Introduction

   In our lab we are using the free-living nematode Caenorhabditis elegans to try to understand eukaryotic regulatory and developmental processes. We are hoping to use the special advantages of a simple model system to draw lessons which can be applied at a higher level in more complex organisms. C. elegans has already proven useful as a genetic system to study oncogene mediated signal transduction, (see Science vol 255, pg 1640 [1992] for example) and we are hoping to develop a molecular picture of a different signal transduction system, that involved in sex determination.

fem-2

   Although sex is determined in C. elegans by the ratio of sex (X) chromosomes to sets of autosomes (A), [X/A ratio], mutations have been isolated which cause the developing animal to completely ignore the chromosomal X/A signal. One such gene is fem-2, which functions at an important branch point in the genetic pathway for the regulation of sex determination in both the soma and germ line. Animals mutant for fem-2 develop as females, regardless of the genotype. We have cloned and characterized the fem-2 gene and its product (Pilgrim et al, 1995). The fem-2 gene product is a protein phosphatase and is thought to function in signal transduction between a cell surface receptor (produced by the tra-2 gene) and a nuclear transcription factor (the tra-1 product). FEM-2 is unusual in having a long amino-terminal extension, relative to other protein phosphatases of it's class (PP2C), and is most similar in its predicted primary structure to a protein involved in abscisic acid signaling in plants (ABI1), and to a human protein of unknown role. We have shown that Fem-2 can functionally complement a yeast deficient in PP2C activity, and that the amino terminus of the protein is not necessary for the phosphatase activity in vitro or in vivo (Hansen and Pilgrim, 1998). At the present time, we are undertaking a molecular, genetic and cell biological characterization of the fem-2 gene and the interaction of the fem-2 product with the other genes in the pathway. We are also beginning to characterize the mammalian FEM-2 homologue, which maps to chromosome 22, near a region associated with developmental disorders. We have recently become interested in the evolution of sex determining proteins. In most systems (worms and flies are particularly well studied) proteins involved in sex determination seem to show a much more rapid rate of change in their sequence than proteins that do not have a role in sex determination. It is not clear if these changes represent positive selection or lack of constraint. However, with the fem-2 gene, we have an excellent system in which to study the rate of change in a sex determining protein compared to its orthologues in the same organism which do not have a sex determining role.

unc-119

   A second project in the lab concerns a gene, unc-119 , which seems to be involved in the development of the nervous system. As foreseen over twenty years ago, the free-living nematode C. elegans is an ideal system with which to study development of the nervous system. C. elegans has a nearly invariant cell lineage, with similar locations and patterns of connectivity of most neurons in every animal. Of the approximately 1000 somatic nuclei in the adult, over 30% comprise the nervous system. The ease of genetic manipulation allows mutations to be isolated and quickly characterized. Mutations in genes involved in the nervous system are often detected by their uncoordinated "Unc" phenotype, which may range from subtle to severe. unc-119 is one of a large number of genes in C. elegans with mutant phenotypes consistent with a defect in the development of the nervous system. These genes often have subtle phenotypes, affecting only a subset of behaviours, such as response to gentle touch, sensitivity to volatile attractants, contractile ability of certain body muscles and constitutive action of (normally) regulated behaviour such as feeding. In some cases, the knowledge of the wiring diagram of the nervous system, coupled with other tools, has allowed the assignment of specific behaviours to particular neurons or classes of neurons. Preliminary evidence indicates that the defect in unc-119 mutants lies in at least two aspects of neuronal function; chemosensation and movement. Recently we have demonstrated a fasciculation defect in unc-119 mutants . We have shown that the human homologue, when expressed from a C. elegans promoter, can rescue all aspects of the unc-119 mutant phenotype, including movement, chemosensation and fasciculation. In addition, we have cloned the Drosophila and zebrafish homologues of UNC-119. Therefore, we feel that we have identified a new family of evolutionarily conserved neuronal proteins. Given the complexity of the vertebrate nervous system, the simpler nervous systems of invertebrates (e.g. Drosophila and C. elegans) have proven to be excellent model systems for the identification and molecular characterization of factors involved in development, differentiation and function. Many gene products first characterized in these model systems have later been shown to have vertebrate homologues with conserved biochemical functions. Since UNC-119, and many of the other identified components of the C. elegans nervous system have direct human homologues, this work may also shed light on the processes and products involved in the development of the human nervous system.

unc-45

   Finally in C. elegans, we have recently begun studying a gene, unc-45, whose product is essential for muscle cell development. Homozygous unc-45 null mutants die as embryos, but conditional viable alleles exist which are paralyzed at the permissive temperature. We have mapped and cloned the unc-45 gene, and shown that the UNC-45 gene product contains tetratricopeptide repeats, and shows limited similarity to yeast proteins hypothesized to regulate cytoskeletal assembly. It is not yet clear what the specific role of UNC-45 protein is in the early differentiation of the muscle cell. Recently we have shown that the UNC-45 protein is a component of muscle thick filaments, and co-localizes with myosin heavy chain B, but not A. We are continuing to investigate the expression of the gene and its product and hope to define a particular role for UNC-45 in development.

Zebrafish

   Largely due to the efforts of Angela Manning (and with lots of help from the fine fishy folks at the University of Oregon) we have established zebrafish in our laboratory, and have them happily breeding. Initially, we hope to examine the expression of UNC-119 homologues during early vertebrate development.. There are at least three zebrafish UNC-119 homologues, and preliminary results suggest that they may be expressed in the developing brain. We have also begun to look at PP2C (FEM-2 homologues) in early zebrafish development as well.