The proper form of a multicellular organism requires precise control of terminal differentiation events. This research will address how a single terminal differentiation event is executed during the development of a simple model organism, the nematode C. elegans. The analysis will focus on the terminal differentiation of lateral hypodermal "seam" cells, which terminally differentiate during the final (4th) molt. Several distinct cellular processes are coordinately controlled during this terminal differentiation event, including cell cycle, cell fusion, and the stage-specific regulation of cuticle gene expression. A gene called lin-29 has been identified as a pivotal component in programming the execution of seam cell terminal differentiation. Loss of lin-29 function causes hypodermal cells of the adult stage to reiterate indefinitely the larval program of cell divisions instead of exiting the cell cycle and differentiating. lin-29 encodes a putative transcription factor of the (Cys)2-(His)2 class, and, thus likely programs these events by regulating the transcription of other genes. The major objective of this work is to identify genes controlled by the lin-29 protein. Three approaches will be employed to identify lin-29-regulated genes. ?1! Interactions of lin-29 protein with predicted target genes: The stage-specific expression of the cuticle collagen genes, col-17 and col-19, will be analyzed. col-17 expression is transcriptionally repressed during the larval/adult switch, whereas col-19 expression is activated. The role of lin-29 in the alternative repression and activation of these genes will be tested. ?2! Differential hybridization screens to identify genes whose expression is influenced by the presence or absence of lin-29 protein: Genes isolated in this fashion will be examined for direct regulation by lin-29, and those directly regulated will be characterized further. ?3! Development of a protein-DNA UV crosslinking method to isolate DNA sequences bound by a specific protein in vivo: In the future, this method will be used to identify genes bound by lin-29 protein in vivo, and should also be applicable to the isolation of genes controlled by other C. elegans gene-regulatory proteins. Genes involved in cell cycle exit and cell fusion, as well as additional stage-specifically expressed cuticle genes, should be identified by these methods. Analysis of the types of genes regulated by lin-29 in vivo will lead to an understanding of how a complex terminal differentiation program is orchestrated at a specific time during development.
