Establishment of the differentiated state begins with cell fate restriction, a process by which subsets of genomic information are made unavailable for use in a particular cell type. New advances in the propagation of human embryonic stem cells in culture raise the possibility of in vitro production of specific cell types to be used as therapeutic agents for damaged or diseased tissues. The production of a desired cell type will require detailed knowledge of how to direct the selection of genomic subsets for expression. This proposal is designed to develop the technology for a genome-wide survey of changes in gene expression during the specification of cell fate in the mouse embryo. Comprehensive genome scanning is now available only for microorganisms with completely sequenced genomes. The NIH will soon produce a comprehensive data base of messenger RNA molecules expressed during early development in the mouse embryo; when this information becomes available, its meaningful use will depend on its application in a coherent context. Described in this proposal is a method for isolating precursor cells to the skeletal muscles of the limbs. Messenger RNA from these cells will be compared to messenger RNA expressed in regions from which these cells migrate, the brachial and inguinal somites. During this migratory transition, the first indication of restriction of cartilage potential in limb muscle precursor cells occurs. While the identity of every mRNA molecule expressed in these two cell populations cannot be known at this time, techniques exist for the isolation of messages that are unique to one population or the other, and for messages that change in abundance during this transition. These """"""""target"""""""" molecules may perform important regulatory functions, and will be isolated and cloned for further experimentation.
