InteractiveFly: GeneBrief

moody: Biological Overview | References

BIOLOGICAL OVERVIEW

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. The G protein-coupled receptor (GPCR) Moody, the G protein subunits Gαi and Galphao, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. The 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. This study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core (Schwabe, 2005).

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 has revealed that septate junctions (SJs) between the epithelial cells are responsible for the insulation of the nerve cord. Fate-mapping studies have shown that the nerve cord is enveloped by glia expressing the glial-specific marker Repo, 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 (Schwabe, 2005).

Several assays were developed to follow the morphogenesis of the surface glial sheath. Due to the onset of cuticle formation, immunohistochemistry becomes unreliable after 16 hr of development. Live imaging of GFP-tagged marker proteins was therefore used to visualize cell shapes, in particular the actin cytoskeleton marker GFP/RFP-Moesin and the SJ marker Neuroglian (Nrg)-GFP. 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 and thus conclusively demonstrating the insulating function of the surface glia. To probe the permeability of the transcellular barrier, fluorescent dye was injected into the body cavity and dye penetration into the nerve cord was quantified by determining mean pixel intensity in sample sections (Schwabe, 2005).

The surface glia are born in the ventrolateral neuroectoderm and migrate to the surface of the developing nerve cord, 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. SJ material is visible as a thin contiguous belt by 18 hr but continues to accumulate until the end of embryogenesis. Similar to other secondary epithelia, the surface glia do not form a contiguous adherens-junction belt (zonula adherens), but only spotty, inconsistent adherens junctions were seen, as visualized by Armadillo-GFP (driven by own promoter). At 16 hr, the fluorescent dye freely penetrates into the nerve cord, but by 20 hr the nerve cord is completely sealed. The completion of the seal thus coincides with the onset of visible movements in the late embryo (Schwabe, 2005).

To further gauge the dye-penetration assay, embryos mutant for known septate-junction components were examined: Neurexin IV, which is required for blood-nerve barrier formation in the PNS, Neuroglian, and the sodium-pump component Nervana 2, for which only a role in the earlier formation of the ectodermal seal has been demonstrated. In all three mutants, severe penetration of dye was found, well after the nerve cord is sealed in wild-type (22 hr). 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 (Schwabe, 2005).

In a genome-wide screen for glial genes, using FAC sorting of GFP-labeled embryonic glia and Affymetrix microarray expression analysis, two novel GPCRs, Moody (CG4322) and Tre1 (CG3171: Trapped in endoderm-1) were identified. Both are orphan receptors belonging to the same novel subclass of Rhodopsin-family GPCRs. Their expression was examined by RNA in situ hybridization; different subtypes of glia in the embryonic nerve cord can be distinguished based on their position and morphology. 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). Both CNS and Peripheral nervous system expression of moody are lost in mutants for the master regulator of glial fate, glial cells missing (gcm^N17), confirming that they are indeed glial. 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. 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 (Schwabe, 2005).

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. Gβ13F and Gγ1 are ubiquitously expressed during embryogenesis. 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. The presence of both Moody and Loco protein in the surface glia is confirmed using immunohistochemistry, but at 17 hr of development, when staining is feasible, the protein levels are still quite low (Schwabe, 2005).

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 (Schwabe, 2005).

To examine protein expression and distribution of the GPCR signaling components in greater detail, third-instar larval nerve cords were examined. By this stage, the surface glia have doubled in size and show robust protein expression of GPCR signaling and SJ components (Schwabe, 2005).

Moody immunostaining is found at the plasma membrane, where it shows strong colocalization with the SJ marker Nrg-GFP. Loco immunostaining is punctate and more dispersed throughout the cytoplasm, with some accumulation at the plasma membrane, where it colocalizes with Moody. To avoid fixation and staining artifacts, fluorescent-protein fusions (Moody-mRFP; Loco-GFP) were generated and expressed using moody-Gal4, which drives weak surface glial expression. In the live nerve-cord preparations, Loco-GFP is much less dispersed and shows strong colocalization with Moody-mRFP at the plasma membrane (Schwabe, 2005).

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, and vertebrate Loco homologs (RGS12/14) have been shown to negatively regulate Gi/Go. In S2 tissue-culture assays, it was found that Loco binds to Gi and Go, but not to Gs and Gq. Double-label immunohistochemistry confirms that both Gi and Go are expressed in the surface glia (Schwabe, 2005).

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 (Schwabe, 2005).

Using dye penetration as the principal assay, whether the GPCR signaling components that are expressed in surface glia play a role in insulation was examined. moody genomic (Δ17; Bainton, 2005) and RNAi mutants show similar, moderate insulation defects. The embryos are able to hatch but show mildly uncoordinated motor behavior and die during larval or pupal stages. The dye-penetration defect of moody^Δ17 is completely rescued by genomic rescue constructs containing only the moody ORF. Both moody splice forms (α and β; (Bainton, 2005) are able to rescue the defect independently, as well as in combination. tre1 genomic (Kunwar, 2003) and RNAi mutants show no significant dye-penetration defect and no synergistic effects when combined with moody using RNAi. 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 (Schwabe, 2005).

loco is expressed both maternally and zygotically. loco zygotic nulls are paralytic and, on the basis of an ultrastructural analysis, a disruption of the glial seal, has been suggested. In a 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. The extant null allele of loco (Δ13) did not yield germline clones; therefore loco RNAi was used to degrade the maternal in addition to the zygotic transcript. In loco RNAi embryos, dye penetration is indeed considerably more severe. 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 (Schwabe, 2005).

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 (Schwabe, 2005).

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. In Gi maternal and zygotic null embryos show a mild dye-penetration defect, which is markedly weaker than that of moody, suggesting redundancy among Gα subunits. To further probe Gi function, the wt protein (Gi-wt) as well as a constitutively active version (Gi-GTP) were overexpressed 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. Only Gi-wt but not Gi-GTP can complex with Gβγ; overexpression of Gi-wt thus forces Gβγ into the inactive trimeric state. This result therefore suggests that the phenotypically crucial signal is not primarily transduced by activated Gi but rather by free Gβγ. Similar results have been obtained in the analysis of Gi function in asymmetric cell division (Schwabe, 2005).

Go null germline clones do not form eggs and do not survive in imaginal discs, indicating an essential function for cell viability (Katanaev, 2005). Therefore animals with glial overexpression of constitutively active (Go-GTP), constitutively inactive (Go-GDP), and wt (Go-wt) Go (Katanaev, 2005) were examined. Overexpression of Go-GDP, which cannot signal but binds free Gβγ, leads to severe dye penetration, again pointing to a requirement for Gβγ in insulation. However, Go-GTP and Go-wt show a moderate effect, suggesting that signaling by active Go does contribute significantly to insulation, in contrast to active Gi (Schwabe, 2005).

Overall, it was found that all four GPCR signaling components expressed in surface glia are required for insulation, further supporting the notion that the four components are part of a common pathway. The phenotypic data suggest that this pathway is complex: two Gα proteins, Gi and Go, are involved, but with distinct roles: activated Go and Gβγ appear to mediate most of the signaling to downstream effectors, while activated Gi seems to function primarily as a positive regulator of Gβγ. The loss of moody appears much less detrimental than the loss of free Gβγ (through overexpression of Gi-wt or Go-GDP); this is inconsistent with a simple linear pathway and points to additional input upstream or divergent output downstream of the G proteins. Finally, it was consistently observed that both loss (moody, Gi null, and Go-GDP) and gain (loco and Go-GTP) of signal are disruptive to insulation, suggesting that the G protein signal or signals have to be localized within the cell (Schwabe, 2005).

These complexities of G protein signaling in insulation preclude an unambiguous interpretation of genetic-interaction experiments and thus the linking of moody to Gi/Go/loco by genetic means. Double-mutant combinations between moody and loco were generated using genomic mutants as well as RNAi, with very complex results: in moody loco genomic double mutants, the insulation defect is worse than that of loco alone, while in moody loco RNAi double mutants the insulation defect is similar to that of moody alone. This strong suppression of loco by moody is also observed in the survival and motor behavior of the RNAi-treated animals. Thus the phenotype of the double-mutant combination is dependent on the remaining levels of moody and loco, with moody suppressing the loco phenotype when loco elimination is near complete (Schwabe, 2005).

To understand how the GPCR signaling components effect insulation at the cellular level, the distribution of different markers in the surface glia was examined under moody and loco loss-of-function conditions and under glial overexpression of Gi-wt. To rule out cell fating and migration defects, the presence and position of the surface glia were determined using the panglial nuclear marker Repo. In all three mutant situations, the full complement of surface glia is present at the surface of the nerve cord, with the positioning of nuclei slightly more variable than in wt (Schwabe, 2005).

In the three mutants, the SJ marker Nrg-GFP still localizes to the lateral membrane compartment, but the label is of variable intensity and sometimes absent, indicating that the integrity of the normally continuous circumferential SJ belt is compromised. Notably, the size and shape of the surface glia are also very irregular. While qualitatively similar, the phenotypic defects are more severe in loco and under Gi-wt overexpression than in moody, in line with the results of functional assays. When examining the three mutants with the actin marker GFP-Moesin, it was found that the cortical actin cytoskeleton is disrupted in varying degrees, ranging from a thinning to complete absence of marker, comparable to the effects observed with Nrg-GFP. However, GFP-positive fibrous structures are present within the cells, indicating that the abnormalities are largely restricted to the cell cortex. The microtubule organization, as judged by tau-GFP marker expression, appears normal in the mutants. The light-microscopic evaluation thus demonstrates that, in the GPCR signaling mutants, the surface glia are positioned correctly and capable of forming a contiguous epithelial sheet as well as septate junctions. Instead, the defects occur at a finer scale -- abnormally variable cell shapes and sizes, and irregular distribution of cortical actin and SJ material (Schwabe, 2005).

