The goals of the proposed experiments are twofold: to understand the genetic control of key regulatory steps in the program of spermatogenesis and to elucidate the molecular mechanisms that mediate changes in cellular and subcellular morphology. We have identified several intriguing male sterile mutants that cause defects in spermatocyte development or early spermatid differentiation in preliminary screens of male sterile mutants induced by mobilization of a single marked P-element. Three mutants (ms(3)sa, can, and aly) identify potential regulatory genes required for the cell cycle transition from G2 to the male meiotic divisions in primary spermatocytes. To determine if these spermatocyte arrest mutants effect known cell cycle control mechanisms, we will assay the expression pattern and modification state of Drosophila cell cycle control genes in mutant testes using molecular and antibody probes. We will clone the three genes, starting with ms(3)sa, and determine if their products resemble kinases, phosphatases, or oncogenes consistent with their proposed role in cell cycle control during male meiosis. To determine if the genes act directly to control entry into meiotic division, we will test if male germline cells are driven into meiosis prematurely by expression of ms(3)sa, can or aly from a heterologous promotor, and we will establish order of action of the three genes by molecular epistasis tests. Two other mutations (fwd and neb) identify potential effector genes involved in specific cytoskeletal-based morphological events. The fwd mutation causes failure of cytokinesis, a microfilament-based event, during male meiosis. We will determine if the contractile ring is formed in fwd homozygotes, and if the product of the fwd gene is a component of the contractile ring in wild type by immunofluorescence microscopy. We will clone the gene and sequence corresponding cDNA(s) to determine if the fwd product resembles a known component of microfilament based motility systems. The neb mutation, which affects the microtubule-based elongation of the mitochondrial derivative, maps near a potential member of the kinesin heavy chain superfamily in Drosophila. We will determine if the neb product is a kinesin-like protein by molecular analysis. To determine if neb encodes a microtubule binding protein or motor, we will test the ability of expressed neb protein to bind to and/or move along microtubules in vitro. Two other genes that appear to affect the same process as neb could encode other components of the machinery that mediates elongation of the mitochondrial derivative, such as kinesin light chains or the proteins that connect the motor to the mitochondrial membrane. We will clone the genes identified by the neb-like mutants, determine the molecular identity of their gene products, and test if they bind to and/or alter the in vitro biochemical properties of the neb protein. Finally, we propose to identify and characterize mutants in additional regulatory and morphogenetic effector genes that act to control cellular and subcellular morphogenesis during spermatogenesis.
