Muscle development is regulated by a family of basic helix-loop-helix proteins. In vertebrates, several myogenic factors, MyoD, Myf5, MRF4 and myogenin are required for differentiation of muscle cells. The C. elegans genome encodes only one of these factors, Ce MyoD, also known as HLH-1. Mutants of
hlh-1 are larval lethal and lumpy. Surprisingly, normal markers of muscle development such as the myosins are expressed in
hlh-1 mutants, but the muscle of mutants is disorganized. Body wall muscle cells arise from four blastomeres in the worm: AB, MS, C and D. CeMyoD/hlh-1 is expressed in all body wall muscle cells and their precursors. Previous studies have identified elements in the promoter and first intron required for
hlh-1 expression in specific lineages. To date, no genes have been identified which regulate
hlh-1 expression. Using molecular and genetic approaches, we are trying to understand the determination of muscle and the regulation of the
hlh-1 gene. We have made an embryonic cDNA expression library from N2 worms, and we are probing the library for DNA-binding proteins with elements of the
hlh-1 gene. In these experiments, only proteins which can bind the probes independently will be identified. We are studying worms from two genetic screens. In the first screen, we looked for mutations affecting
hlh-1 expression.
lin-2 Egl animals carrying an
hlh-1::gfp integrated array were mutagenized, and F2 animals carrying temperature-sensitive embryonic lethal mutations were isolated. Those mutant lines showing reduced or altered
hlh-1::gfp expression were selected for study. We are mapping mutations which produce embryos with relatively normal morphology to avoid mutations whose primary defect is in early morphological events. The difficulty we have encountered is the low penetrance of lethality, and the variable effects on
hlh-1::gfp expression. We have performed a new screen for mutations leading to lumpy larvae such as seen in
hlh-1 mutants. From 13000 haploid genomes, we have isolated about 30 mutations. In general, the worms hatch, but have limited ability to move and generally die in the early larval stages. They are short, and look as though the embryo failed to elongate. We are studying these mutant lines by staining for muscle markers. We are roughly mapping candidate mutations to help eliminate pat (paralyzed, arrested elongation at two-fold) genes, which have similar phenotypes.