The changes in cell shape and actin distribution that were observed in the three mutants might simply be a secondary consequence of abnormalities in the SJ belt; to test this possibility, how a loss of the SJ affects the morphology and the actin cytoskeleton of the surface glia was examined. SJ components are interdependent for the formation and localization of the septa, and lack of a single component, such as Nrg, leads to nearly complete loss of the junction and severe insulation defects. In Nrg mutants, the surface glial cell shape and cortical actin distribution show only mild abnormalities. Thus, in contrast to the GPCR signaling mutants, the complete removal of the SJ causes only weak cytoskeletal defects, strongly arguing against an indirect effect. It is concluded that GPCR signaling most likely functions by regulating the cortical actin cytoskeleton of the surface glia, which in turn affects the positioning of SJ material along the lateral membrane (Schwabe, 2005).

More detailed insight into the nature of the defects in GPCR signaling mutants is afforded by electron microscopy. The surface glia in nerve cords of first-instar wild-type and mutant larvae were examined. Initially, dye penetration into the nerve cord was tested using ruthenium red. In wild-type, the dye diffuses only superficially into the surface glial layer, while in moody and loco mutants the dye penetrates deep into the nerve cord, in concordance with light-microscopic data. Tissue organization and SJ morphology were examined under regular fixation in randomly selected transverse sections. It has been reported that the surface glial sheath is discontinuous in loco mutant nerve cords, but this analysis was carried out at 16 hr of development, i.e., at a time when, even in wild-type, SJs are not yet established and the nerve cord is not sealed. In contrast to these findings, in the current study it was observed that, in loco as well as moody mutants, the glial sheath is in fact contiguous at the end of embryonic development. The ultrastructure of individual septa and their spacing also appear normal, indicating that moody and loco do not affect septa formation per se. However, the global organization of the junctions within the glial sheath appears perturbed: in wild-type, the surface glia form deep interdigitations, and the SJs are extended, well-organized structures that retain orientation in the same plane over long distances. In moody and loco mutants, the SJs are much less organized; they are significantly shorter in length and do not form long planar extents as in wild-type (Schwabe, 2005).

Taken together, the light- and electron-microscopic evaluations of the GPCR signaling mutants both show defects in the organization of the surface glial epithelium. The reduction in SJ length is consonant with the variability and local disappearance of the Nrg-GFP marker. Since the sealing capacity of the junction is thought to be a function of its length, the reduction in mean SJ length in the mutants provides a compelling explanation for the observed insulation defect (Schwabe, 2005).

Therefore, in addition to a reduction of the insulating SJs, this analysis of the GPCR signaling mutants revealed irregular cell shape and size, as well as weaker and variable accumulation of cortical actin in the surface glia. These data suggest that the primary defect in the mutants lies with a failure to stabilize the cortical actin, whose proper distribution is required for the complex extended morphology of the glia, which then affects SJ formation as a secondary consequence. Several lines of evidence exclude the reverse chain of causality, that is, a primary SJ defect resulting in destabilization of cortical actin and cell-shape change. Surface glia coalesce into a contiguous sheath and show strong accumulation of cortical actin before SJ material accumulates and sealing is completed. In the GPCR signaling mutants, there is misdistribution of SJ material along the cell perimeter, but the junctions do form. Finally, the GPCR signaling mutants show cell-shape and cortical actin defects that are much more severe than those observed in the near complete absence of SJ (Schwabe, 2005).

Compared to the columnar epithelia of the ectoderm and the trachea, the surface glial sheath is very thin. Compensating for their lack in height, surface glia form deep 'tongue-and-groove' interdigitations with their neighbors. This increases the length of the intercellular membrane juxtaposition and thus of the SJ, which ultimately determines the tightness of the seal. It is proposed that the surface glial interdigitations are the principal target of regulation by GPCR signaling. In GPCR signaling mutants, a loss of cortical actin leads to diminished interdigitation and thus to a shortening of the SJ, resulting in greater permeability of the seal. This model integrates all the observations at the light- and electron-microscopic levels (Schwabe, 2005).

moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila

moody was identified in a genetic screen for Drosophila mutants with altered cocaine sensitivity. Hypomorphic mutations in moody cause an increased sensitivity to cocaine and nicotine exposure. In contrast, sensitivity to the acute intoxicating effects of ethanol is reduced. The moody locus encodes two novel GPCRs, Moody-α and Moody-β. While identical in their membrane-spanning domains, the two Moody proteins differ in their long carboxy-terminal domains, which are generated by use of alternative reading frames. Both Moody forms are required for normal cocaine sensitivity, suggesting that they carry out distinct but complementary functions. Moody-α and Moody-β are coexpressed in surface glia that surround the nervous system, where they are actively required to maintain the integrity of the blood-brain barrier in the adult fly. It is proposed that a Moody-mediated signaling pathway functions in glia to regulate nervous system insulation and drug-related behaviors (Bainton, 2005).

To identify novel molecules that may regulate the nervous system's sensitivity to drugs of abuse, a genetic screen was carried out for Drosophila mutants with altered responses to volatilized freebase cocaine. Behavior was quantified using a simple assay that measures drug-induced loss of negative geotaxis. Upon exposure to moderate doses of cocaine, flies show a series of unusual motor behaviors, including reduced locomotion and vigorous circling, which interfere with negative geotaxis, a robust innate behavior of Drosophila. A collection was screened of 400 fly strains, each carrying an insertion of the EP element on the X chromosome; five mutants were identified with a reduced cocaine sensitivity (corresponding to three genes) and seven mutants with an increased drug sensitivity (corresponding to six genes. This study describes the phenotypic and molecular characterization of EP1529, identified by its increased sensitivity to cocaine. EP1529 flies also exhibit increased sensitivity to the effects of volatilized nicotine exposure. In contrast, EP1529 flies are resistant to the acute intoxicating effects of ethanol, manifested as an increased mean elution time (MET) in the inebriometer, an assay that measures ethanol-induced loss of postural control. EP1529 flies absorb ethanol normally, are fully viable, and show normal baseline behaviors, such as climbing and locomotion. The EP1529 insertion is responsible for the aberrant cocaine sensitivity, as precise excision of the EP element restores normal drug sensitivity. Sequence analysis by the Berkeley Drosophila Genome Project showed that the EP1529 element is inserted between two predicted genes, CG4322 and CG4313. Further analysis (see below) revealed that the gene disrupted by EP1529 is CG4322. moody encodes a member of a group of three highly related orphan GPCRs, which also include CG4313 and Tre1. Tre1 has been shown to be involved in transepithelial migration of germ cells (Kunwar, 2003), while the function of CG4313 is unknown (Bainton, 2005).

Moody-α and -β differ in their long C-terminal domains. Both forms of Moody are coexpressed in glia that surround and insulate the nervous system and are both found at regions of cell-cell contact. Partial loss of moody function causes increased sensitivity to cocaine and nicotine, and reduced sensitivity to ethanol-induced loss of postural control. Complete loss of function results in lethality due to defective insulation of the nervous system (Schwabe, 2005). Transient inhibition of moody expression in the adult fly causes a reversible disruption of the BBB, indicating that moody function is continuously required to insulate the nervous system. The role of moody in drug sensitivity can, however, be dissociated from its role in nervous system insulation: while either Moody-α or -β are sufficient for normal blood-brain barrier formation, both protein forms are needed for flies to show normal cocaine sensitivity. It is postulated that a Moody-mediated signaling pathway functions in surface glia to regulate both nervous system insulation and drug-related behaviors (Bainton, 2005).

The moody locus encodes two proteins that differ in their C-terminal domains; these predicted cytoplasmic regions are 271 and 230 amino acids long for Moody-α and -β, respectively. The two Moody proteins are found in nearly equal amounts and are coexpressed during development and adulthood in glia that ensheath the nervous system. Interestingly, coexpression is accomplished by an unusual alternative splicing event that leads to translation from overlapping reading frames. This mechanism likely insures stoichiometric amounts of the two protein products, which in turn may be important for normal function. Although either Moody protein is sufficient for viability and normal blood-brain barrier formation, both proteins are needed for flies to respond normally to acute cocaine exposure. This likely reflects the high degree of sensitivity of behavioral outputs to changes in organismal physiology. Why would normal drug sensitivity require both Moody forms? It is possible that the two proteins interact with distinct downstream signaling pathways or that their optimal function, maturation, and/or stability requires the formation of heterodimers. Regardless of the exact functional significance of the two Moody forms, their existence is likely to be important, as their presence is conserved in Drosophila pseudoobscura, a species that diverged from Drosophila melanogaster some 30 million years ago. The Moody-α C-terminal domain shows 58% identity and 63% similarity, while the Moody-β-specific domain shows 48% identity and 61% similarity between the two Drosophila species (Bainton, 2005).

