Characterization of molecular interactions regulating glial development and cytoskeletal structure

Astrocytes are glial cells of the central nervous system (CNS) that have a number of key functions including structural support of CNS cells and providing guidance for neuronal axon migration during development. This study describes the isolation and characterization of protein-protein interactions that are important in contributing to glial cell fates in the developing Drosophila embryo and those that maintain astrocyte cytoskeletal stability. The master glial fate determinant in Drosophila melanogaster is the transcription factor, glial cells missing (gcm). In flies, Gcm instructs progenitor cells to adopt glial fates. In the absence of gcm, progenitor cells assume a neuronal cell fate and a lack of glial cells results in mutant gcm embryos. We show that Gcm binds related forkhead-domain containing proteins, sloppy paired (Slp) 1 and S1p2. Slp1 binding to Gcm represses its transcriptional activation capacity. In a Drosophila model, we demonstrate that ectopic Slp1/2 expression in embryos leads to a severe reduction in gcm and glial cell numbers. In addition, we find a concomitant increase in neurons and further demonstrate that axonogenesis is disrupted. In contrast, mutant slp1/2 null embryos display a marked increase in gcm expression and glial cell number. These results demonstrate a novel mechanism of gcm regulation by Slp1 and Slp2. The second protein-protein interaction characterized relates to astrocyte cytoskeletal stability. Glial fibrillary acidic protein (GFAP) is an astrocyte-specific intermediate filament protein. One of its functions is to maintain cytoskeletal cell integrity within the astrocyte. A novel interaction between the GFAP and the actin-bundling protein, fascin is demonstrated. We show that GFAP and fascin colocalize in glial cell lines and normal astrocytes. Because fascin also binds to actin we propose that fascin links the actin cytoskeleton to GFAP, and that this interaction provides structural integrity to the astrocyte. Taken together, the studies presented offer insight into glial cell commitment during embryogenesis and structure.