loco

Transcriptional Regulation

To test whether CNS glia expression of loco depends on the function of pointed, beta-Galactosidase expression directed by the rC56 enhancer was assayed in a pointed mutant background. Initially expression directed by the rC56 element at early stage 12 in the progeny of the longitudinal glioblast appears to be slightly weaker in the mutant. During later stages of CNS development, a reduction in the expression level as well as in the number of cells expressing the rC56 reporter gene can be detected. In stage 16 mutant embryos, the activity of the rC56 enhancer is most prominently reduced or absent in the longitudinal glial cells, whereas A and B glial cells and the VUM glial cells appear relatively unaffected. The influence of pointed on the rC56 enhancer is less pronounced in the posteriormost 2-3 neuromeres. Furthermore, the rC56 reporter can be ectopically activated by ectopic expression of pointed P1. This suggests that the lacZ gene located in the rC56 P-element is under at least partial control of a pointed-dependent genomic enhancer element (Granderath, 1999).

In Drosophila, lateral glial cell development is initiated by the transcription factor encoded by glial cells missing. gcm activates downstream transcription factors such as repo and pointed, which subsequently control terminal glial differentiation. The gene loco has been identified as a potential target gene of pointed and is involved in terminal glial differentiation. It encodes an RGS domain protein expressed specifically by the lateral glial cells in the developing embryonic CNS. The loco promoter and the control of the glial-specific transcription pattern has been analyzed. Using promoter-reporter gene fusions, a 1.9 kb promoter element capable of directing the almost complete loco gene expression pattern has been identified. Sequence analysis suggests the presence of Gcm and Pointed DNA binding sites. Following in vitro mutagenesis of these sites their relevance in vivo has been demonstrated. The expression of loco is initially dependent on gcm. During subsequent stages of embryonic development Gcm and Pointed appear to activate loco transcription synergistically. In addition, at least two other factors appear to repress loco expression in the ectoderm and in the CNS midline cells (Granderath, 2000).

Two alternative modes are presented as to how loco transcription might be regulated. In the simple model, a linear array of transcriptional regulators results in the correct expression of loco. gcm acts on top of this cascade and activates pointed, which in turn leads to glial-specific loco expression. Alternatively, loco gene activation might be biphasic. Initially gcm concomitantly activates both loco and pointed. In a second phase, gcm and pointed act synergistically on the loco promoter to mediate high levels of glial-specific loco expression. The data favour the latter model (Granderath, 2000).

The 1.9 kb Rrk promoter element is capable of directing expression of a lacZ reporter in the complete loco expression domain. The Rrk fragment itself appears to contain more than one crucial regulatory element. The US1 construct, which overlaps the Rrk fragment, which harbors two gcm binding sites located in the 5' part of the Rrk fragment, directs glial expression resembling the expression of loco in a pointed mutant background. The 3' sequences of the Rrk fragment are found in the Nrk fragment. This promoter fragment, which harbours one gcm and one pointed binding site, is not able to confer any glial expression. Only the complete Rrk fragment is able to direct the entire loco transcriptional profile, pointing to synergistic effects of proteins binding to the 3' and 5' portions of the Rrk element. This notion is supported by the observation that pointed cannot activate the Rrk element when both Gcm binding sites GBS1 and GBS3 are deleted. Ectopic expression of either gcm or pointed alone within the neuroectoderm leads to sporadic activation of the Rrk enhancer, suggesting the presence of both gcm and pointed responsive elements. Coexpression of gcm and pointed in the rhomboid expression pattern shows two interesting results: (1) it is evident that cells within neuroectoderm activate the Rrk reporter fragment very strongly, showing that the two transcription factors act synergistically; (2) it is important to note that although comparably high levels of gcm and pointed are found in the CNS midline and the mesodermal cells, they never activate the Rrk reporter (Granderath, 2000).

