The aim of this work is to understand the timing mechanisms that govern specific cell fate decisions during metazoan development. The heterochronic genes of the nematode C. elegans are global temporal regulators that control the sequence and timing of diverse events during post embryonic development This research will investigate how one of these events, the terminal differentiation of lateral hypodermal """"""""seam"""""""" cells, becomes temporally restricted to the final molt in wild-type animals. Mutations in the heterochronic genes cause seam cell terminal differentiation to occur earlier or later than normal. Nearly a dozen heterochronic genes have been identified and they can be roughly divided into two classes based upon their known or proposed times of action. The early acting genes specify developmental progression through the first three larval stages (L1-L3) and then the late acting genes take over and direct the remainder of development (late L3-Adult). This research will investigate the molecular roles of four heterochronic genes, the early acting genes, lin-42 and lin-58 and the late acting genes, lin-57 and lin-29. The experiments described in this proposal will seek to understand the functions of these genes in the context of the other genes that comprise the heterochronic gene pathway. Genetic and molecular epistasis experiments will be performed and the expression patterns of these genes will be determined. lin-42 encodes a protein with similarity to Drosophila Period, a protein involved in a second type of biological timing mechanism, control of circadian rhythms. Proposed experiments will investigate the functional relevance of this sequence homology and the yeast two-hybrid system will be employed to discover UN-42-interacting proteins. lin-58 will be cloned, and the information gleaned from its identity will be used to design molecular tests of its function. Because only a single, hypomorphic allele of lin-57 exists, genetic screens will be performed to identify lin-57(null) mutations. These alleles will be characterized and the hypothesis that lin.57 is temporally controlled by the 21 nt let-7 regulatory RNA will be tested. lin-29 plays a key role as the most downstream regulator of seam cell terminal differentiation. The molecular mechanism that times lin-29 activity will be determined and sequences critical for lin-29 regulation will be defined. Finally, genetic screens will be employed to search for addition heterochronic genes. Loss of lin-29 function causes cells of the adult stage to indefinitely reiterate the larval program of cell divisions rather than exiting the cell cycle and differentiating. Thus, lin-29 can be thought of in general terms as an anti-oncogene: a gene whose function is required for cell cycle exit Temporal control of lin-29 provides a model for how cells within an organism are instructed to cease dividing at a specific time in development and differentiate. This knowledge should aid in our understanding of the problems that occur when growth controls go awry, such as the inappropriate resumption of cell divisions that occurs in some cancers.
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