Molecular genetic studies in Caenorhabditis elegans have revealed that heterochronic genes explicitly define and interpret temporal information during development. These genes control the temporal sequence of the C. elegans postembryonic cell lineage by generating a temporal molecular gradient of the two nuclear proteins that are the products of the heterochronic gene lin-14. We propose to dissect how the lin-14 gene generates and interprets temporal signals to control C. elegans development. We will establish how transcriptional and posttranscriptional regulation of lin-14 gene activity generates the temporal gradient reverse genetic analysis of mutations in lin-14 regulatory regions. By monitoring the regulation of the lin-14 gene in a variety of heterochronic mutants, we will dissect how these genes conspire with lin-14 to generate or interpret temporal information. We will explore the lin-14 biochemical function in the nucleus by testing whether lin-14 proteins can bind to the DNA or RNA of the other cloned heterochronic genes that genetic and molecular epistasis analysis suggests they may regulate. These interactions will be analyzed with various lin-14 protein mutants constructed in vitro. We will test whether each of the two lin-14 proteins specify distinct cell fates by expressing each inappropriately and scoring their effect on the fates of cells that express them. We will characterize a newly identified set of heterochronic genes that control timing of the neuron-regulated dauer developmental pathway. These heterochronic genes may control timing in the specific neuronal cells that control entry into the dauer stage, or they may be more general developmental timing genes. We will explore how these new heterochronic genes fit into the heterochronic gene epistasis pathway and into the dauer epistasis pathway to discern which genes they regulate or vice versa. We will generate transposon insertion mutations in these genes to facilitate their cloning and molecular analysis. The heterochrony observed in phylogeny is very reminiscent of that we have dissected in C. elegans and suggests that heterochronic genes like lin-14 may be major players in evolutionary change and developmental control across phylogeny. Based on numerous examples of conservation of gene structure and function between species, molecular and genetic explorations of these genes in C. elegans promises to illuminate the functions and mechanisms of their human homologues. Mutations in these human temporal control genes may be the molecular basis for some human genetic diseases (for example, particular cancers) that lead to temporal transformations in cell fate.
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