The investigator is interested in how the self renewing asymmetric division of stem cells is achieved and regulated. He is using the female germ line of Drosophila as a model system to study this problem. As a postdoctoral fellow with Allan Spradling the PI developed an in vivo culture system for the germ line stem cells which are located near the end of the germarium. He combined this with laser ablation to show that each germarium contained 2-3 stem cells that are in contact with somatic cells that form the terminal filament. He identified a novel """"""""organelle"""""""" that he named the spectrosome that contained both spectrin and the adducin like protein product of the hts gene. The spectrosome is located between the stem cell nuclei and the terminal filament. During mitosis the spectrosome remains next to the terminal filament. Following centriole duplication one centriole stays near the spectrosome, and one aster and one pole of the mitotic spindle becomes associated with the spectrosome. The asymmetric association of the spectrosome with the mitotic spindle reveals the asymmetry of the stem cell division. The hts mutation results in a loss of the spectrosome and abnormal egg chamber development. In other experiments the PI found that ablation of terminal filament cells resulted in increased cell division of the stem cells suggesting that the terminal filament cells regulated cell proliferation of the stem cells. The PI further identified a number of enhancer traps that are expressed in the cells of the anterior region of the germarium and he obtained evidence that a number of segment polarity genes such as hh, wg and en are involved in regulating development of the egg chamber. Finally the PI identified two genes defined by P insertions that appear to be essential for the asymmetric division of the stem cells. One of these, ovarette (ovt), results in ovarioles mostly with two-three clusters of germ line cells that appear similar to one another. There is no evidence for differentiation. However, a small number of ovariole contain only two-three mature eggs but no other germ cells. The PI interprets this as suggesting that the mutation prevents the asymmetric division of the stem cells. The second gene is called piwi. Mutations in this gene result in ovarioles with two or fewer developing egg chambers. The PI suggests that this mutation results in a failure of the stem cells to be maintained in an undifferentiated state (i.e., they lose the quality of stemness). There are 5 specific aims proposed. The first is to systematically examine the role of the various somatic cells in regulating stem cell division. A photochemical activation ablation method will be used to ablate different sets of somatic cells located at the anterior end of the germaria. The ablated germaria would then be cultured in vivo to assay the stem cells. The second specific aim is to examine the stem cell division in the absence of the spectrosome. This will be done by examining hts mutant germaria via confocal microscopy. The third specific aim is to define the role of ovt and piwi in the somatic induction mechanism. The PI will use pole cell transplantation to generate genetic mosaics to determine whether the mutations act in the germ line or soma. The PI will also examine the expression of these two genes at both the RNA and protein level. Of particular interest is which cells express each of the genes. The fourth specific aim is to analyze the molecular structure of these two genes and to determine the nature of their protein products. The final specific aim is to analyze the cellular behavior of the two proteins.
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