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• W2024255736 abstract "The blood-brain barrier of Drosophila is established by surface glia, which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions. The mechanisms underlying the formation of this barrier remain obscure. Here, we show that the G protein-coupled receptor (GPCR) Moody, the G protein subunits Gαi and Gαo, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. Our data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. Our study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core. The blood-brain barrier of Drosophila is established by surface glia, which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions. The mechanisms underlying the formation of this barrier remain obscure. Here, we show that the G protein-coupled receptor (GPCR) Moody, the G protein subunits Gαi and Gαo, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. Our data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. Our study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core. The complex nervous systems of higher animals are insulated from the body fluid by an impenetrable blood-brain barrier. In Drosophila, as in other insects, this barrier serves primarily as a shield against the high potassium levels of the hemolymph: if the barrier is compromised, action potentials can no longer propagate, and the animal is paralyzed. The barrier is established at the end of embryonic development by a thin layer of epithelial cells, which are thought to be glia derived from the neural ectoderm, named surface glia. This glial epithelium ensheathes the entire nerve cord and generates an ionic seal by forming intercellular septate junctions (SJs) (Carlson et al., 2000Carlson S.D. Juang J.L. Hilgers S.L. Garment M.B. Blood barriers of the insect.Annu. Rev. Entomol. 2000; 45: 151-174Crossref PubMed Scopus (113) Google Scholar, Edwards et al., 1993Edwards J.S. Swales L.S. Bate M. The differentiation between neuroglia and connective tissue sheath in insect ganglia revisited: the neural lamella and perineurial sheath cells are absent in a mesodermless mutant of Drosophila.J. Comp. Neurol. 1993; 333: 301-308Crossref PubMed Scopus (50) Google Scholar). A similar process occurs in the PNS, where peripheral glia form a single-cell tube that envelops the nerve and is sealed by autic SJs (Auld et al., 1995Auld V.J. Fetter R.D. Broadie K. Goodman C.S. Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila.Cell. 1995; 81: 757-767Abstract Full Text PDF PubMed Scopus (220) Google Scholar, Baumgartner et al., 1996Baumgartner S. Littleton J.T. Broadie K. Bhat M.A. Harbecke R. Lengyel J.A. Chiquet-Ehrismann R. Prokop A. Bellen H.J. A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.Cell. 1996; 87: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). The cellular and molecular processes involved in the ensheathment of the nervous system are generally not well understood. In the CNS, the study of the blood-brain barrier has been hampered by technical difficulties. The surface glia are extremely thin and delicate and complete their seal only at the very end of embryogenesis, making their visualization and phenotypic analysis challenging. In the PNS, Rho family GTPases and a PAK-like serine-threonine kinase (Fray) have been shown to be required for establishing or maintaining the glial ensheathment of peripheral nerves (Leiserson et al., 2000Leiserson W.M. Harkins E.W. Keshishian H. Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment.Neuron. 2000; 28: 793-806Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, Sepp and Auld, 2003Sepp K.J. Auld V.J. RhoA and Rac1 GTPases mediate the dynamic rearrangement of actin in peripheral glia.Development. 2003; 130: 1825-1835Crossref PubMed Scopus (54) Google Scholar). By contrast, SJ formation has been studied extensively, but mostly in columnar epithelia such as the ectoderm and the trachea (for review, see Tepass et al., 2001Tepass U. Tanentzapf G. Ward R. Fehon R. Epithelial cell polarity and cell junctions in Drosophila.Annu. Rev. Genet. 