The long range goal of this project is to understand the mechanisms of early determinative decisions in embryos of the simple model metazoan Caenorhabditis elegans. Using a combined molecular, cellular, and genetic approach, we will continue to focus on two major aspects of this problem: I) determination of sexual identity, and II) determination of cell fate and pattern during early embryogenesis. I. 1) We will continue with analysis of the molecular structure and expression of her-1, the initial major switch gene in the regulatory pathway that controls sex determination in response to the ratio of X chromosomes to autosomes (X/A ratio; XX animals are normally hermaphrodite, XO animals male). We have recently identified genomic clones and sex-specific transcripts of her-1. We will now sequence cDNA and genomic clones, and determine normal temporal and spatial patterns of mRNA accumulation during development, by gel blot analysis and in situ hybridization to mRNA in embryos, respectively. If sequencing confirms that the gene codes for a polypeptide, we will use an expression vector for synthesis of a fusion protein to allow production of a suitable antibody that can be used for immunofluorescence analysis of her- 1 gene product localization and purification of her-1 polypeptide(s). We will also analyze patterns of expression in several her-1 mutants and other sex-determination mutants to characterize regulatory relationships suggested by previous genetic analysis. In addition, we will continue two other investigations related to her-1 regulation: (2) genetic and molecular analysis of at least three loci, identified by suppressors of a dominant her-1 mutation, that appear to be either components of the X/A ratio or involved in its assessment, and (3) analysis of the extent to which the sex determination decision is cell-autonomous, by laser ablation experiments as well as by characterization of sexual phenotypes at the cell-lineage level in two types of sexually mosaic animals. II. To investigate the genetic control of early embryogenesis, we will (4) continue characterization of maternal-effect embryonic lethal mutations, particularly a novel class with dominant effects, and (5) initiate a search for non-maternal-effect mutations representing embryonically expressed genes required for early pattern formation, using two different screening methods: analysis of defects in embryos homozygous for known deficiencies, and characterization of mutants isolated after psoralen mutagenesis under newly developed conditions that yield a high frequency of non-maternal-effect embryonic-lethal mutations. Finally, we will (6) exploit recent advances in techniques for manipulating and maintaining embryonic cells in culture outside of the eggshell to explore the importance of cell- cell interactions on early cell fate and pattern determination.
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