The long-term goal of this research is to describe genetic control of complex events underlying the development of the mouse embryo. In a program specifically designed to generate and transfer random insertional mutations into mice, cultures of ES cells predetermined as carrying high copy numbers of proviral sequences were used to make germ line chimeras. Three different mutations have been identified. These include (1) a recessive lethal mutation acting in the mid-term embryo, (2) a recessive mutation affecting tail morphology and (2) a mutation which affects primary sex determination in the XY embryo. A major goal of this proposal is to identify specific retroviral integration site(s) associated with each mutation. Using the proviral sequence as a probe, we will molecularly clone the locus of insertion. The cellular defect(s) associated with each mutation will be described. We will make of pluripotential embryonic stem cell lines (ES cells) to investigate the cell biology of the developmental failure, and to determine whether the mutant phenotype(s) is attributable to defects in specific cell lineages. In addition, cDNA libraries constructed from undifferentiated ES cells and from ES cells induced to differentiate in culture, have been screened for transcripts which are developmentally regulated. Three such transcripts have been identified and partially characterized. The temporal and spatial expression of these genes will be examined in the developing embryo. These studies will use in situ hybridisation techniques and antibodies directed against the protein products encoded by these genes. To test for a developmental role of specific genes, we plan to experimentally manipulate expression in transgenic chimeras constructed using ES cell lines. These studies will hopefully identify genes responsible for regulating processes of mammalian development.