2001; 35: 747-784Crossref PubMed Scopus (400) Google Scholar). SJs contain regularly spaced, electron-dense septa that give them a ladder-like appearance. The septa are thought to serve as a series of filters that impede the penetration of small molecules through the intercellular cleft; the more septa are arrayed, the tighter the seal (Abbott, 1991Abbott N.J. Permeability and transport of glial blood-brain barriers.Ann. N Y Acad. Sci. 1991; 633: 378-394Crossref PubMed Scopus (19) Google Scholar). The SJ consists of a large complex of transmembrane and intracellular proteins, including Neurexin IV, Neuroglian, Contactin, Coracle, and the sodium pump. It is not clear to what extent the glial SJ mirrors the ectodermal SJ; to date, two of the molecular components of the ectodermal SJ have been shown to be functional in peripheral glia. The fly SJ shows striking structural, molecular, and functional similarity to the vertebrate paranodal junction, which is formed between neurons and myelinating glial cells (Poliak and Peles, 2003Poliak S. Peles E. The local differentiation of myelinated axons at nodes of Ranvier.Nat. Rev. Neurosci. 2003; 4: 968-980Crossref PubMed Scopus (457) Google Scholar, Salzer, 2002Salzer J.L. Nodes of Ranvier come of age.Trends Neurosci. 2002; 25: 2-5Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). G protein-coupled receptors (GPCRs) are a large and diverse superfamily of receptors that share a seven-transmembrane-domain structure and interact with a wide range of extracellular ligands. They transduce their signal mostly through trimeric G proteins, which consist of three subunits (α, β, and γ). Upon ligand binding, the GPCR catalyzes the exchange of GDP for GTP at Gα, leading to dissociation of the complex into Gα and Gβγ. Once separated, Gα-GTP and Gβγ can each interact with downstream effectors. Signaling is terminated by GTP hydrolysis, which is stimulated by RGS (regulator of G protein signaling) molecules; reassociation of Gα-GDP with Gβγ completes the cycle (Neer, 1995Neer E.J. Heterotrimeric G proteins: organizers of transmembrane signals.Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1266) Google Scholar). Our understanding of the role of GPCRs and trimeric G proteins in metazoan development is limited to relatively few examples, including germ-cell migration; asymmetric cell division; and, most recently, Wnt and planar polarity signaling (Katanaev et al., 2005Katanaev V.L. Ponzielli R. Semeriva M. Tomlinson A. Trimeric G protein-dependent frizzled signaling in Drosophila.Cell. 2005; 120: 111-122Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, Knoblich, 2001Knoblich J.A. Asymmetric cell division during animal development.Nat. Rev. Mol. Cell Biol. 2001; 2: 11-20Crossref PubMed Scopus (237) Google Scholar, Kunwar et al., 2003Kunwar P.S. Starz-Gaiano M. Bainton R.J. Heberlein U. Lehmann R. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.PLoS Biol. 2003; 1: e80https://doi.org/10.1371/journal.pbio.0000080Crossref PubMed Scopus (81) Google Scholar, Schier, 2003Schier A.F. Chemokine signaling: rules of attraction.Curr. Biol. 2003; 13: R192-R194Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). A role for G protein signaling in Drosophila blood-brain barrier formation was first suggested by the identification of the RGS loco, which is expressed in the surface glia and shows locomotion defects as a mutant (Granderath et al., 1999Granderath S. Stollewerk A. Greig S. Goodman C.S. O’Kane C.J. Klambt C. loco encodes an RGS protein required for Drosophila glial differentiation.Development. 