The goal of this application is to study mechanisms underlying the development of non-skelatal muscle using the pharyngeal muscle of C. elegans as a model system. Non-skeletal muscle, such as cardiac or smooth muscle, do not use MyoD-like proteins to control their differentiation. The nature of the molecules involved is unknown and the goal of this application is to identify those molecules. As a first step to identify these proteins, Dr. Okkema has studied the regulation of the pharyngeal muscle-specific myosin heavy chain gene myo-2 during his postdoctoral work. Expression of this gene is controlled by an enhancer containing three subelements, A, B and C, that are inactive individually but can enhance transcription when dimerized or in various combinations. B activates transcription in a subset of pharyngeal muscles, whereas C is active in all cells of the pharynx. A might activate transcription in pharyngeal cells not controlled by B. A homeobox protein named CEH-22 has been identified; this protein interacts with a specific sequence of the B element and belongs to the NK subclass of homeobox proteins. Ectopic expression of CEH-22 in body wall muscles causes activation of myo-2 expression, but mutations in the ceh-22 gene do not affect transcription of myo-2. A 2.5 kb enhancer responsible for ceh-22 expression has been identified. A second putative regulator of myo-2 expression has also been found; this protein belongs to the basic leuzine zipper family and binds to the B subelement of the myo-2 enhancer, adjacent to the CEH-22 binding site. The experiments proposed in the application have three major aims.
The first aim i s to understand how CEH-22 participates in paharingeal muscle development. Dr. Okkema will isolate loss of function mutations in the ceh-22 gene, including a null allele, and their phenotype will be analyzed in detail. He will also express CEH-22 under the control of heat shock promoters to test whether this protein can activate a complete program of pharyngeal myogenesis, and whether it requires other muscle-specific factors. Pharyngeal muscle differentiation will be followed using early markers such as 3NB12; inhibition of body wall muscle differentiation will also be followed using specific markers such as UNC-54, MYO-3, and HLH-1. Effects on non-muscle cells will be similarly analyzed. Interactions with other genes involved in pharyngeal development, such as pha-1 and pha-4, will be analyzed to identify other members of the pathway; mutations in these two genes block differentiation of all cell types in the pharinx. The second part of the proposal describes experimetns aimed at understanding the regulation of the ceh-22 gene. Cis-acting sequences will be delineated further, and proteins that bind to these sequences will be identified. Finally, the last part of the proposal describes an analysis of the C subelement of the myo-2 enhancer. This element specifies transcription n an organ-specific fashion, and Dr. Okkema proposes to identify factors that bind to these sequences by screening cDNA libraries for clones encoding proeins that bind to specific sequences.
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