Li et al., 2012; Priglinger et al., 2013). expressing driving Bsg-RNAi in the perineurial glia with the septate junctions (SJs) labeled using an antibody against core SJ component SDZ 220-581 Nervana 2.1 was reconstructed in 3D from a stack. The SJs between opposing membranes of the subperineurial glia appear convoluted in regions of glial compression, but the strands remain continuous. sup_ns-JN-RM-1397-19-s02.mp4 (680K) DOI:?10.1523/JNEUROSCI.1397-19.2020.video.2 Movie 3: 3D reconstruction of the perineurial glial actin cytoskeleton shows breakage of actin filaments in regions of glial compression. A peripheral nerve from a third SDZ 220-581 instar larvae expressing driving expression of fluorescent actin marker lifeact::GFP and Bsg-RNAi in the perineurial glia was reconstructed in 3D from a stack. In regions of glial compression, the actin filaments appear discontinuous, and GFP-positive puncta accumulate in the tips of the compressions. sup_ns-JN-RM-1397-19-s03.mp4 (477K) DOI:?10.1523/JNEUROSCI.1397-19.2020.video.3 Abstract The nervous system SDZ 220-581 is ensheathed by a layer of outer glial cells, the perineurial glia, and a specialized extracellular matrix, the neural lamella. The function of perineurial glial cells and how they interact with the extracellular matrix are just beginning to be elucidated. Integrin-based focal adhesion complexes link the glial membrane to the extracellular matrix, but little is known about integrin’s regulators in the glia. The transmembrane Ig domain protein Basigin/CD147/EMMPRIN is highly expressed in the perineurial glia surrounding the larval nervous system. Here we show that Basigin associates with integrin at the focal adhesions to uphold the structure of the glia-extracellular matrix sheath. Knockdown of Basigin in perineurial glia using RNAi results in significant shortening of the ventral nerve cord, compression of the glia and extracellular matrix in the peripheral nerves, and reduction in larval locomotion. We determined that Basigin is expressed in close proximity to integrin at the glial membrane, and that expression of the extracellular integrin-binding domain of Basigin is sufficient to rescue peripheral glial compression. We also found that a reduction in expression of integrin at the membrane rescues the ventral nerve cord shortening, peripheral glial compression, and locomotor phenotypes, and that reduction in the integrin-binding protein Talin can partially rescue glial compression. These results identify Basigin as a potential negative regulator of integrin in the glia, supporting proper glial and extracellular matrix ensheathment of the nervous system. SIGNIFICANCE STATEMENT The glial cells and extracellular matrix play important roles in supporting and protecting the nervous system, but the interactions between these components have not been well characterized. Our study identified expression of a conserved Ig superfamily protein, Basigin, at the glial membrane of where it associates with the integrin-based focal adhesion complexes to ensure proper ensheathment of the CNS and PNS. Loss of Basigin in the glia results in an overall compression of the nervous system due to integrin dysregulation, which causes locomotor defects in the animals. This underlies the importance of glia-matrix communication for structural and functional support of the nervous system. glia during nervous system development, and in particular, its interaction with integrin. At the third instar larval stage, the nervous system is ensheathed by the perineurial glia, which interact with the overlying ECM. We found that Bsg is expressed in the perineurial glial in close proximity with integrin. Knockdown of Bsg in the perineurial glia results in reduced SDZ 220-581 larval locomotion, significant shortening of the VNC, and compression of the glia and ECM in the peripheral nerves. Loss-of-function mutants of Integrin and Talin rescue the Bsg knockdown phenotype. Together, our results show that Bsg and Integrin interact in the glia to maintain the morphology of the nervous system. Materials and Methods Fly strains and genetics The (Riedl et al., 2008), (Wright, 1960), (Wieschaus et al., 1984), and (Brown et al., 2002) lines were gifts from Guy Tanentzapf. The GFP-tagged UAS-Bsg mutant constructs and (Besse et al., 2007) were gifts from Anne Ephrussi, and (Curtin et al., 2005) was a gift from Bruce Reed. Bsg::cherry was generated using Recombinase-Mediated Cassette Exchange (Bateman et al., 2006) where a pseudo-exon carrying an in-frame mCherry coding sequence was spliced into the gene using the Minos-Mediated Integration Cassette system (Venken et al., 2011; Nagarkar-Jaiswal et al., 2015). DNA injection into flies and screening of mCherry-positive flies was done by Rainbow Transgenic Flies. The following fly strains were from the Bloomington Stock Center: (Sepp et al., 2001); (Lee and Luo, 1999); (E. Gavis, personal communication; http://flybase.org/reports/FBrf0207858); (Dietzl et al., 2007); (Morin et al., ATP1B3 2001); Viking::GFP (Morin et al., 2001); and (Morin et.