Coexpression of gcm and pointed can also direct expression of the Nrk reporter. Within the Nrk fragment only one Gcm and one Pointed binding site are found, 370 bp apart. gcm is the master regulatory gene controlling lateral glial cell development. The gene pointed is not expressed in mutant gcm embryos suggesting that pointed expression depends on gcm. However, only coexpression of pointed and gcm leads to an efficient activation of the Rrk enhancer, indicating that gcm can not efficiently activate pointed transcription in the neuroectoderm. Despite the fact that pointedP1 is thought to act as a transcriptional activator it appears that cofactors such as gcm are required to allow full activation. This observation parallels results obtained in vertebrate systems, where it has been suggested that the binding of cofactors is a mechanism to relieve auto-inhibition of ETS proteins. pointed is expressed in many tissues during development and activates very different sets of genes (e.g., depending on the cells in which pointedP1 is expressed it activates tracheal, epidermal, neuronal or glial development). Thus, interaction with different tissue-specific coactivators might be an important step in selecting the appropriate downstream target genes (Granderath, 2000). Direct coactivation of glial target genes by both gcm and pointedP1 is possibly not confined to loco; the analysis of a second pointed-dependent enhancer element has revealed the presence of putative binding sites for both gcm and pointed (Granderath, S. and Klambt, C., unpublished data cited in Granderath, 2000).

The synergistic activation of loco by Gcm and Pointed could suggest that Pointed might be able to recruit or stabilize Gcm at the regulatory regions of terminal differentiation genes. This would lead to an increased expression of the respective genes but concomitantly could also disrupt the positive auto-regulatory feedback loop found for the gcm gene. This would provide a possible mechanism as to how the positive auto-regulation of gcm is terminated. How loco expression is maintained in vivo remains to be addressed (Granderath, 2000).

Terminal differentiation of glial cells is controlled by pointed. Two different isoforms are generated from the pointed locus, PointedP1 and PointedP2. They share the DNA binding domain and during embryonic CNS development they are expressed in the lateral glia (PointedP1) or the midline glia (PointedP2). Despite the common DNA binding activity, the two factors activate non-overlapping sets of target genes in the different glial cell types. The mechanism by which the selection of glial PointedP1 and PointedP2 target genes occurs appears to be complex. A simple model would be to postulate that specific, as yet unidentified cofactors are expressed either in the neuroectoderm or the CNS midline cells. However, in the midline, PointedP2 function can be substituted by PointedP1. This might be explained by postulating that PointedP1 is able to interact with a pointedP2 coactivator. Besides Gcm, additional factors appear to be required to specify PointedP1 target genes, because the coexpression of PointedP1 and Gcm in the CNS midline is not sufficient to evoke any Rrk reporter gene expression. Alternatively, the discrimination of PointedP1 and PointedP2 target genes might be mediated by transcriptional repressors. Two such proteins are known to be expressed in the CNS midline: Single minded and Abrupt. No potential Single minded binding sites were found in the Rrk construct. One potential Abrupt binding site (CTTAATTAA at position 1537-1547 of the Rrk fragment) was predicted by DNA sequence analysis. However, disruption of this site does not alter the reporter gene expression directed by the Rrk fragment in vivo. Thus, if Abrupt directly acts on the lococ1 promoter, it must bind to a different site in the Rrk fragment. Abrupt apparently represses Rrk-mediated expression (and possibly expression of other gcm-dependent genes) only in the apodemata, which might explain the muscle attachment defects observed in abrupt mutant embryos. In the CNS midline, however, the function of abrupt is not required for the repression of loco. Thus, additional experiments are required to determine which mechanisms are used in vivo to discriminate between lateral and midline glial gene expression (Granderath, 2000).

