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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053996-02
Application #
2415348
Study Section
Genetics Study Section (GEN)
Project Start
1996-05-01
Project End
2001-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Clary, Lynn M; Okkema, Peter G (2010) The EGR family gene egrh-1 functions non-autonomously in the control of oocyte meiotic maturation and ovulation in C. elegans. Development 137:3129-37
Ray, Paramita; Schnabel, Ralf; Okkema, Peter G (2008) Behavioral and synaptic defects in C. elegans lacking the NK-2 homeobox gene ceh-28. Dev Neurobiol 68:421-33
Roy Chowdhuri, Sinchita; Crum, Tanya; Woollard, Alison et al. (2006) The T-box factor TBX-2 and the SUMO conjugating enzyme UBC-9 are required for ABa-derived pharyngeal muscle in C. elegans. Dev Biol 295:664-77
Franks, Dawn M; Izumikawa, Tomomi; Kitagawa, Hiroshi et al. (2006) C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans. Dev Biol 296:409-20
Okkema, Peter G; Krause, Michael (2005) Transcriptional regulation. WormBook :1-40
Fernandez, Anthony P; Gibbons, Jack; Okkema, Peter G (2004) C. elegans peb-1 mutants exhibit pleiotropic defects in molting, feeding, and morphology. Dev Biol 276:352-66
Vilimas, Tomas; Abraham, Alin; Okkema, Peter G (2004) An early pharyngeal muscle enhancer from the Caenorhabditis elegans ceh-22 gene is targeted by the Forkhead factor PHA-4. Dev Biol 266:388-98
Beaster-Jones, Laura; Okkema, Peter G (2004) DNA binding and in vivo function of C.elegans PEB-1 require a conserved FLYWCH motif. J Mol Biol 339:695-706
Thatcher, J D; Fernandez, A P; Beaster-Jones, L et al. (2001) The Caenorhabditis elegans peb-1 gene encodes a novel DNA-binding protein involved in morphogenesis of the pharynx, vulva, and hindgut. Dev Biol 229:480-93
Thatcher, J D; Haun, C; Okkema, P G (1999) The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx. Development 126:97-107

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