In insects, including Drosophila, the nervous system is insulated from the humoral environment through a glial-dependent blood-brain and blood-nerve barrier, which plays a crucial role in its electrical and chemical insulation. Septate junctions between the surface glial cells are believed to form the structural basis for these barriers and to be functionally equivalent to vertebrate paranodal junctions found at nodes of Ranvier. Indeed, many molecular components of septate junctions -- including the cell adhesion molecules gliotactin (a member of the neuroligin family), Neurexin IV, and Contactin -- are also found at paranodal junctions of myelinated nerves. The GPCR Moody was identified by two completely independent means: as a gene expressed in embryonic glia that ensheath the nervous system (Kunwar, 2003; Schwabe, 2005) and as a mutation that alters the sensitivity of adult flies to acute cocaine administration (this study). Schwabe (2005) postulate that the Moody GPCR signals through trimeric G proteins, which, in turn, regulate the cortical actin cytoskeleton, the proper development of septate junctions, and the formation of the blood-brain barrier (Bainton, 2005).

Interestingly, this study found that Moody continues to be expressed in the surface glia of the adult fly, where it functions to maintain the integrity of the BBB; transient reduction of moody expression causes a reversible disruption in nervous system insulation. It is therefore believed that, in addition to its role in establishment of the BBB, moody functions continuously to regulate its degree of permeability. What signals would Moody normally respond to in order to carry out its functions? One possibility is that the ligand is delivered via the hemolymph that bathes the nervous system. The hemolymph is not only rich in certain ions (such as K⁺), but also contains nutrients, amino acids, hormones, neuropeptides, and various proteins involved in clotting and the immune response. Alternatively, the ligand may be produced by the underlying neurons, or by neighboring glia. In mammals, the permeability of the blood-brain barrier can be altered by hypoxia-ischemia and by various substances released under pathological conditions, including the amino acids glutamate and aspartate, ATP, histamine, serotonin, and various peptides. Several of these substances signal via receptors of the GPCR superfamily, although the mechanisms by which this signaling leads to altered BBB function remain poorly understood. The identification of the Moody ligand and downstream signaling pathway, and the physiological conditions that modulate their function, should provide interesting new insights into the mechanisms that regulate nervous system insulation (Bainton, 2005).

In addition to moody, behavioral screens for drug sensitivity mutants identified a hypomorphic allele of loco (7-88). 7-88 flies show reduced sensitivity to acute cocaine administration. loco encodes an RGS (regulator of G protein signaling) protein whose normal function is to terminate signaling by GPCRs. Interestingly, loco has been implicated in nervous system insulation and, more recently, shown to function together with moody in the development of the BBB (Schwabe, 2005). The observation that mutations in moody and loco cause opposite cocaine sensitivity defects is therefore consistent with their molecular functions. Interestingly, the drug resistance seen with the loco 7-88 mutation (and flies heterozygous for the null allele loco^Δ13) suggests that overactivation of the Moody-GPCR pathway can cause the opposite effect as its inhibition, implying that the pathway is under both positive and negative control, providing a broad range of physiological and behavioral regulation. The ability to discern such subtle physiological changes likely reflects the exquisite sensitivity of behavioral phenotypes to changes in organismal physiology, such as alterations in BBB function, and further demonstrates the utility of psychoactive compounds as probes into CNS function (Bainton, 2005).

Could the increased cocaine (and nicotine) sensitivity seen with the EP1529 moody mutation be caused by an altered drug accessibility to the nervous system? As in vertebrate systems the Drosophila respiratory system is the most accessible route for drug entry in acute exposure paradigms; drug volatilization ensures quick and relatively homogeneous delivery to a population of flies. Drugs enter the tracheal system that is connected to the environment through spiracles at the cuticular surface. The tracheal network then divides into smaller tubes, or tracheoles, which make links throughout the organism to the hemolymph and to end organs, such as the brain. There is no reason to believe that moody mutations affect the delivery of drugs via the tracheal system, since moody does not appear to be expressed in these cells, and the tracheal system is therefore expected to function normally in moody mutants. moody does, however, play a role in the development and function of surface glia that insulate the nervous system from the hemolymph that bathes it. Drugs delivered via the hemolymph would need to cross this barrier to access the nervous system. It is not believed, however, that increased drug accessibility -- caused by a dysfunctional BBB -- is the cause of drug phenotypes observed with moody mutations. (1) Molecules such as cocaine and nicotine freebase -- neutral compounds with molecular weights (MW) of 303 and 163 Da, respectively -- readily cross the blood-brain barrier in mammals, and the same is thought to be true in flies. Indeed, it was found that Rhodamine B (a neutral compound with a MW of 600 Da) readily crosses the BBB of wild-type flies as assayed by the dye-injection assay, while FITC (a negatively charged compound of MW 450 Da) is excluded. Rhodamine B's ability to penetrate into the CNS is not due to a toxic effect of the compound on the BBB, as FITC is still excluded when coinjected with Rhodamine B. (2) FITC exclusion from the CNS in the behavioral mutants (moody^Δ17 flies carrying either the gen-α or gen-β transgenes) is indistinguishable from wild-type, when ascertained by either dye-injection or dye-feeding assays. (3) EP1529 flies and moody^Δ17 flies carrying either the gen-α or gen-β transgenes, are resistant to the acute intoxicating effects of ethanol. Since ethanol readily crosses cell membranes, a defect in the BBB should have no effect on ethanol's ability to access its targets in the nervous system. Indeed, it was found that ethanol absorption is completely normal in EP1529 flies. Finally, downregulation of moody expression in flies carrying the UAS-moody-RNAi and hsGAL4 transgenes, a manipulation that clearly disrupts BBB integrity, causes resistance, not sensitivity, to acute cocaine exposure; the bases for this resistance is currently not understood. Regardless, the data listed above argue strongly that the behavioral defects observed in moody flies are not caused by altered drug accessibility to its sites of action in the nervous system (Bainton, 2005).

Rather, it is postulated that in the behavioral mutants the 'state' or responsiveness of the nervous system has changed due to a chronic yet subtle alteration in blood-brain barrier function. This could be caused, for example, by a chronic alteration in the ionic composition of the CNS or changes in the concentrations of various small molecules (such as neurotransmitters) that may leak into or out of the CNS. Interestingly, these proposed adaptations have opposite effects on the flies' response to cocaine and nicotine (increased sensitivity) and ethanol (reduced sensitivity). This divergence is not too surprising, since unbiased genetic screens have identified several mutants that differentially affect the response to psychostimulants and ethanol. Thus, a particular set of changes in the nervous system -- caused either by its altered insulation or by single-gene mutation -- can have distinct effects on the organism's response to drugs. Further studies of the mechanisms of Moody signaling and its downstream effects in glia should begin to reveal how the BBB and the molecules that regulate its permeability interact with the nervous system to regulate behavior. Interestingly, a recent study in Drosophila identified a signaling pathway -- involving the neuropeptide Amnesiac and the neurotransmitter transporter Inebriated -- that functions to regulate the growth of peripheral perineurial glia in response to signals from motorneurons (Yager, 2001). It is possible that reciprocal interactions occur between surface glia and the mature nervous system to regulate behavioral responses to drugs of abuse. In mammals, claudin-5, a cell-adhesion molecule found in tight junctions of epithelial cells that form the BBB, has been implicated in normal BBB function. Specifically, claudin-5-deficient mice show an increased permeability to small molecules. Interestingly, the human claudin-5 locus (CLDN5) has been associated with vulnerability to schizophrenia. Taken together with this study, these observations warrant a closer examination of the role of the BBB in nervous system function and the etiology of mental illness (Bainton, 2005 and references therein).

Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression

In the Drosophila embryonic CNS several subtypes of glial cells develop, which arrange themselves at characteristic positions and presumably fulfil specific functions. The mechanisms leading to the specification and differentiation of glial subtypes are largely unknown. By DiI labelling in glia-specific Gal4 lines the lineages of the lateral glia in the embryonic ventral nerve cord were clarified and each glial cell was linked to a specific stem cell. For the lineage of the longitudinal glioblast, it was shown to consist of 9 cells, which acquire at least four different identities. A large collection of molecular markers (many of them representing transcription factors and potential Gcm target genes) reveals that individual glial cells express specific combinations of markers. However, cluster analysis uncovers similar combinatorial codes for cells within, and significant differences between the categories of surface-associated, cortex-associated, and longitudinal glia. Glial cells derived from the same stem cell may be homogeneous (though not identical; stem cells NB1-1, NB5-6, NB6-4, LGB) or heterogeneous (NB7-4, NB1-3) with regard to gene expression. In addition to providing a powerful tool to analyse the fate of individual glial cells in different genetic backgrounds, each of these marker genes represents a candidate factor involved in glial specification or differentiation. This was demonstrated by the analysis of a castor loss of function mutation, which affects the number and migration of specific glial cells (Beckervordersandforth, 2008).

This report provides a comprehensive description of marker gene and enhancer trap expression in CNS glial cells of late Drosophila embryos. The markers include many transcription factors known to be involved in cell fate specification, as well as a number of still unknown factors. They were chosen for this analysis either because they were known to be expressed in subsets of glial cells or because they were known to be involved in cell fate determination in the nervous system. All together, more than 50 markers were tested, 39 of which showed expression in glial cells and hence were described in detail. Their specific expression patterns, though in many cases not restricted to glia, enable identification of groups of cells, as well as individual cells (Beckervordersandforth, 2008).