1999; 126: 1781-1791PubMed Google Scholar). However, Loco’s cellular function has not been elucidated, nor has it been placed in a genetic pathway. In a reverse genetic screen for factors with glial expression and function, we identified two GPCRs of a small novel Rhodopsin family, moody and tre1, as well as loco. Here we show that moody, loco, and the Gα genes Gi and Go are (differentially) expressed in the surface glia; Moody and Loco colocalize at the plasma membrane, and Loco physically interacts with both Gi and Go, suggesting that the four proteins are part of a common signaling pathway. Using dye penetration into the nerve cord as an assay, we show that all four factors are required for proper insulation of the nervous system. Interestingly, loss and gain of signal cause qualitatively similar insulation defects, strongly suggesting that the signal is graded or localized within the cell. Using live imaging and transmission electron microscopy, we examine the cellular function of the signaling components in the morphogenesis of the surface glia and in the establishment of the intercellular SJs that generate the seal. The Drosophila nerve cord is ensheathed by a thin single-layer epithelium, which in turn is surrounded by an acellular layer of extracellular matrix material. Ultrastructural analysis had revealed that SJs between the epithelial cells are responsible for the insulation of the nerve cord (Carlson et al., 2000Carlson S.D. Juang J.L. Hilgers S.L. Garment M.B. Blood barriers of the insect.Annu. Rev. Entomol. 2000; 45: 151-174Crossref PubMed Scopus (113) Google Scholar, Edwards et al., 1993Edwards J.S. Swales L.S. Bate M. The differentiation between neuroglia and connective tissue sheath in insect ganglia revisited: the neural lamella and perineurial sheath cells are absent in a mesodermless mutant of Drosophila.J. Comp. Neurol. 1993; 333: 301-308Crossref PubMed Scopus (50) Google Scholar). Independent fate-mapping studies showed that the nerve cord is enveloped by glia expressing the glial-specific marker Repo (Halter et al., 1995Halter D.A. Urban J. Rickert C. Ner S.S. Ito K. Travers A.A. Technau G.M. The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster.Development. 1995; 121: 317-332PubMed Google Scholar, Ito et al., 1995Ito K. Urban J. Technau G.M. Distribution, classification, and development of Drosophila glial cells in the late embryonic and early larval ventral nerve chord.Rouxs Arch. Dev. Biol. 1995; 204: 284-307Crossref Scopus (295) Google Scholar, Schmidt et al., 1997Schmidt H. Rickert C. Bossing T. Vef O. Urban J. Technau G.M. The embryonic central nervous system lineages of Drosophila melanogaster. II. Neuroblast lineages derived from the dorsal part of the neuroectoderm.Dev. Biol. 1997; 189: 186-204Crossref PubMed Scopus (349) Google Scholar), but to date there has been no direct proof that it is these surface glia that form intercellular SJs and thus the insulating sheath. Moreover, the time course for the formation of the sheath and of the SJ-mediated seal has not been established. We developed several assays to follow the morphogenesis of the surface glial sheath. Due to the onset of cuticle formation, immunohistochemistry becomes unreliable after 16 hr of development. We therefore used live imaging of GFP-tagged marker proteins to visualize cell shapes, in particular the actin cytoskeleton marker GFP/RFP-Moesin (Edwards et al., 1997Edwards K.A. Demsky M. Montague R.A. Weymouth N. Kiehart D.P. GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in Drosophila.Dev. Biol. 1997; 191: 103-117Crossref PubMed Scopus (219) Google Scholar) and the SJ marker Neuroglian (Nrg)-GFP (Morin et al., 2001Morin X. Daneman R. Zavortink M. Chia W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.Proc. Natl. Acad. Sci. USA. 2001; 98: 15050-15055Crossref PubMed Scopus (558) Google Scholar). We find that Nrg-GFP expressed under its own promoter and RFP-Moesin driven by repo-Gal4 are colocalized in the same cells, establishing that the SJ-forming cells are repo positive (Figure 1N ) and thus conclusively demonstrating the insulating function of the surface glia. To probe the permeability of the transcellular barrier, we injected fluorescent dye into the body cavity and quantified dye penetration into the nerve cord by determining mean pixel intensity in sample sections (see Experimental Procedures). The surface glia are born in the ventrolateral