ron is required for trophoblast function by examining the morphology and function of trophoblast cells surrounding Ron-/- embryos during implantation, determining whether Ron-/- embryos can be rescued with wild-type extraembryonic tissue, and elucidating the molecular mechanisms involved in ligand induced Ron activation in trophoblast cell lines.
In Specific Aim 3 we will test the hypothesis that HGFL-/- mice develop normally because of the presence of either constitutively active forms of Ron or additional ligands for Ron during implantation by determining whether plasminogen and/or livertine are ligands for Ron, identifying other HGF-like proteins and/or ligands for Ron, and determining if the constitutively active forms of Ron promote implantation and development in the mouse.