The lateral CNS glial cells have been assigned to three categories, according to their spatial distribution and morphology: the surface-, the cortex-, and the neuropile-associated glial cells. Categories were further divided into subgroups, as for example the surface-associated glial cells into subperineural glial cells and channel glia. Several of the molecular markers described in this study exhibit expression patterns, which correspond to the spatial/morphological definition of glial categories or subgroups. For example, moody or svp-lacZ are expressed in all surface-associated glial cells, whereas P101-lacZ is only expressed in the SPG-subgroup and engrailed only in the CG-subgroup. In addition, nearly each individual glial cell expresses a specific combination of markers indicating that they develop unique identities. Yet, nothing is known about how these identities are acquired. Comparing subtype affiliation or lineage ancestry of all glial cells with their respective marker gene expression patterns, it becomes obvious that glial cell specification is a process occurring on the level of individual cells. Cells might have a predisposition for a particular subtype laid down by lineage (e.g. NB5-6 derived cells to become subperineurial glia). In contrast, a temporal cascade within a lineage could determine individual cell identities (as it might be the case for the NB7-4 lineage) (Beckervordersandforth, 2008).

DiI labelling of the lineages of various progenitor cells in combination with cell-specific enhancer trap lines revealed that the composition of glial progeny within the lineages is invariant. Clonally related glia cells often express similar combinations of marker genes. The LGs, a prominent subgroup of the neuropile-associated glia and the only interface glia in the embryonic VNC, have been defined as the progeny of the LGB, which become aligned along the longitudinal connectives. However, there has been confusion about the size and composition of the LGB-lineage. By means of DiI labelling and marker gene expression, the size of this lineage was determined to be 9 cells. Although all cells of the LGB-lineage express a similar set of markers, a few markers are restricted to only parts of the lineage. Based on such markers, as well as positional criteria, the group of LGs was further subdivided. One of the cells, the LP-LG, is located slightly more lateral than the other LGs, and seems to be geared towards the ISN. Since it lies close to, and expresses a similar combination of markers as the M-ISNG, it would be justified to assign this cell to the group of nerve root glia; however, this was not done in order to avoid conflicts with the established nomenclature. Despite of their similarities, these two cells are of different origin: the LP-LG derives from the LGB, and the M-ISNG is generated by NB1-3 (Beckervordersandforth, 2008).

Most of the NBs that arise at corresponding positions and times in thoracic and abdominal segments (called serially homologous NBs) acquire the same fate, i.e. they generate the same lineages expressing corresponding sets of markers. However, some of these serially homologous lineages develop characteristic, tagma-specific differences with regard to cell number and/or cell types. Tagma-specific characteristics of these lineages have been shown to be under the control of Hox genes (Beckervordersandforth, 2008).

Although the total number of CNS glial cells is identical in thoracic and abdominal neuromeres, there are some differences in their origin and distribution of subtypes. This is due to tagma-specific differences among serially homologous lineages of NBs 1-1, 2-2, 5-6, and 6-4, which give rise to CBGs and SPGs. NB6-4A (A, abdominal) generates only two CBGs: MM-CBG and M-CBG, whereas the NB6-4T (T, thoracic) lineage comprises an additional MM-CBG2 and a neuronal sublineage. NB1-1T generates only neurons, whereas NB1-1A produces three SPGs (A-SP-G, B-SPG, and LV-SPG) in addition to neurons. In the thorax, the LV-SPG is presumably generated by NB5-6T, a cell at the position of A-SPG is produced by NB2-2T, and a cell at the B-SPG position is missing. Despite their different origin, the NB1-1A- and NB2-2T-derived SPGs specifically express hkb-lacZ and mirr-lacZ. Furthermore, the NB1-1A- and the NB2-2T-derived A-SPG appear to assume the same identity, as they express the same set of markers (including castor, which is not found in the abdominal B-SPG. Taken together, the differences between thorax and abdomen are restricted to only few glial cells, most of which acquire similar cell fates in thorax and abdomen (as judged by marker gene expression) irrespective of their progenitor (Beckervordersandforth, 2008).

The collection of marker genes and enhancer trap lines presented in this study provides powerful tools for the identification of specific glial cells in different genetic backgrounds. Each of markers also represents a candidate factor involved in glial subtype specification and/or differentiation. Many of these genes encode transcription factors known to be involved in cell fate specification, like fushi tarazu, mirror, and muscle segment homeobox. Other genes encode factors involved in cell signalling, e.g moody and CG11910, or enzymes like CG7433 and CG6218 (Beckervordersandforth, 2008).

moody is expressed in all cells belonging to the surface-associated glia. At the end of embryogenesis, surface-associated glial cells form a thin layer ensheathing the entire CNS, thereby establishing the blood-brain barrier. Moody is a G-protein coupled receptor, which acts in a complex pathway to regulate the cortical actin, thereby stabilizing the extended morphology of the surface-glia. This is necessary for the formation of septate junctions to achieve proper sealing of the nerve cord (Bainton, 2005; Daneman, 2005; Schwabe, 2005). Moody therefore represents a protein, which is essential for establishing and maintaining a specific function of surface glia (Beckervordersandforth, 2008).

Two of the markers analyzed represent metalloproteases: Neprilysin4 (Nep4) and Invadolysin. Invadolysin has been described to play a role in mitotic progression and in migration of germ cells (McHugh, 2004), but as for Nep4, its function in the nervous system is unknown. In vertebrates it has been shown that metalloproteases are involved in various processes in the CNS: they are associated with neurite outgrowth, migration of neurons and myelination of axons. For one matrix-metalloprotease, MMP-12, it has been shown that it is expressed in oligodendrocytes, where it functions in maturation and morphological differentiation of OL lineages (Larsen, 2004; Larsen, 2006). It has been postulated that LGs are analogous to vertebrate oligodendrocytes (Hidalgo, 2000), as both groups of cells enwrap axonal projections in the CNS, although to different degrees (no myelination in Drosophila). The two metalloproteases analysed are exclusively expressed in neuropile- (LGs) and cortex-associated glial cells (CBGs). Thus, both Nep4 and Invadolysin may possibly be involved in the differentiation of LGs. In invadolysin loss of function mutants, the specification of lateral glial cells does not seem to be affected, but the LGs show a very subtle phenotype in their positioning. An explanation for the subtle phenotype may be redundant function of both enzymes. Indeed, in vertebrates it has been shown that metalloproteases have many overlapping substrates in vitro, and redundancy and compensation has been shown for matrix-metalloproteases (MMPs) in double mutants. Furthermore, it has been shown for members of the neprilysin family of metalloendopeptidases in Caenorhabditis elegans and Drosophila melanogaster, that many of the enzymatic properties have been conserved during evolution (Beckervordersandforth, 2008).

Making use of the molecular markers, this study characterized the phenotype of a cas loss of function mutation. Cas is a transcription factor, which acts in temporal cell fate specification. Together with Pdm, Cas is involved in the determination of late progeny cells in CNS lineages. In late embryonic stages, cas is specifically expressed in four glial cells per hemisegment, the V-CG and D-CG, the A-SPG and the LV-SPG, as well as in many neurons. The A- and LV-SPG, which are late progeny of the NB1-1A, are not affected in cas mutants, whereas the NB7-4-derived CGs seem mislocalized, with the medial migration of both CGs being impaired in cas mutants. This points to different functions of Cas in distinct NB lineages. As can be deduced from Repo stainings, general aspects of glial differentiation do not seem to be affected in cas mutants. Further analysis will have to clarify whether the role of Cas in NB7-4 derived glial cells is on the level of cell fate determination and/or whether it directly acts on specific aspects of differentiation (migration, motility). It also remains to be shown whether Cas acts cell-autonomously in this process (Beckervordersandforth, 2008).

Sex-specific signaling in the blood-brain barrier is required for male courtship in Drosophila

Soluble circulating proteins play an important role in the regulation of mating behavior in Drosophila melanogaster. However, how these factors signal through the blood-brain barrier (bbb) to interact with the sex-specific brain circuits that control courtship is unknown. This study shows that male identity of the blood-brain barrier is necessary and that male-specific factors in the bbb are physiologically required for normal male courtship behavior. Feminization of the bbb of adult males significantly reduces male courtship. The bbb-specific G-protein coupled receptor Moody and bbb-specific Go signaling in adult males are necessary for normal courtship. These data identify sex-specific factors and signaling processes in the bbb as important regulators of male mating behavior (Hoxha, 2013).

It is worth noting that the integrity of the bbb was not affected by feminization or by any other of our manipulations using a standard approach to examine bbb barrier function, although small defects cannot be ruled out. Therefore, the observed effects support the interpretation that feminization affects physiological sex- specific processes within the bbb. This study shows that moody GPCR signaling is one of these processes. Normal courtship requires both moody isoforms, α and β, similar to the previously reported response to alcohol and cocaine. As has been described, moody appears to have two distinct roles: While either one of the moody isoforms is sufficient for a functional and intact barrier, both isoforms are required for adult signaling processes. RNA sequencing data suggest that the two isoforms are not present in equal abundance and that the ratio of the two isoforms is sex-specifically regulated. It is not clear at present why two forms of the moody protein are required in behavior. It is unlikely that a strict stoichiometric ratio of the two isoforms is required, since normal courtship was observed in wild-type flies that express additional Moody-β. The two isoforms differ in their intracellular domain, which could indicate that they interact with different effector molecules that are both contributing to the behavioral response. Courtship defects were observed when either isoform is missing, indicating that both forms have a role in regulating courtship. Interestingly, it was observed that the ratio of the two isoforms is under the control of the sex-specific splicing factor TraF, raising the possibility that the moody pre-mRNA is a target for splicing regulation by TraF or one of its downstream effectors. It will be of interest to identify other sex-specific factors in the bbb and examine their contribution to the regulation of male courtship (Hoxha, 2013).

