Molecular Genetics of Nematode Development and Behavior

Our major strategy is to identify mutations that make cells or animals misbehave and then to study the functions of the genes defined by these mutations, using a combination of molecular cloning and genetic analysis

Our focus recently has been on the development and behavior of the C. elegans male. C. elegans has two sexes, males and hermaphrodites, which essentially are females that make sperm and thus can fertilize eggs without copulation with males. We have identified the sensory neurons that control each step in male mating behavior: response to contact with a hermaphrodite, turning just before reaching the end of the hermaphrodite, location of the vulva, insertion of the copulatory spicules, and transfer of sperm.

By screening for mutant males that cannot mate and analyzing these mutations by classical genetic analysis, we identified a number of genes necessary for normal development and behavior. Some of these genes are involved in the development of the copulatory spicules; others are involved in their function. One of the copulation-defective strains is a loss-of-function mutation in goa-1, the gene encoding the C. elegans homologue of the heterotrimeric G protein Go alpha subunit, which we are studying as part of the Human Frontier Science Program project. This goa-1 mutation results in a defect in both turning and spicule insertion.

We have evidence that a signal from the hermaphrodite uterus is required for the male to transfer sperm, and we are screening for candidate mutants in which the hermaphrodites do not make this signal. One of the spicule sensory neurons prevents sperm transfer and thus is the likely target of this hermaphrodite signal: the signal inhibits the inhibitory neuron, thus allowing sperm transfer.

We are analyzing how the combination of intercellular signals controls cell fates during development. In the developing male, either of two cells signals precursor cells to generate particular sets of spicule cells. This inductive signaling process requires the lin-2 gtrowth factor, the let-23 tyrosine kinase, the let-60 Ras protein, and the lin-45 Raf protein encoding genes we cloned over the last several years.

In a project supported by the National Institutes of Health, we showed that lin-3 encodes the inductive signal for the hermaphrodite vulva. This gene also acts as an inductive signal for proper spicule development. In addition to this inductive signal, there are antagonistic signals. By removing the source of one of these antagonistic signals in various mutants, we discovered that one signal acts on spicule preculsor cells downstream of LIN-45 Raf in the signal transduction pathway.

Another focus is on negative regulators of the LET-23-mediated signalling pathway. The types of mutations that activate LET-23-mediated signaling are analogous to mutations that contribute to human tumors by activating proto-oncogenes. Thus, mutations that abolish gene function and result in increased LET-23-mediated signaling will define negative regulators and are predicted to be analogous to tumor-suppressor genes. Our analysis of one of these hegative regulators, SLI-1, implicates the human homologue, a proto-oncogene called CB1, in signal transdution by transmembrane tyrosine kinases. By analysis of these negative regulators, we hope to understand better the role of these potential tumor-suppressor genes. These projects are supported by grans from the U.S. Army Breast Cancer Research Program, the National Institutes of Health, and the March of Dimes Birth Defects Foundation.

We are analyzing the several functions of the anchor cell. Besides inducing the vulva, the hermaphrodite anchor cell induces nearby uterine precursor cells to become specialized. We find that this induction occurs via a conserved signaling pathway mediated by the receptor protein LIN-12. The egl-29 gene, identified by Robert Horvitz (HHMI, Massachusetts Institute of Technology), appears necessary for activation of LIN-12 in this aspect of development. Later in development, the anchor cell invades the epidermis and forms a specific connection with the developing vulva. We have identified a new gene, necessary for the connection of the anchor cell to the vulva, and an analogous process in the male, the connection of the linker cell to the developing cloaca.