The long-term goal of this application is to understand the molecular mechanisms underlying the regulation of early mammalian embryogenesis, specifically gastrulation. Mammalian development is controlled by genes which are differentially expressed in a temporal and spatial manner. The goal of this proposal is to identify and isolate genes which control developmental processes during gastrulation, using the mouse as a model system. To achieve this goal, the applicant proposes to employ a genetic screen using """"""""gene trap"""""""" retroviral vectors in pluripotent mouse embryonic stem cells (ES cells). These vectors contain a reporter gene, Escherichia lacZ, whose expression is dependent on transcription initiated from cis- acting regulatory sequences of a cellular """"""""tagged"""""""" gene. Therefore, genes potentially important for developmental processes can be identified based upon the patterns of the reporter gene expression disrupting the normal expression of the cellular gene, resulting in abnormal developmental process. Finally, the integrated provirus serves as a """"""""tag"""""""" to isolate the disrupted cellular gene. To use this strategy effectively, for genetic screening in developing mouse embryos, the applicant proposes to pre-screen a large number of ES cell clones for desired retroviral insertions in gastrulation- specific genes by monitoring the pattern of lacZ expression (i) upon differentiation of ES cells in vitro into embryoid bodies, and (ii) in vivo in developing chimeric embryos. Transgenic mice will be generated and analyzed for developmental abnormalities associated with the mutated gene(s). Finally, the investigator proposes to isolate the disrupted endogenous gene(s). Successful identification of gastrulation-specific genes will be crucial for elucidating the regulation of early mammalian embryogenesis and, more generally, the molecular basis of developmental abnormalities in humans.
