phalloidine and 5-bromo-4-chloro-3-indolyl-beta-galactoside

In a phenotypic screen in mice using a gene trap approach in embryonic stem cells, we have identified a recessive loss-of-function mutation in the mgcRacGAP gene. Maternal protein is present in the oocyte, and mgcRacGAP gene transcription starts at the four-cell stage and persists throughout mouse pre-implantation development. Total mgcRacGAP deficiency results in pre-implantation lethality. Such E3.5 embryos display a dramatic reduction in cell number, but undergo compaction and form a blastocoel. At E3.0-3.5, binucleated blastomeres in which the nuclei are partially interconnected are frequently observed, suggesting that mgcRacGAP is required for normal mitosis and cytokinesis in the pre-implantation embryo. All homozygous mutant blastocysts fail to grow out on fibronectin-coated substrates, but a fraction of them can still induce decidual swelling in vivo. The mgcRacGAP mRNA expression pattern in post-implantation embryos and adult mouse brain suggests a role in neuronal cells. Our results indicate that mgcRacGAP is essential for the earliest stages of mouse embryogenesis, and add evidence that CYK-4-like proteins also play a role in microtubule-dependent steps in the cytokinesis of vertebrate cells. In addition, the severe phenotype of null embryos indicates that mgcRacGAP is functionally non-redundant and cannot be substituted by other GAPs during early cleavage of the mammalian embryo.

Topics: Animals; Blotting, Northern; Brain; Cell Nucleus; Cloning, Molecular; DNA, Complementary; Embryo, Mammalian; Female; Galactosides; Genotype; GTP Phosphohydrolase Activators; GTPase-Activating Proteins; Heterozygote; Homozygote; In Situ Hybridization; Indoles; Male; Mice; Models, Genetic; Mutation; Phalloidine; Phenotype; Recombinant Fusion Proteins; RNA, Messenger; Time Factors; Tissue Distribution; Transcription, Genetic