GENETIC ANALYSIS OF MESODERM INDUCTION IN MICE

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DESCRIPTION (adapted from investigator's abstract): This is a revised application to study the function of the msd locus in mouse development. The msd mutant phenotype consists of a failure to form mesoderm and the locus is defined by a series of overlapping deletions within the albino complex. The broad goal of the proposed research is to understand the genetic regulation of mesoderm differentiation. The specific aims of the proposal are to 1) determine if the msd mutation disrupts growth factor signaling. 2) To assess whether msd has other roles during development besides its involvement in mesoderm formation, and 3) to clone the msd gene. For specific aim 1, marked wild type ES cells will be injected into msd mutant blastocysts to determine whether msd endoderm is able to produce a mesoderm inducing signal(s). In addition, the expression patterns of Nodal, BMP-4 and Bmpr will be examined in mutant msd embryos to determine if msd functions by blocking production or reception of known mesoderm inducing signals. In a related series of experiments, regulated ectopic expression of known downstream components of mesoderm signaling pathways such as Mad1 , T and Map kinase will be examined for their ability to bypass the mesoderm block in msd mutants. In section 2 additional characterization of the msd phenotype will include experiments to address the cell-cycle length during early gastrulation, in situ hybridization studies to determine if early markers of anteroposterior axis formation are expressed in msd mutant embryos, production of chimeric embryos containing mutant msd cells in a wildtype background, and isolation of new ENU induced mutations in the msd gene. In the third section of the grant, approaches to clone the msd region are described. They include the isolation of a contig spanning the msd region and identification of transcription units within the contig by cDNA selection. The msd gene or genes will be identified by rescue of mutant embryonic stem cells after transfection with either individual cDNA or multiple cDNA clones.