Molecular genetic studies in C. elegans have revealed that heterochronic genes explicitly control the temporal sequence of cell lineage by generating a temporal molecular gradient of the nuclear protein products of the heterochronic gene lin-14. Conserved elements in the lin-14 3' UTR that are complementary to the RNA products of the regulatory gene lin-4 generate this temporal gradient. By analysis of mutations that perturb the structure and placement of the multiple lin-4/lin- 14 RNA duplexes and other conserved elements, the applicant proposes to dissect how the lin-14 3' UTR generates the temporal gradient. He will assay the mutant lin-4/lin-14 RNA duplexes in an in vitro translation system which partially recapitulates lin-4 in vivo regulation of lin-14 3' UTR. He will correlate the in vivo and in vitro effects. He will use genetic screens to identify gene products that recognize the lin-4/lin-14 RNA duplex as well as other conserved elements to mediate temporal gradient formation. He will clone those genes that act in concert with lin-4 to down-regulate lin-14. He will determine the function of each LIN-14 protein by a combination of observation and ectopic expression experiments. He will use other genetic screens that will identify genes that act in combination with lin-14 or downstream of lin-14 to control the temporal fates of cells. Genes that act downstream of lin-14 will be cloned and the molecular mechanisms by which they are regulated by lin-14 will be studied. He will screen for proteins that interact with LIN-14 and characterize their function in temporal pattern formation by reverse genetic analysis. Regulation of pattern formation gene expression by 3' UTR regulation has been found in a number of systems, especially those involving the temporal and spatial cascade of maternal mRNA translational control. The involvement of an antisense RNA in this process is at this point unique in metazoans but likely to be the first example of a more generally used mechanism. In addition, the mechanisms and indeed the genetic components of the lin-14 temporal gradient pathway promise to allow the identification of homologues from other organisms such as man. The applicant posits that such homologues will perform very similar functions in temporal pattern formation or translational control. The recent discoveries of universal components in developmental control by, for example, HOX clusters and signalling pathways endorse such a view of universality. If the gene pathway is so conserved, mutations in human heterochronic genes may be molecular bases for various oncogenic and endocrine genetic diseases that transform cells to precursor or descendent fates.