neuroectoderm and migrate to the surface of the developing nerve cord (Ito et al., 1995Ito K. Urban J. Technau G.M. Distribution, classification, and development of Drosophila glial cells in the late embryonic and early larval ventral nerve chord.Rouxs Arch. Dev. Biol. 1995; 204: 284-307Crossref Scopus (295) Google Scholar, Schmidt et al., 1997Schmidt H. Rickert C. Bossing T. Vef O. Urban J. Technau G.M. The embryonic central nervous system lineages of Drosophila melanogaster. II. Neuroblast lineages derived from the dorsal part of the neuroectoderm.Dev. Biol. 1997; 189: 186-204Crossref PubMed Scopus (349) Google Scholar), where they spread until they touch their neighbors (17 hr of development). The glia then join to form a contiguous sheet of square or trapezoidal cells, tiled to form three-cell corners (Figures 1A–1C). SJ material is visible as a thin contiguous belt by 18 hr but continues to accumulate until the end of embryogenesis (Figures 1D–1F). Similar to other secondary epithelia, the surface glia do not form a contiguous adherens-junction belt (zonula adherens), but only spotted adherens junctions, as visualized by Armadillo-GFP (driven by own promoter; McCartney et al., 2001McCartney B.M. McEwen D.G. Grevengoed E. Maddox P. Bejsovec A. Peifer M. Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin.Nat. Cell Biol. 2001; 3: 933-938Crossref PubMed Scopus (140) Google Scholar; Figure 1M). At 16 hr, the fluorescent dye freely penetrates into the nerve cord, but by 20 hr the nerve cord is completely sealed (Figures 1G–1I). The completion of the seal thus coincides with the onset of visible movements in the late embryo. To further gauge our dye-penetration assay, we examined embryos mutant for known septate-junction components: Neurexin IV, which is required for blood-nerve barrier formation in the PNS (Baumgartner et al., 1996Baumgartner S. Littleton J.T. Broadie K. Bhat M.A. Harbecke R. Lengyel J.A. Chiquet-Ehrismann R. Prokop A. Bellen H.J. A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.Cell. 1996; 87: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar); Neuroglian; and the sodium-pump component Nervana2, for which only a role in the earlier formation of the ectodermal seal has been demonstrated (Genova and Fehon, 2003Genova J.L. Fehon R.G. Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function in Drosophila.J. Cell Biol. 2003; 161: 979-989Crossref PubMed Scopus (178) Google Scholar). In all three mutants, we found severe penetration of dye well after the nerve cord is sealed in wild-type (22 hr, Figures 1J–1L). These findings provide further evidence that the sealing of the nerve cord is achieved by SJs and suggest that the components of the ectodermal SJs are required for the function of surface glial SJs as well. In a genome-wide screen for glial genes, using FAC sorting of GFP-labeled embryonic glia and Affymetrix microarray expression analysis (H. Courvoisier, D. Leaman, J. Fak, N. Rajewsky, and U.G., unpublished data), we identified two novel GPCRs, Moody (CG4322; Bainton et al., 2005Bainton R.J. Tsai L.T.-Y. Schwabe T. DeSalvo M. Gaul U. Heberlein U. moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila.Cell. 2005; 123 (this issue): 145-156Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar [this issue of Cell]; Freeman et al., 2003Freeman M.R. Delrow J. Kim J. Johnson E. Doe C.Q. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function.Neuron. 2003; 38: 567-580Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, Kunwar et al., 2003Kunwar P.S. Starz-Gaiano M. Bainton R.J. Heberlein U. Lehmann R. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.PLoS Biol. 2003; 1: e80https://doi.org/10.1371/journal.pbio.0000080Crossref PubMed Scopus (81) Google Scholar) and Tre1 (CG3171; Kunwar et al., 2003Kunwar P.S. Starz-Gaiano M. Bainton R.J. Heberlein U. Lehmann R. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.PLoS Biol. 2003; 1: e80https://doi.org/10.1371/journal.pbio.0000080Crossref PubMed Scopus (81) Google Scholar). Both are orphan receptors belonging to the same novel subclass of Rhodopsin-family GPCRs (Kunwar et al., 2003Kunwar P.S. Starz-Gaiano M. Bainton R.J. Heberlein U. Lehmann R. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.PLoS Biol. 2003; 1: e80https://doi.org/10.1371/journal.pbio.0000080Crossref PubMed Scopus (81) Google Scholar). We examined their expression by RNA in situ hybridization; different subtypes of glia in the embryonic nerve cord can be distinguished based on their position and morphology (Ito et al., 1995Ito K. Urban J. Technau G.M. Distribution, classification, and development of Drosophila glial cells in the late embryonic and early larval ventral nerve chord.Rouxs Arch. Dev. Biol. 1995; 204: 284-307Crossref Scopus (295) Google Scholar). In the CNS, moody is expressed in surface glia from embryonic stage 13 onward (10 hr); in addition to cells surrounding the nerve cord (subperineurial glia), this includes cells lining the dorsoventral channels (channel glia). moody is also expressed in the ensheathing glia of the PNS (exit and peripheral glia) (Figure 2A ). Both CNS and PNS expression of moody are lost in mutants for the master regulator of glial fate, glial cells missing (gcmN17; Jones et al., 1995Jones B.W. Fetter R.D. Tear G. Goodman C.S. glial cells missing: a genetic switch that controls glial versus neuronal fate.Cell. 1995; 82: 1013-1023Abstract Full Text PDF PubMed Scopus (389) Google Scholar), confirming that they are indeed glial (Figure 2B). tre1 is expressed in all longitudinal glia and a subset of surface glia, as well as in cells along the midline. As expected, the (lateral) glial expression is lost in gcm mutants, while midline expression is not (Figures 2C and 2D). Both moody and tre1 are also expressed outside the nervous system in a largely mutually exclusive manner, specifically in the germ cells, the gut, and the heart. Several additional G protein signaling components are found in the surface glia. The six extant Gα genes show broad and overlapping expression in embryogenesis, with three of them (Go, Gq, and Gs) expressed throughout the nervous system and Gi expressed more specifically in surface glia (Figures 2G and 2H; Parks and Wieschaus, 1991Parks S. Wieschaus E. The Drosophila gastrulation gene concertina encodes a G alpha-like protein.Cell. 1991; 64: 447-458Abstract Full Text PDF PubMed Scopus (235) Google Scholar, Quan et al., 1993Quan F. Wolfgang W.J. Forte M. A Drosophila G-protein alpha subunit, Gf alpha, expressed in a spatially and temporally restricted pattern during Drosophila development.Proc. Natl. Acad. Sci. USA. 1993; 90: 4236-4240Crossref PubMed Scopus (24) Google Scholar, Wolfgang et al., 1990Wolfgang W.J. Quan F. Goldsmith P. Unson C. Spiegel A. Forte M. Immunolocalization of G protein alpha-subunits in the Drosophila CNS.J. Neurosci. 1990; 10: 1014-1024Crossref PubMed Google Scholar); Gβ13F and Gγ1 are ubiquitously expressed during embryogenesis (Schaefer et al., 2001Schaefer M. Petronczki M. Dorner D. Forte M. Knoblich J.A. Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system.Cell. 2001; 107: 183-194Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, Yarfitz et al., 1988Yarfitz S. Provost N.M. Hurley J.B. Cloning of a Drosophila melanogaster guanine nucleotide regulatory protein beta-subunit gene and characterization of its expression during development.Proc. Natl. Acad. Sci. USA. 1988; 85: 7134-7138Crossref PubMed Scopus (38) Google Scholar). Finally, the RGS loco is uniformly expressed in early embryos due to a maternal contribution but is then transcriptionally upregulated in surface and longitudinal glia, as well as in other tissues outside the nervous system. The nervous-system expression of loco is lost in gcm mutants (Figures 2E and 2F; Granderath et al., 1999Granderath S. Stollewerk A. Greig S. Goodman C.S. O’Kane C.J. Klambt C. loco encodes an RGS protein required for Drosophila glial differentiation.Development. 