Identification of novel septate junction components through genome-wide glial screens

Epithelial barriers are central to the development of metazoans by compartmentalizing the body in distinct chemical milieus essential for the function of many organs. One such barrier is the blood-brain barrier, which isolates the nervous system from the body fluid to maintain its ionic homeostasis and ensure nerve pulse transmission. In Drosophila, the blood-brain barrier is formed late in embryogenesis by a thin epithelium of subperineurial glia that ensheath the nervous system. Similar to other epithelia, subperineurial glia seal the paracellular space by forming large multiprotein complexes at the lateral membrane, the septate junctions (SJs), which impede free diffusion and mediate barrier function. To identify novel genes required for blood-brain barrier formation, we followed a genome-wide in vivo RNAi approach. We initially screened almost the whole genome for genes required in glia for adult viability and impressively identified 3679 potential candidates. Subsequently, we tested these candidates for requirement in subperineurial glia for adult survival and identified 383 genes. At a last step, we directly asked if blood-brain barrier formation is compromised in the knock-down of the genes by performing the embryonic dye penetration assay in a selection of candidates and identified five genes that play a role during barrier development. Three of these genes, macroglobulin complement-related (mcr) and the previously uncharacterized pasiflora1 and pasiflora2 are further characterized in the context of this thesis. Here we show that all three proteins are novel components of the Drosophila SJ. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetra-spanning membrane proteins, while Mcr was reported to be a secreted protein in S2 cells required for phagocytosis and clearance of specific pathogens. Through detailed phenotypic analysis we demonstrate that the mutants show leaky blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins, phenotypes that are characteristic of SJ mutants. Consistent with the observed phenotypes, the genes are co-expressed in SJ-forming embryonic epithelia and glia and are required cell-autonomously to exert their function. In columnar epithelia, the proteins localize at the apicolateral membrane compartment, where they colocalize with other SJ proteins, and similar to known SJ components, their restricted localization depends on other complex members. Using fluorescence recovery after photobleaching experiments, we demonstrate for Pasiflora proteins that they are core SJ components, as they are required for complex formation and themselves show restricted mobility within the membrane of wild-type epithelial cells, but fast diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral SJ components and implicate a new family of tetraspan proteins in the development of cell junctions. In addition, we find a new unexpected role for Mcr as a transmembrane SJ protein, which raises questions about a potential intriguing link between epithelial barrier function, phagocytosis and innate immunity and has potential implications for the function of occluding junctions