Dr. Steven L'Hernault requests funding to continue his studies of the genetics of C. elegans morphogenesis, which has been supported by a FIRST Award.The goals of this project are to understand how gene products are localized to specialize morphological structures during cellular differentiation. The development of the C. elegans sperm is being studied. Sperm are not required for viability of the animal, so it has been easy to identify genes that have defective sperm; over 58 genes that affect spermatogenesis have been identified. Homogenous sperm can be obtained for biochemical and cell biological studies. The goals of this project are to understand the targeting of specific proteins to organelles and specific regions of cells, the morphogenesis of those organelles, and the genetic control of organelle structure. The FB-MO complex is segregated exclusively into the spermatid during its formation and excluded from the residual body that is left behind at the end of spermatogenesis. Thus, the FB-MO complex behavior depends on an asymmetric division that gives rise to the spermatids and the residual body. In the spermatids, the membranous body eventually fuses with the plasma membrane. The major sperm proteins are associated with the fibrous body.By contrast, all ribosomes and virtually all of the actin, tubulin, and myosin of the spermatocyte end up in the residual body. Disruption of the FB-MO structure appears to occur in many spermatogenesis defective mutants. Three genes involved in spermatogenesis will be studied. Those known as spe-4 and spe-5 appear to encode integral membrane proteins based on sequence. Immunocytochemistry will be used to determine which sperm membranes contain spe-4. spe-4 antisera will be used to examine other spe mutants to determine which genes regulate spe-4 expression or distribution. Ultrastructural studies of spe-5 mutants will be conducted to examine morphogenesis of the sperm and antisera will be generated to the spe-5 gene product. New alleles of spe-4 and spe-5 will be sought, particularly temperature sensitive or conditional alleles that could be used to dissect morphological events of spermatogenesis. Site directed mutagenesis may be conducted to find new alleles if conventional genetics is unsuccessful. Suppressors of spe-4 mutations will be sought, either by reverting putative temperature sensitive mutations or by reverting missense mutations. Allele-specific suppressors will be studied in most detail. The gene spe-17 encodes a novel hydrophilic protein. Antisera will be used to localize the spe-17 gene product in developing sperm.New spe-17 alleles will also be sought, mainly by site-directed mutagenesis since spe-17 is a small gene. The spe-17 phenotype will be analyzed. In spe-17 mutants, ribosomes segregate to the spermatids in association with the FB-MO (they are excluded in the normal case) and the spermatids are abnormal in their structure. Developing spe-17 spermatids will be treated with puromycin to see whether spermatids are rescued if ribosomes are released from the FB- MO. Double mutants between spe-17 and other sperm morphogenesis mutants will also be made. Patch clamp analysis of ion channels in sperm will be conducted. Ion channels are found preferentially in the residual body and not in the spermatid. Several pharmacological treatments affect the development of sperm. The patch clamp analysis will be used to determine which treatment is most analogous to the normal sperm activation program. In addition, electrophysiological analysis of some mutant sperm will be conducted. Maturation of the spermatid into the spermatozoon, the last step of sperm development, will be examined.
