Genomic Screening for Bristle Defect Enhancement in Drosophila melanogaster

David Yan, Kathryn G. Miller, Department of Biology, Washington University in St. Louis.

Our lab studies the cytoskeleton. It gives cells structure, integrity, and shape; and it plays important roles in cell development, differentiation and specialization. Our main interest is one of the three types of cytoskeleton structure systems - Actin. Actin is an abundant protein present in all eukaryotic cells. Actin monomers polymerize to form actin filaments, which in turn are cross-linked by proteins to form many types of organized networks (cytoskeleton) that run through the cytoplasm. The most well-known function of actin is its critical role in skeletal muscle contraction. However, actin is important in all cells providing a 3D structural framework that gives cells shape, forming the contractile ring in cell division, allowing cell movement, and in developing cell specializations. Our lab’s goal is to understand how actin structure assembly is organized in cells and how different actin-binding proteins modify the assembly of actin structures in cells.

Bristle growth in Drosophila is dependent on actin filaments. The bristles are formed by elongation of a long tube of cytoplasm from an epithelial cell with the help of the actin cytoskeleton. Bristles grow to a great length and their shapes depend on actin organization. If actin cytoskeleton becomes disorganized, abnormally shaped bristles result. The fruit fly, Drosophila melanogaster is excellent system to study the actin cytoskeleton. This is primarily because that Drosophila stocks are easy and inexpensive to keep, they are easy to handle, they have a fast generation time, its entire genome has been sequenced and available to us, and many genetic methods can be used on them.

For our genetic screen, a sensitized background was created using a modified genotype, B11 Gal4 UAS Chic that causes flies to have abnormal bristles. In this background, small changes in amounts of actin regulators cause changes in bristle shape. No change would be observable in a wild-type genetic background. This allows us to sensitively detect genes that work to regulate actin. This stock is then crossed with various Deficiency mutant strains, which are missing certain segments of their genetic code. In this cross, reduced amounts of certain proteins that regulate actin assembly will cause the fly to have bristles with enhanced defects. By examining the genes within the deficiencies that causes enhancement, we can deduce which genes encode regulators of interest.

Fourteen deficiencies were examined in this study. These 14 deficiencies were previously selected as enhancing the B11 Gal4 UAS Chic bristle defects. My studies were designed to confirm and extend this previous work. From these 14 strains, a few of the most prominent enhancer strains were selected for a second screen, in which a quantitative scoring was performed on specific bristle groups (Scutellars, Dorso-centrals, Sternopleurals and the Head-verticals). The results from both the quantitative data record and the qualitative observations pointed to two chromosome regions that might contain genes that regulate actin. The next step in data collection would be look at abnormal bristles by immunostaining and to identify and test candidate genes that lie within the deficiency regions

Through this study, we hope to identify genes that produce proteins that regulate actin assembly and organization during development. Since all cells of all eukaryotic organisms have actin and other associated proteins, our results will provide information applicable across species.