No courtship phenotype was observed when dominant mutants were expressed for Gs and Gq that have been shown to act as dominant negative mutations in developmental processes. Likewise, expression of Gi-RNAi or concertina-RNAi did not result in reduced courtship either. This suggests that these G proteins do not play a significant role in courtship in this layer. In contrast, courtship defects were observed when Go signaling was compromised. Two approaches were employed to show that the heterotrimeric protein Go is required for male courtship behavior: inhibition by PTX and mRNA reduction by RNAi. The S1 subunit of PTX from Bordetella pertussis catalyzes the transfer of an ADP-ribose onto the Gα subunit of the heterotrimeric G protein. In contrast to mammals, where PTX inhibits both Go and Gi, in Drosophila PTX is a specific inhibitor for Go, since the only Gi present (Gⁱ65A) does not contain the PTX recognition site. PTX will only ribosylate heterotrimers (not individual alpha subunits), and the consequence of this ribosylation is inhibition of the heterotrimer activation. The inhibition of Go signaling by PTX is therefore very specific; since the ADP-ribosylated Go heterotrimers cannot be activated, they do not generate ectopic Gβγ subunits, nor do they sequester free Gβγ subunits away from other Gα subunits. Conditional induction of PTX only in adult mature flies, as well as conditional adult reduction of Gαo by RNAi reduced male courtship. This demonstrates that physiological signaling through Go is an important signaling pathway that regulates courtship in the bbb. Given these findings it is likely that moody signals through Go to exert its function in courtship. In embryos, Go, Gi, moody and loco mutations each disrupt the formation of the bbb and lead to bbb leakiness, as shown by dye penetration. In contrast, dye penetration was not observed in the PTX and Go-RNAi mutants that were generated, further evidence that the developmental and physiological roles of moody and Go signaling differ in their mechanisms (Hoxha, 2013).

It is not knowm what the downstream pathways are that are mediating the Go action. Few Go effectors have been demonstrated and its α or βγ subunits could be mediating the signal. In many cases in vertebrates it is the βγ subunits that are responsible for actuating signaling. In neurons, presynaptic voltage-gated Ca²⁺ channels have been shown to represent an effector for Go. Studies of the role of Go in learning and memory in Drosophila have suggested that Go signaling does not occur through the rut adenylyl cyclase. Signaling through Go is not generally thought to occur through PKA, consistent with the finding that disruption of PKA signaling in the bbb did not affect courtship. It is unknown whether potential downstream signaling molecules like loco, Gγ13F or PKC are sex-specifically expressed in the blood-brain barrier and might have a courtship role in this layer. In whole heads, PKC98E is male-preferentially expressed. It is unknown what the ligand is for Moody and it remains to be seen what the exact role is for moody bbb signaling in courtship. Hemolymph factors that influence courtship could conceivably do so by initiating signaling pathways at the bbb, or by passage and transport through the bbb. Moody could be playing a role in signaling, as well as through a possible effect on transport, perhaps in processes similar to its previously demonstrated effects on the actin cytoskeleton during development (Hoxha, 2013).

This study has demonstrated that sex-specific molecules in the bbb are important regulators of male courtship behavior in Drosophila. The Moody GPCR and Go signaling in this layer are an important part of this regulation. It will be of importance to identify the ligand(s) and downstream signaling events that ultimately interact with the brain circuits that control male courtship behavior (Hoxha, 2013).

Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila

During development, many epithelia are formed by a mesenchymal-epithelial transition (MET). This study examined the major stages and underlying mechanisms of MET during blood-brain barrier (BBB) formation in Drosophila. Contact with the basal lamina was shown to be essential for the growth of the barrier-forming subperineurial glia (SPG). Septate junctions (SJs), which provide insulation of the paracellular space, are not required for MET, but are necessary for the establishment of polarized SPG membrane compartments. In vivo time-lapse imaging reveals that the Moody GPCR signalling pathway regulates SPG cell growth and shape, with different levels of signalling causing distinct phenotypes. Timely, well-coordinated SPG growth is essential for the uniform insertion of SJs and thus the insulating function of the barrier. This is the first dynamic in vivo analysis of all stages in the formation of a secondary epithelium and of the key role trimeric G protein signalling plays in this important morphogenetic process (Schwabe, 2017).

This study of Drosophila BBB development represents the first dynamic in vivo study of MET and secondary epithelium formation. The data shed particular light on the roles of the basal lamina and of the insulating SJs, as well as on the function of GPCR signaling in this important morphogenetic process. Once SPG reach the CNS surface, contact with the basal lamina is essential for the extensive growth of the SPG during epithelium formation. Previous in vitro studies have shown that adhesion to basal lamina components is necessary for cell spreading and proliferation, however this study is the first to demonstrate in vivo that attachment to the basal lamina is essential for non-proliferative cell growth and ensheathment. Attachment to the ECM occurs primarily through focal adhesions and integrins, which in turn can activate MAPK signaling, triggering cell proliferation and growth. In addition, adhesion to the ECM has been shown to provide traction, which facilitates cell spreading. Contact to the ECM may thus provide the SPG with both growth signals and attachment sites. Being highly expressed on the basal lamina facing side of SPG, Dystroglycan (Dg) is an excellent candidate for mediating ECM attachment. However, zygotic mutants of Dg show no BBB defects and germline clones could not be analyzed due to Dg's role in oogenesis (Schwabe, 2017).

Beyond supporting SPG growth, contact with the basal lamina likely provides an important cue for polarizing the cells, as judged by their strong enrichment of Dg at the basal lamina facing (basal) membrane compartment. Previous studies have shown that Dg and its ligand Pcan/Trol are required for the establishment of polarity in follicle cells. However, when the basal lamina and thus its ligand Pcan are depleted, Dg is still expressed and polarized in the SPG, suggesting that glial polarity can be supported by the residual basal lamina or that additional polarizing signals exist (Schwabe, 2017).

Once SJs have formed, the GPCR Moody and the Mdr65 transporter are asymmetrically distributed within the SPG, further demonstrating that these cells possess distinct apical and basal membrane compartments. This polarized distribution is coincident with and dependent on the presence of SJs, demonstrating for the first time that SJs serve a function in cell polarity. By acting as a fence and preventing diffusion of membrane proteins across the lateral compartment, the SJs maintain asymmetric protein distributions, which could result from polarized exocytosis or endocytosis. Intriguingly, a separate study has identified PKA as a crucial antagonistic effector of Moody signaling. PKA has been shown to regulate polarized exocytosis at the trans-Golgi network in different types of epithelia. Apical-basal polarity plays an important morphogenetic role in the continued growth of the SPG epithelium during larval stages and in the function of the BBB (Schwabe, 2017).

Signaling by the GPCR Moody plays a critical role both in regulating the growth of individual SPG and in synchronizing this process across the entire SPG cell population. In Moody pathway mutants, glial growth behavior is more erratic, and more variable between cells. This increased variability of glial cell shape, size, and growth causes a significant delay of epithelial closure of up to 1.5 hrs. This delay is not caused by an earlier delay in glial migration or by a delay in SJ formation (Schwabe, 2017).

The detailed dynamic analysis reveals that, in moody and loco mutants, the spatio-temporal coordination of cell spreading is impaired. Spreading cells, like other motile cells, show fluctuating exploratory motions of the leading edge visible as cycles of protrusion and retraction. This complex process can be broken down into discrete steps: actin protrusion of the leading edge, adhesion to the ECM, and myosin-driven contraction against adhesions. Time-lapse recordings indicate that Moody signaling has its most pronounced effect on the stabilization of protrusions, as evidenced by an increase in the ratio of retractions to extensions, and the marked shift of cell contours over time. The destabilization of protrusions might be due to weaker integrin-mediated interaction of focal adhesions with the ECM, but also due to impaired stress-mediated maturation of focal adhesions. The fact that both under- and overactivity of the Moody pathway impair protrusion stabilization may be due to the feedback between actin-myosin and focal adhesion, which also causes the well-known biphasic response of migration speed to adhesion strength of migrating cells. While the loss of moody has no significant effects on the other parameters that were measured, the loss of loco also reduces the frequency and size of protrusions, suggesting that actin polymerization may be specifically affected by increased GPCR signaling activity. Cumulatively, these impairments in protrusion/retraction behavior lead to retarded, non-isometric growth of SPG and to the irregular cell shapes observed in moody and loco mutants (Schwabe, 2017).

Interestingly, PKA, Rho1 and MLCK have been identified as important downstream effectors of Moody signaling. All three factors are well known to control actin-myosin contraction; Rho1 and MLCK as positive regulators, PKA as a negative regulator. More recently, Rho1 activity has been shown to also drive actin polymerization at the leading edge, and a PKA-RhoGDI-Rho1 regulators feedback loop has been suggested to act as a pacemaker of protrusion- retraction cycles (Schwabe, 2017).