1999; 126: 1781-1791PubMed Google Scholar). The presence of both Moody and Loco protein in the surface glia is confirmed using immunohistochemistry (Figures 2I and 2J), but at 17 hr of development, when staining is feasible, the protein levels are still quite low. In sum, the GPCR Moody, the RGS Loco, and Gi are differentially expressed in surface glia. This expression precedes and accompanies the morphogenesis and sealing of the surface glial sheath. To examine protein expression and distribution of the GPCR signaling components in greater detail, we turned to third-instar larval nerve cords. By this stage, the surface glia have doubled in size and show robust protein expression of GPCR signaling and SJ components. Moody immunostaining is found at the plasma membrane, where it shows strong colocalization with the SJ marker Nrg-GFP (Figure 3C ). Loco immunostaining is punctate and more disperse throughout the cytoplasm, with some accumulation at the plasma membrane, where it colocalizes with Moody (Figure 3A). To avoid fixation and staining artifacts, we generated fluorescent-protein fusions (Moody-mRFP; Loco-GFP) and expressed them using moody-Gal4, which drives weak surface glial expression (see Figure S1 in the Supplemental Data available with this article online). In the live nerve-cord preparations, Loco-GFP is much less disperse and shows strong colocalization with Moody-mRFP at the plasma membrane (Figure 3B). In the absence of a known ligand, the coupling of G proteins to receptors is difficult to establish, but their binding to RGS proteins is readily determined. Loco physically binds to and negatively regulates Gi (Granderath et al., 1999Granderath S. Stollewerk A. Greig S. Goodman C.S. O’Kane C.J. Klambt C. loco encodes an RGS protein required for Drosophila glial differentiation.Development. 1999; 126: 1781-1791PubMed Google Scholar, Yu et al., 2005Yu F. Wang H. Qian H. Kaushik R. Bownes M. Yang X. Chia W. Locomotion defects, together with Pins, regulates heterotrimeric G-protein signaling during Drosophila neuroblast asymmetric divisions.Genes Dev. 2005; 19: 1341-1353Crossref PubMed Scopus (73) Google Scholar), and vertebrate Loco homologs (RGS12/14) have been shown to negatively regulate Gi/Go (Cho et al., 2000Cho H. Kozasa T. Takekoshi K. De Gunzburg J. Kehrl J.H. RGS14, a GTPase-activating protein for Gialpha, attenuates Gialpha- and G13alpha-mediated signaling pathways.Mol. Pharmacol. 2000; 58: 569-576Crossref PubMed Scopus (75) Google Scholar, Snow et al., 1998Snow B.E. Hall R.A. Krumins A.M. Brothers G.M. Bouchard D. Brothers C.A. Chung S. Mangion J. Gilman A.G. Lefkowitz R.J. Siderovski D.P. GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain.J. Biol. Chem. 1998; 273: 17749-17755Crossref PubMed Scopus (177) Google Scholar). In S2 tissue-culture assays, we find that Loco binds to Gi and Go, but not to Gs and Gq, in line with the previous results (Figure 3F). Double-label immunohistochemistry confirms that both Gi and Go are expressed in the surface glia (Figures 3D and 3E). Thus, Loco physically interacts with Gi and Go and shows subcellular colocalization with Moody, suggesting that the four signaling components are part of a common molecular pathway. Using dye penetration as our principal assay, we examined whether the GPCR signaling components that are expressed in surface glia play a role in insulation. moody genomic (Δ17; Bainton et al., 2005Bainton R.J. Tsai L.T.-Y. Schwabe T. DeSalvo M. Gaul U. Heberlein U. moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila.Cell. 2005; 123 (this issue): 145-156Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and RNAi mutants show similar, moderate insulation defects (Figures 4A–4C ; see Experimental Procedures). The embryos are able to hatch but show mildly uncoordinated motor behavior and die during larval or pupal stages. The dye-penetration defect of moodyΔ17 is completely rescued by genomic rescue constructs containing only the moody ORF. Both moody splice forms (α and β; Bainton et al., 2005Bainton R.J. Tsai L.T.