Identifying Transcription Co-Regulators of Gcm/Repo in the Development of Glia in \u3cem\u3eDrosophila Melanogaster\u3c/em\u3e

The molecular mechanism mediating the differentiation of neural progenitors to either glia or neurons is guided by the gene glial cells missing (gcm), a master regulator of glial cell differentiation. Knockout of this transcription factor (TF) and its homolog gcm2 in the fruit fly Drosophila Melanogaster results in embryos that develop neurons in place of lateral glia. Conversely, overexpression of gcm in those neural progenitors results in embryos that have substantially fewer neurons and excess glia(Jones, et.al, 1995; Hosoya, et.al, 1995; and Vincent, et.al, 1996). Additionally, gcm has also been shown to be involved in the differentiation of blood cells, tendon cells, and lamina cells, suggesting that it interacts with other transcriptional regulators to exert different influences in different contexts (Jones, 2005; Johnson, et al.,2012). A Yeast One Hybrid screen was used to identify such co-factors (Nipper, 2014). Two protein candidates that shoa strong interaction with Gcm were Groucho (Gro) and the B-prime regulatory subunit of the widerborst (wdb) gene (Nipper, 2014). In order to verify these interactions and further investigate them, it is necessary to develop a protocol where the genes of these proteins can be co-expressed in S2 cells. The goal of this thesis was to establish a protocol that outlines the proper conditions for gcm expression in S2 cells, and the subsequent staining of its protein product on a Western blot. Several steps along this process were optimized to achieve this result. This protocol can now be used in the Jones lab to investigate these protein candidates further. A co-immunoprecipitation of Gcm with the aforementioned protein candidates could confirm interaction. It would be of interest to investigate how the protein candidates affect a key glial gene–repo–whose regulatory DNA is controlled by Gcm and potentially other transcription factors