The role of Moody pathway signaling in directed and well-coordinated cell growth is strikingly similar to the function of trimeric G protein signaling in other contexts. In Dictyostelium, G protein signaling is essential for directed cell migration: When all G protein signaling is abolished, cells are still mobile and actively generate protrusions, however these protrusions form in random directions, with the result that the cells lose their directionality. During gastrulation in Drosophila, signaling by the G₁₂ ortholog Concertina and the putative GPCR ligand Folded Gastrulation synchronizes apical actin-myosin constrictions of mesodermal precursor cells, and thereby effects their concerted invagination. Thus, a major role of G protein signaling during development may be to modulate basic cellular behaviors such as cell growth, protrusion, or contraction, and reduce variability within cells and between neighboring cells, with the goal of generating uniform patterns and behaviors (Schwabe, 2017).

Synchronized growth behavior of SPG is not only important for rapid epithelial closure but, ultimately, also for generating an evenly sealed BBB. All the evidence supports the notion that the defects in SJ organization that are responsible for the BBB failure are a secondary consequence of the morphogenetic function of the GPCR pathway. Cell contacts precede and are necessary for SJ formation, and the growth of cell contacts and SJ accumulation are strongly correlated. Delayed and more erratic cell- cell contact formation, as is the case in Moody pathway mutants, is likely to result in uneven circumferential distribution of SJ material later on; conversely, the timing of SJ formation per se is not affected by the pathway, arguing against a direct effect. Since the insulating function of SJs depends on their length, a decrease in the length in some local areas will result in insulation defects. Moreover, since SJs are known to form very static complexes, any irregularity in SJ distribution may be retained for long periods of time (Schwabe, 2017).

Although under- and overactivity of the Moody pathway lead to globally similar outcomes, impaired epithelium formation and failure of BBB insulation, the data point to subtly different subcellular effects of the two types of pathway modulation. During MET, loco mutants (which this study confirms as inducing pathway overactivity) show predominantly retarded growth, presumably as a result of curtailed protrusive activity, while moody mutants show severe fluctuation and variability in growth. It will be very interesting to investigate these distinct outcomes of Moody pathway misregulation in greater detail (Schwabe, 2017).

The Drosophila blood-brain barrier adapts to cell growth by unfolding of pre-existing septate junctions

The blood-brain barrier is crucial for nervous system function. It is established early during development and stays intact during growth of the brain. In invertebrates, septate junctions are the occluding junctions of this barrier. This study used Drosophila to address how septate junctions grow during larval stages when brain size increases dramatically. Septate junctions are preassembled as long, highly folded strands during embryonic stages, connecting cell vertices. During subsequent cell growth, these corrugated strands are stretched out and stay intact during larval life with very little protein turnover. The G-protein coupled receptor Moody orchestrates the continuous organization of junctional strands in a process requiring F-actin. Consequently, in moody mutants, septate junction strands cannot properly stretch out during cell growth. To compensate for the loss of blood-brain barrier function, moody mutants form interdigitating cell-cell protrusions, resembling the evolutionary ancient barrier type found in primitive vertebrates or invertebrates such as cuttlefish (Babatz, 2018).

This study has dissected the mechanisms underlying growth of occluding septate junctions in non-dividing Drosophila cells. In primary and secondary epithelia, septate junction strands are mostly generated during embryonic stages. Septate junction protein complexes show a remarkable stability and persist over several days. In primary epithelia, newly synthesized linear and unbranched septate junction strands are added basally to the apically localized adherens junctions. In contrast, in secondary epithelial cells of the Drosophila blood-brain barrier, such newly synthesized septate junction strands are added on both sides of the pre-existing septate junctions. This process depends on the G-protein coupled receptor Moody and F-actin formation to integrate preformed vesicular septate junctions into existing strands. In addition, it was shown that moody mutants activate compensatory mechanisms to cope with the loss of junctional integrity. moody mutant cells activate expression of septate junction proteins and form an excess of membrane cell-cell interdigitations, which establish sufficient barrier function (Babatz, 2018).

Septate junctions are assembled by an astonishing number of proteins in an almost crystalline appearance with considerable stability. This study has demonstrated that septate junctions are indeed stable for several days and that most of the septate junction proteins are generated during early larval development. This is also confirmed by analyzing the developmental expression profile of all septate junction proteins. Moreover, FRAP data are consistent with the notion that septate junctions are arranged in unbranched, long lines connected to tricellular junctions (TCJs). Nevertheless, it is important to note that septate junction strands appear to mature during larval development, and, for example, the loosely associated protein Fas3 is constantly added to the growing septate junctions. Finally, septate junctions of the blood-brain barrier are more stable compared to those found in primary epithelia and use a different mechanism of new strand integration, suggesting alternative characteristics of septate junctions in different tissues (Babatz, 2018).

When epithelial cells grow or divide, the occluding septate junctions have to be adjusted to the changing cell geometry. During growth of the lateral cell membrane, this could imply an opening of existing strands and the subsequent insertion of new septate junction proteins. However, in freeze fracture studies of reorganizing tissues of Rhodnius or hydra, no evidence for such processes was detected. Consequently, it was postulated that existing septa can be redistributed in the plane of the membrane in cell contact areas, meaning septate junction strands stretch across the entire domain upon changes in cell geometry. This study used high-resolution imaging to show that indeed septate junctions are formed in a wavy manner during embryonic stages and unfold during the larval growth period (Babatz, 2018).

The corrugated organization of septate junctions appears to be a more general feature. Salivary gland cells have a conical shape, and the perimeter of the cell increases about two times from apical to basal. Similar to the growing subperineurial glial cells, septate junctions are formed as folded structures at the apical side and stretch out as they move to more basal positions. Likewise, during Drosophila oocyte development, follicle cells initially form corrugated adherens junctions that become straight when cells expand. A similar unfolding of tig (Babatz, 2018).

The unfolding of the bicellular septate junction strands requires force and possibly anchoring of strands to a fixed point. Such a fixed point could be a tricellular junction, which also allows force generation during cytokinesis. Classical freeze fracture studies as well as FRAP data suggest that bicellular occluding junctions are connected to tricellular junctions. This connection might be mediated by the tricellular junction proteins Bark beetle (Anakonda) and Gliotactin, which can also interact with bicellular septate junction proteins (Babatz, 2018).

During epithelial development, tissue convergence and extension correlate with adherens junction dynamics due to local actomyosin-based contractions. However, the initial corrugated-appearing septate junction strands suggest that the initial mechanical forces do not act on the establishment of septate junctions. Only when the cell grows and thus expands its lateral plasma membrane, the pre-established junctions are straightened. When subperineurial glial growth is suppressed, the initial corrugated shape of the septate junctions is not changed. When additional growth of the subperineurial glia is triggered by, for example, excessive neuroblast proliferation as seen in l(2)gl mutant animals, only rare ruptures of septate junction strands were noticed. In contrast, additional septate junction strands appear to be added to compensate for the increased cell growth. This suggests that the growing subperineurial glia senses barrier integrity, and any leakage results in the addition of new septate junction strands. This plasticity appears to depend on gene activity, since upon block of polyploidization in subperineurial glial cells, septate junctions are interrupted. The ability of subperineurial glial cells to react to changing barrier requirements can also be seen in moody mutants, which show an increase of NrxIV expression (Babatz, 2018).

The G-protein coupled receptor Moody is initially found along the entire plasma membrane, but after formation of septate junctions, its localization is restricted to the cell membrane facing the cortex glia with some enrichment at the cell-cell contact sites. Septate junctions form in the absence of Moody, but their arrangement in long, corrugated, and possibly anchored strands fails. Recently, special actin-rich structures have been observed along the lateral borders of the subperineurial glia that are regulated by Moody signaling. Gene dose experiments presented in this study indicate that Moody affects septate junction formation by the local activation of F-actin formation possibly to provide the physical force to integrate septate junction building blocks preformed in vesicular structures (Babatz, 2018).

As the brain grows in size, septate junctions cannot expand in moody mutants. In consequence, the blood-brain barrier established by the subperineurial glial cells becomes leaky, which triggers the expression of additional septate junction proteins, which, however, cannot be properly integrated into the existing strands. Remarkably, moody mutant subperineurial glia also form interdigitating membrane folds, which partially restore barrier integrity. A similar excess of cell interdigitation is induced upon reduction of NrxIV expression, specifically in the subperineurial glia. Interestingly, this type of barrier is also found in several other invertebrates and primitive vertebrates. Thus, moody mutants disclose the principles of an evolutionary ancient form of the blood-brain barrier. The efficient blood-brain barrier based on long, uninterrupted septate junction strands induced by Moody then allowed the evolution of more compact nervous systems, as found in Drosophila (Babatz, 2018).

The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation

The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previous work identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. This study identified cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. PKA is enriched at the basal side of the SPG cell, and this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. It is proposed that Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity (Li, 2021).

Previous studies implicated a novel GPCR signaling pathway in the formation of the Drosophila BBB in late embryos. This work also revealed that besides the GPCR Moody two heterotrimeric G proteins (Gαiβγ, Gαoβγ5) and the RGS Loco participate in this pathway. This study provides a comprehensive molecular and cellular analysis of the events downstream of G protein signaling using a candidate gene screening approach. New, more sensitive methods for phenotypic characterization are presented, and the analysis was extended to beyond the embryo into larval stages. This work identifies, together with some of its targets, as crucial antagonistic effectors in the continued cell growth of SPG and maintenance of the BBB sealing capacity. This role is critical to ensure proper neuronal function during BBB formation and maturation (Li, 2021).

