InteractiveFly: GeneBrief

moody: Biological Overview | References

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 Nervana2, 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).

Search PubMed for articles about Drosophila Moody

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 Citation: 16213219

Beckervordersandforth, R. M., Rickert, C., Altenhein, B. and Technau, C. 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 Citation: 18296030

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

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

Katanaev, V. L., et al. (2005). Trimeric G protein-dependent Frizzled signaling in Drosophila. Cell 120: 111-122. PubMed Citation: 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 Citation: 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 Citation: 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 Citation: 16495447

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

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

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 Citation: 11517334

date revised: 12 May 2009

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.