We are interested in understanding the function of the CeTwist protein in the mesoderm as a model for cell-fate specification during development. Previous characterization of a null allele in the CeTwist gene,
hlh-8 (
nr2061), showed that this protein plays a role in non-striated muscle development (1). Animals with a null mutation in
hlh-8 do not properly form sex muscles, which leads to an egg-laying defective phenotype (1). A second semidominant allele
hlh-8 (
n2170) has also been characterized (2). These animals are also egg-laying defective but the cellular defects suggest an earlier blockage in sex muscle development when compared with
hlh-8 (
nr2061) animals. A suppressor screen is currently underway to identify mutations that can compensate for defects caused by the
n2170 allele. We expect this screen will identify proteins that physically interact with CeTwist or are downstream of CeTwist. CeTwist is a member of the basic helix-loop-helix (bHLH) family of transcription factors. CeTwist can function as a heterodimer with CeE/DA, the product of the
hlh-2 gene, to turn on
egl-15 and
ceh-24 (1,3). We have recently identified
arg-1 (
apx-1 related gene) as another CeTwist target. In order to reveal a complete pathway of CeTwist function in C. elegans mesoderm development, it will be necessary to ultimately identify all downstream target genes of
hlh-8. Using DNA microarrays, it is possible to observe global changes in gene expression of nearly every mRNA during development (4). Since
hlh-8 is expressed in at most 2% of cells at any given time during development, it may be difficult to obtain meaningful data using DNA microarrays by comparing mRNA from
hlh-8 null animals to mRNA from wild type animals. To circumvent this difficulty, we will overexpress both
hlh-8 and
hlh-2 from heat shock promoters and compare mRNA isolated from this strain to mRNA isolated from isogenic wild type animals. The three known targets genes of CeTwist:
egl-15,
ceh-24, and
arg-1 are overexpressed when both
hlh-8 and
hlh-2 are overexpressed from the heat shock promoter, and we predict unknown targets will also be overexpressed. To avoid measuring a secondary cascade of mRNA expression, the time of heat shock treatment will be optimized by studying the kinetics of mRNA induction of
egl-15,
ceh-24, and
arg-1 by RT-PCR after heat shock. We are currently building the appropriate strains for this study.