Multiple lines of evidence demonstrate a role of PKA for proper sealing of the BBB: loss of PKA activity leads to BBB permeability defects, irregular growth of SPG during epithelium formation, reduced membrane overlap, and a narrower SJ belt at SPG cell-cell contacts. The role of PKA as an effector of the Moody signaling pathway is further supported by dominant genetic interaction experiments, which show that the dye penetration phenotype of PkaC1 heterozygous mutant embryos was partially rescued by removing one genomic copy of Gβ13F or loco. Moreover, the analysis of the larval phenotype with live SJ and cytoskeleton markers shows that PKA gain of function behaved similarly to Moody loss of function. Conversely, PKA loss of function resembled the overexpression of GαoGTP, which mimics Moody gain-of-function signaling (Li, 2021).

The results from modulating PKA activity suggest that the total cell contact and SJ areas are a major function of PKA activity: low levels of activity cause narrow contacts, and high levels give rise to broad contacts. Moreover, the analysis of various cellular markers (actin, microtubules, SJs, vesicles) indicates that the circumferential cytoskeleton and delivery of SJ components respond proportionately to PKA activity. This, in turn, promotes the changes in cell contact and junction areas coordinately at the lateral side of SPG. These experiments demonstrate that the modulation of the SPG membrane overlap by PKA proceeds, at least in part, through the regulation of actomyosin contractility, and that this involves the phosphorylation targets MLCK and Rho1. This suggests that crucial characteristics of PKA signaling are conserved across eukaryotic organisms (Li, 2021).

At the ultrastructural level, ssTEM analysis of the larval SPG epithelium clarifies the relationship between the inter-cell membrane overlaps and SJ organization and function. Across different PKA activity levels, the ratio of SJ areas to the total cell contact area remained constant at about 30%. This proportionality suggests a mechanism that couples cell contact with SJ formation. The primary role of Moody/PKA appears in this process to be the control of membrane contacting area between neighboring cells. This is consistent with the results of a temporal analysis of epithelium formation and SJ insertion in late embryos of WT and Moody pathway mutants, which shows that membrane contact precedes and is necessary for the appearance of SJs. The finding that the surface area that SJs occupy did not exceed a specific ratio, irrespective of the absolute area of cell contact, suggests an intrinsic, possibly steric limitation in how much junction can be fitted into a given cell contact space. While most phenotypic effects are indeed a major function of Moody and PKA activity, the discontinuity and shortening of individual SJ strands is not. It occurred with both increased and decreased signaling and appears to cause the leakiness of the BBB in both conditions. ssTEM-based 3D reconstruction thus demonstrates that the total area covered by SJs and the length of individual contiguous SJ segments are independent parameters. The latter appears to be critical for the paracellular seal, consistent with the idea that Moody plays a role in the formation of continuous SJ stands (Li, 2021).

The asymmetric localization of PKA that was observed sheds further light on the establishment and function of apical-basal polarity in the SPG epithelium. Prior to epithelium formation, contact with the basal lamina leads to the first sign of polarity. Moody becomes localized to the apical surface only after epithelial closure and SJ formation, suggesting that SJs are required as a diffusion barrier and that apical accumulation of Moody protein is the result of polarized exocytosis or endocytosis. This study now shows that the intracellular protein PKA catalytic subunit-PkaC1 accumulates on the basal side of SPG, and that this polarized accumulation requires (apical) Moody activity. Such an asymmetric, activity-dependent localization has not previously been described for PKA in endothelium, and while the underlying molecular mechanism is unknown, the finding underscores that generating polarized activity along the apical-basal axis of the SPG is a key element of Moody pathway function (Li, 2021).

An intriguing unresolved question is how increased SPG cell size and SJ length can keep up with the expanding brain without disrupting the BBB integrity during larva growth. The SJ was found to grow dramatically in length (0.57 ± 0.07 μm vs. 2.16 ± 0.14 μm, about 3.7-fold) from the late embryo to third instar larva, which matches the increased cell size of SPG (about fourfold). During the establishment of the SPG epithelium in the embryo, both increased and decreased Moody signaling resulted in asynchronous growth and cell contact formation along the circumference of SPG, which in turn led to irregular thickness of the SJ belt. Therefore, a similar relationship may exist during the continued growth of the SPG epithelium in larvae, with the loss of continuity of SJ segments in Moody/PKA mutants resulting from unsynchronized expansion of the cell contact area and an ensuing erratic insertion of SJ components. Since SJs form relatively static complexes, any irregularities in their delivery and insertion may linger for extended periods of time. The idea that shortened SJ segments are a secondary consequence of unsynchronized cell growth is strongly supported by the finding that disruption of actomyosin contractility in MLCK and Rho1 mutants compromises BBB permeability (Li, 2021).

Collectively, these data suggest the following model: polarized Moody/PKA signaling controls the cell growth and maintains BBB integrity during the continuous morphogenesis of the SPG secondary epithelium. On the apical side, Moody activity represses PKA activity (restricting local cAMP level within the apial-basal axis in SPG) and thereby promotes actomyosin contractility. On the basal side, which first adheres to the basal lamina and later to the PNG sheath, PKA activity suppresses actomyosin contractility via MLCK and Rho1 phosphorylation and repression. Throughout development, the SPG grow continuously while extending both their cell surface and expanding their cell contacts. The data suggest that the membrane extension occurs on the basolateral surface through insertion of plasma membrane and cell-adhesive proteins, with similar behavior in epithelial cell, but regulated by a distinct polarized Moody/PKA signaling in SPG. In analogy to motile cells, the basal side of the SPG would thus act as the 'leading edge' of the cell, while the apical side functions as the 'contractile rear'. According to this model, Moody/Rho1 regulate actomyosin to generate the contractile forces at the apical side to driving membrane contraction, which directs the basolateral insertion of new membrane material and SJs. In this way, differential contractility and membrane insertion act as a conveyor belt to move new formed membrane contacts and SJ from the basolateral to apical side. Loss of Moody signaling leads to symmetrical localization of PKA and to larger cell contact areas between SPG due to diminished apical constriction. Conversely, loss of PKA causes smaller cell contact areas due to increased basal constriction (Li, 2021).

These results may have important implications for the neuron-glia interaction in the nervous system and the development and maintenance of the BBB in vertebrates. SJs have several structural and functional components in common with paranodal junctions, which join myelinating glial cells to axons in the vertebrate nervous system, and they share similar regulation mechanisms. The vertebrate BBB consists of a secondary epithelium with interdigitations similar to the ones between the Drosophila SPG . While the sealing is performed by TJs, it will be interesting to investigate whether there are similarities in the underlying molecular and cellular mechanisms that mediate BBB function (Li, 2021).

The Drosophila blood-brain barrier regulates sleep via Moody G protein-coupled receptor signaling

Sleep is vital for most animals, yet its mechanism and funwction remain unclear. This study found that permeability of the BBB (blood-brain barrier)-the organ required for the maintenance of homeostatic levels of nutrients, ions, and other molecules in the brain-is modulated by sleep deprivation (SD) and can cell-autonomously effect sleep changes. Increased BBB permeability was observed in known sleep mutants as well as in acutely sleep-deprived animals. In addition to molecular tracers, SD-induced BBB changes also increased the penetration of drugs used in the treatment of brain pathologies. After chronic/genetic or acute SD, rebound sleep or administration of the sleeping aid gaboxadol normalized BBB permeability, showing that SD effects on the BBB are reversible. Along with BBB permeability, RNA levels of the BBB master regulator moody are modulated by sleep. Conversely, altering BBB permeability alone through glia-specific modulation of moody, g&slpha;o, loco, lachesin, or i>neuroglian -each a well-studied regulator of BBB function-was sufficient to induce robust sleep phenotypes. These studies demonstrate a tight link between BBB permeability and sleep and indicate a unique role for the BBB in the regulation of sleep (Axelrod, 2023).

This study establish the BBB as a sleep regulatory center in Drosophila. It was observed that the BBB dynamically and rapidly—both functionally and molecularly—responds to acute as well as chronic-genetic SD and that altering BBB function alone, through mutations of the moody signaling pathway, can affect sleep, suggesting that the BBB senses sleep need and responds to it by altering its permeability. The correct BBB permeability state seems to be required for normal sleep, as disrupting BBB function causes sleep loss (Axelrod, 2023).

In normal physiology, the BBB controls influx and efflux of molecules in and out of the brain, including ions, amino acids, neurotransmitters, vitamins, proteins, carbohydrates, lipids, and hormones. Transport is facilitated via various mechanisms which include active transporters, transcytosis, and passive diffusion. The function of the BBB is not static but is affected by circadian rhythms, neuronal activity, and the metabolism. the data add sleep as an additional parameter modulating BBB function, as has been suggested by studies in mammals, and this study found that the relationship between the BBB and sleep is reciprocal. The results show that the BBB rapidly and dynamically responds to SD by increasing its permeability and that permeability increases conversely cause sleep loss. It is conceivable that BBB permeability changes allow the passage of one or multiple molecules that affect sleep into or out of the brain. Defective xenobiotic exclusion of steroid hormones from the brain via the BBB has been shown to affect sleep in Drosophila, demonstrating that molecules outside the CNS can affect sleep. Future studies may determine whether the BBB mediates a rapid global switch from a sleeping to an awake brain, for example, by controlling ionic diffusion and potassium concentrations, which could broadly raise neuronal activation thresholds and lead to the observed extracellular ionic changes in the sleeping mammalian brain (Axelrod, 2023).

