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.