-Y. Schwabe T. DeSalvo M. Gaul U. Heberlein U. moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila.Cell. 2005; 123 (this issue): 145-156Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) are able to rescue the defect independently, as well as in combination (Figure 4E). tre1 genomic (Kunwar et al., 2003Kunwar P.S. Starz-Gaiano M. Bainton R.J. Heberlein U. Lehmann R. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.PLoS Biol. 2003; 1: e80https://doi.org/10.1371/journal.pbio.0000080Crossref PubMed Scopus (81) Google Scholar) and RNAi mutants show no significant dye-penetration defect and no synergistic effects when combined with moody using RNAi (data not shown). Thus, despite the close sequence similarity of the two GPCRs and their partially overlapping expression in surface glia, only moody plays a significant role in insulation. Overexpression of moody causes intracellular aggregation of the protein (data not shown). loco is expressed both maternally and zygotically. Granderath et al., 1999Granderath S. Stollewerk A. Greig S. Goodman C.S. O’Kane C.J. Klambt C. loco encodes an RGS protein required for Drosophila glial differentiation.Development. 1999; 126: 1781-1791PubMed Google Scholar had shown that loco zygotic nulls are paralytic and suggested, on the basis of an ultrastructural analysis, a disruption of the glial seal (see below). In our dye-penetration assay, loco zygotic null mutants show a strong insulation defect, which can be rescued by panglial expression of Loco in its wt or GFP-tagged form (Figures 4A, 4B, and 4E). The extant null allele of loco (Δ13) did not yield germline clones; we therefore used loco RNAi to degrade the maternal in addition to the zygotic transcript. In loco RNAi embryos, dye penetration is indeed considerably more severe (Figures 4B and 4C). Overall, insulation as well as locomotor behavior is affected much more severely in loco than in moody and is close in strength to the SJ mutants. Overexpression of loco is phenotypically normal (data not shown). Thus, positive (moody) and negative (loco) regulators of G protein signaling show qualitatively similar defects in loss of function, suggesting that both loss and gain of signal are disruptive to insulation. Such a phenomenon is not uncommon and is generally observed for pathways that generate a localized or graded signal within the cell (see Discussion). Both Gi and Go have a maternal as well as a zygotic component. Gi zygotic null flies survive into adulthood but show strong locomotor defects (Yu et al., 2003Yu F. Cai Y. Kaushik R. Yang X. Chia W. Distinct roles of Galphai and Gbeta13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions.J. Cell Biol. 2003; 162: 623-633Crossref PubMed Scopus (95) Google Scholar). In our assay, Gi maternal and zygotic null embryos show a mild dye-penetration defect, which is markedly weaker than that of moody (Figures 4A and 4B), suggesting redundancy among Gα subunits. To further probe Gi function, we overexpressed the wt protein (Gi-wt) as well as a constitutively active version (Gi-GTP) (Schaefer et al., 2001Schaefer M. Petronczki M. Dorner D. Forte M. Knoblich J.A. Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system.Cell. 2001; 107: 183-194Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar) in glia using repo-Gal4; such overexpression presumably leads to a masking of any local differential in endogenous protein distribution. Expression of Gi-wt results in very severe dye penetration, while overexpression of Gi-GTP is phenotypically normal (Figures 4A and 4B). Only" @default.
• W2024255736 created "2016-06-24" @default.
• W2024255736 creator A5018040690 @default.
• W2024255736 creator A5039265523 @default.
• W2024255736 creator A5039719329 @default.
• W2024255736 creator A5041401064 @default.
• W2024255736 creator A5052325233 @default.
• W2024255736 date "2005-10-01" @default.
• W2024255736 modified "2023-10-18" @default.
• W2024255736 title "GPCR Signaling Is Required for Blood-Brain Barrier Formation in Drosophila" @default.
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