The BBB creates a highly controlled microenvironment in the brain, perturbations of which affect neuronal function, as evidenced by neuropathologies at least in part caused by BBB disruption including stroke, Alzheimer's, Multiple Sclerosis, epilepsy and brain tumor metastasis. BBB disruption also occurs during normal aging, under chronic stress and during systemic inflammation and infection; however, whether the relationship between BBB disruption and these pathologies is causal or merely correlative is less clear. The magnitude of BBB changes in disease states can be an order of magnitude larger than the observed changes after sleep loss described in this study, illustrating the physiological relevance of these experiments. All of the pathologies listed above are also marked by sleep disruption, which in some instances increases the risk for developing the disease in the first place and the severity of which correlates with disease severity. The fly ortholog of a human autism gene has been reported to act in the BBB and elicit sleep fragmentation via developmental hyperserotonemia. However, a direct involvement of the BBB in sleep regulation had not been shown until this present study (Axelrod, 2023).

BBB and sleep disruptions have been, so far independently, associated with causing illness, and this study provides additional evidence for the notion that the two processes are linked. If BBB permeability and sleep regulation are fundamentally connected as part of the same system, it will be interesting to study how they relate to disease risk, development, and prognosis, and whether interventions that affect one also augment the other (Axelrod, 2023).

Adequate drug delivery to the brain is a problem plaguing the treatment of many neuropathologies. This puts strong importance on finding possible regulatory sites for controlling the BBB as a means of finding better ways to deliver drugs to the brain. The BBB has distinct mechanisms to exclude molecules from the brain including xenobiotic transporters in the cell membrane that efflux most small hydrophobic molecules taken up transcellularly through the membrane, and the paracellular barrier formed by junctions between the BBB forming cells. The latter creates a diffusion barrier for hydrophilic molecules in a size-dependent manner with a maximum passage size of about 0.5 kDa. Due to these mechanisms, the BBB poses a major obstacle to treatment of brain pathologies, because 100% of large molecules (>0.5 kDa), like the brain cancer drug vinblastine (molecular weight: 1 kDa), and 98% of small molecule drugs (<0.5 kDa), like the non-steroidal anti-inflammatory drug paracetamol, cannot penetrate it in therapeutic amounts. Current solutions to this problem include pharmacologically increasing the diffusion barrier permeability, implantation of ultrasonic devices to temporarily permeabilize the diffusion barrier, fusing molecules to receptor ligands to engage the transcellular route, or intracranial administration via injection. Provided the observed effects are conserved in humans, a patient's sleep and nighttime administration of drugs could have a significant effect on drug penetration into the CNS and possibly treatment outcomes (Axelrod, 2023).

In Drosophila, the BBB's subperineurial glia form a layer of polarized epithelial cells, with septate junctions between them creating a seal for water-soluble molecules over 500 Da. The observation that SD rapidly induces increases in BBB permeability raises the question: What are the molecular and cellular mechanisms that underlie the remodeling of septate junctions on the scale of hours? The Moody GPCR pathway has been shown to play a key role in the development of the epithelial barrier, as well as its maintenance in adulthood. Moody activates a signaling cascade which, via PKA activation, controls the developmental assembly of the septate junction belt in a highly coordinated spatiotemporal manner that involves rearrangements of the actin cytoskeleton and vesicular trafficking. This study has shown that RNAi-based suppression of moody pathway genes causes increased BBB permeability. Strong downregulation of moody was observed in every genetic sleep mutant tested. These findings suggest that altered moody pathway function may be primarily responsible for the leaky barriers of these mutants (Axelrod, 2023).

During SD and recovery, it is conceivable that similar processes occur. Indeed, it has been shown that perturbation of endocytosis at the Drosophila BBB alters sleep, possibly interfering with Moody-activated remodeling during sleep/wake cycles. Scute SD has been shown to rapidly depress moody expression in wild-type Drosophila, which is accompanied by increased BBB permeability. Moody is expressed on the neuronal-facing apical cell surface of the subperineurial glia. While its ligand is not known, this topology could permit neuronal states related to sleep to be communicated to glia via this GPCR pathway. As many known sleep genes in Drosophila have been shown to be expressed in neurons and this study observed BBB changes upon sleep-suppressing neuronal activation, neuron-glia signaling seems likely to be part of the process, and it will be interesting to elucidate the involved signaling molecules including Moody's ligand (Axelrod, 2023).

The two-step model of sleep regulation posits that circadian rhythm and sleep homeostasis control the timing of sleep. Sleep homeostasis is a process that controls sleep duration to meet an organism's daily sleep need. Rebound sleep after SD is interpreted as proof of the existence of sleep homeostasis. The physiological and cellular nature of the sleep homeostat is-like the nature of sleep itself-the subject of current research. Sleep homeostasis has been shown to be affected by oxidative stress, neuronal, glial, and microglial activity, neuropeptide signaling, infection, the circadian clock, temperature, and starvation. Synaptic downscaling, glymphatic clearance of toxins, peripheral glucose homeostasis, reactive oxygen species clearance, and inhibition of motor function have been proposed as processes occurring during sleep homeostasis, constituting functions of sleep. As at least some of these processes must involve yet to be identified body-brain signals, future studies will reveal how the BBB changes shown here affect known or novel functions of sleep (Axelrod, 2023).

Together the data indicate a system where an increase of sleep need is measured by the BBB, resulting in an adjustment of Moody levels, an opening of the BBB, and the initiation of rebound sleep. This process is likely to involve neuronal to BBB signaling (dopaminergic SD also alters the BBB, and many of the sleep mutants are expressed in neurons) as well as yet-to-be-characterized molecular exchange across the BBB (Axelrod, 2023).

Search PubMed for articles about Drosophila Moody

Axelrod, S., Li, X., Sun, Y., Lincoln, S., Terceros, A., O'Neil, J., Wang, Z., Nguyen, A., Vora, A., Spicer, C., Shapiro, B., Young, M. W. (2023). The Drosophila blood-brain barrier regulates sleep via Moody G protein-coupled receptor signaling. Proc Natl Acad Sci U S A, 120(42):e2309331120 PubMed ID: 37831742

Babatz, F., Naffin, E. and Klambt, C. (2018). The Drosophila blood-brain barrier adapts to cell growth by unfolding of pre-existing septate junctions. Dev Cell 47(6):697-710. PubMed ID: 30482667

Bainton, R. J. et al. (2005). moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila. Cell 123: 145-156. PubMed ID: 16213219

Beckervordersandforth, R. M., Rickert, C., Altenhein, B. and Technau, G. M. (2008). Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression. Mech. Dev. 125: 542-557. PubMed ID: 18296030

Daneman, T. and Barres, B. A. (2005). The blood-brain barrier -- lessons from moody flies. Cell 123: 9-12. PubMed ID: 16213208

Hidalgo, A. and Booth, G. E. (2000). Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS. Development 127: 393-402. PubMed ID: 10603355

Hoxha, V., Lama, C., Chang, P. L., Saurabh, S., Patel, N., Olate, N. and Dauwalder, B. (2013). Sex-specific signaling in the blood-brain barrier is required for male courtship in Drosophila. PLoS Genet 9: e1003217. PubMed ID: 23359644

Katanaev, V. L., et al. (2005). Trimeric G protein-dependent Frizzled signaling in Drosophila. Cell 120: 111-122. PubMed ID: 15652486

Kunwar, P. S., et al. (2003). Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells. PLoS Biol. 1: e8. PubMed ID: 14691551

Larsen, P. H. and Yong, V. W. (2004). The expression of matrix metalloproteinase-12 by oligodendrocytes regulates their maturation and morphological differentiation. J. Neurosci. 24: 7597-7603. PubMed ID: 15342725

Larsen, P. H., et al. (2006). Myelin formation during development of the CNS is delayed in matrix metalloproteinase-9 and -12 null mice. J. Neurosci. 26: 2207-2214. PubMed ID: 16495447

Li, X, Fetter, R., Schwabe, T., Jung, C., Liu, L., Steller, H. and Gaul, U. (2021). The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation. Elife 10. PubMed ID: 34382936

McHugh, B., et al. (2004). Invadolysin: a novel, conserved metalloprotease links mitotic structural rearrangements with cell migration. J. Cell Biol. 167: 673-686. PubMed ID: 15557119

Schwabe, T., et al. (2005). GPCR signaling is required for blood-brain barrier formation in Drosophila. Cell 123: 133-144. PubMed ID: 16213218

Schwabe, T., Li, X. and Gaul, U. (2017). Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila. Biol Open 6(2):232-243. PubMed ID: 28108476

Yager, J., et al. (2001). Control of Drosophila perineurial glial growth by interacting neurotransmitter-mediated signaling pathways. Proc. Natl. Acad. Sci. 98: 10445-10450. PubMed ID: 11517334

date revised: 25 April 2024

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