The
hlh-8 gene encodes a basic helix-loop-helix (bHLH) transcription factor that is involved in mesoderm development.
hlh-8 function is related to postembryonic M lineage patterning and enteric muscle development 1 . The M lineage cells differentiate into egg laying and body wall muscles as well as ceolomocytes. The
hlh-8 gene is composed of 5 exons with a 2Kb intron after the first exon. We are currently characterizing a mutant,
hlh-8(
tm726) , with a large deletion (646 bp) in the large first intron of the gene. Two previous mutants have been characterized:
hlh-8(
n2170) 2 , a semidominant point mutation, and
hlh-8 (
nr2061) 1 , a presumptive null mutation. These mutations occur in the bHLH coding region and result in a phenotype that disrupts the proper development of the egg-laying and enteric muscles. Due to the improper muscle development, the previously characterized mutations result in constipated and egg laying deficient phenotypes. However,
hlh-8(
tm726) causes a constipated phenotype, but the animals are still able to lay eggs. Thus, we hypothesize that this phenotype may be due to a dosage-specific issue in the enteric muscles but not in the M lineage. We plan to introduce gfp constructs into
tm726 animals to determine if the known downstream targets of HLH-8 are still activated. We will investigate the
hlh-8 intron using a
pes-10::gfp construct.
pes-10::gfp contains the basic promoter machinery and can be activated in a variety of tissues by juxtaposition to a tissue-specific enhancer. Expression of a
pes-10 construct made with intron 1, will yield insight into the enhancer elements of the intron region of
hlh-8 . Real Time PCR will also give a quantitative examination of the dosage of HLH-8 in the deletion mutation. Finally, Reverse transcription will be utilized to determine if the mutant mRNA differs from that of wild type
hlh-8 mRNA. 1 Corsi, A.K., Kostas, S.A., Fire, A. and Krause, M. (2000). Caenorhabditis elegans Twist plays an essential role in non-striated muscle development. Development . 127, 2041-2051. 2 Corsi, A.K., Brodigan, T.M., Jorgensen, E.M., Krause, M. (2002). Characterization of a dominant negative C. elegans Twist mutant protein with implications for human Saethre-Chotzen syndrome. Development . 129, 2761-2772.