View Program - 63rd Annual Drosophila Research Conference

Septate junctions (SJ) within the subperineurial glia (SPG) form permeability barriers in Drosophila that protect central and peripheral neurons from exposure to the hemolymph; the blood-brain and blood-nerve barriers. The ladder-like structure (or septa) of SJs forms “rails” running parallel to two adjacent cell membranes between neighboring cells or autotypically in the same cell along the length of the glia, and these create the physical permeability barrier. Core components of SJs (such as NeurexinIV, Neuroglian and the Na/K ATPase pump) are defined as proteins that create the physical junction and are essential for SJ integrity, where the absence of any one leads to a compromised permeability barrier. Another critical component of the SJ is discs large (dlg1), a polarity protein necessary for SJ scaffolding. Though how dlg1 functions to ensure SJ development and maturation is not known. Dlg1 plays wide range of cellular roles including neuromuscular junction formation, establishing polarity in epithelial cells and neuroblasts, and scaffolding various protein complexes to the cytoskeleton. The dlg1 gene organization is complex with two start sites and alternatively spliced exons capable of expressing at least 21 transcripts with a variety of functional domains including S97, 3 PDZs, SH3 and GUK domains. I have found that existing loss of function dlg1 alleles or RNAi-mediated knockdown disrupts the morphology and integrity of the glial SJs. However it is unclear which dlg1 isoforms and which domains are required in glial SJ formation. I am using a two-part strategy to investigate the which dlg1 isoforms are present in SPG and which dlg1 isoforms are necessary for SPG SJ function. I am using RNAseq approaches to identify dlg1 transcripts from isolated and purified SPG mRNA. I am using CRISPR based approaches to generate complete deletion alleles that remove the two transcriptional start sites (S97 start site or the dlgA start site). I plan to test the requirement of each SPG dlg1 isoform by expressing dlg1 transgenes in these null backgrounds to determine which subset is able to rescue the SJ defects. In this way I will determine which domains of dlg1 are necessary for the formation of the Drosophila blood-nerve and blood-brain barriers.

Identifying subperineurial glia-specific dlg1 isoforms required for septate junction function