The Drosophila gene dead ringer (dri) [also known as retained (retn)] encodes a nuclear protein with a conserved DNA-binding domain termed the ARID domain (AT-rich interaction domain). dri is expressed in a subset of longitudinal glia in the Drosophila embryonic central nervous system and dri forms part of the transcriptional regulatory cascade required for normal development of these cells. Analysis of mutant embryos reveals a role for dri in formation of the normal embryonic CNS. Longitudinal glia arise normally in dri mutant embryos, but they fail to migrate to their final destinations. Disruption of the spatial organization of the dri-expressing longitudinal glia accounts for the mild defects in axon fasciculation observed in the mutant embryos. The axon phenotype includes incorrectly bundled and routed connectives, and axons that sometimes join the wrong bundle or cross from one tract to another. Consistent with the late phenotypes observed, expression of the glial cells missing (gcm) and reversed polarity (repo) genes was found to be normal in dri mutant embryos. However, from stage 15 of embryogenesis, expression of locomotion defects (loco) and prospero (pros) was found to be missing in a subset of LG. This suggests that loco and pros are targets of Dri transcriptional activation in some LG. It is concluded that dri is an important regulator of the late development of longitudinal glia (Shandala, 2003).

What is the molecular basis of the mutant phenotype found in dri mutants? Dri is a transcription factor, so the link between loss of dri function and the failure to differentiate properly is likely to be indirect, mediated through misregulation of dri targets required for normal longitudinal glial development. The most informative data came from an analysis of the position of dri in the glial transcriptional regulatory cascade. In general terms, dri activity was found to be downstream of gcm and repo, and independent of pnt and cut. It was also found to be upstream of two genes, loco and pros, which are essential for normal development of some glial cells. In this developmental context dri acts as an activator of downstream targets (Shandala, 2003).

The requirement for Dri in the activation of loco is unexpected. loco has been found to be a transcriptional target of Pnt but not of Repo, while dri expression depends on Repo and not on Pnt. It is possible that expression of loco is co-dependent on Pnt and Dri in some cells and that the reduced level of dri expression observed in repo mutants is enough to permit loco expression (Shandala, 2003).

The genetic analysis presented here strengthens the hypothesis that there are different genetic controls for different subsets of dorsal glia. For example, dri expression in all glial cells requires GCM activation, but only some of them requires Repo. The Repo-independent dri-positive cells, two per hemineuromere, appear to correspond to the A and B subperineural glia (A/B SPG). These derive from neuroglioblast NB1.1, suggesting that Repo is required for the expression of dri only in cells derived from the lateral glioblasts. Unlike dri, pnt and its downstream target loco are not expressed in the medialmost cell body glia, which do not have a lateral glioblast origin. This suggests that there are different pathways for pnt and dri induction downstream of gcm (Shandala, 2003).

At least some of these hierarchical transcriptional interactions may explain the phenotypes observed. The axon and mild positional defects of glia in dri mutants resemble phenotypes of other known late gliogenesis factors, such as those observed in pnt, repo, loco or pros embryos. It is known that early distribution of the glycoprotein Neuroglian is perturbed in pros mutant embryos. loco encodes a regulator of G-protein signalling (RGS) that has been shown to bind to a Gαi-subunit and could regulate a G-protein signalling pathway involved in LG migratory behavior. In addition, expression of the Drosophila FGF receptor Heartless in LG, and similarities between the loco and heartless mutant phenotypes, leaves open the possibility that FGF could trigger final migration of glia along the longitudinal connectives. This hypothesis is strengthened by the recent finding that subcellular redistribution of Neuroglian from the plasma membrane to cytoplasm, which normally happens during final glial migration to enwrap axon bundles, is disrupted in heartless mutants. Alternatively, it remains possible that additional targets of dri mediate the role of this gene in longitudinal glial differentiation (Shandala, 2003).

These studies add dri to the list of genes, including pnt, repo, loco and pros, that exhibit phenotypes that are much milder than those of the gcm, glide2 and Drop/Ltt genes at the head of the dorsal glia hierarchy. It appears that diversification of these downstream regulators produces different types of glial cells. Nonetheless, each plays an essential role in driving the required behavior of glial cells during CNS development. In the case of the Dri transcription factor, this role includes fine tuning the cell shape and migration characteristics of longitudinal glia that enable them to establish a normal axon scaffold (Shandala, 2003).

Protein Interactions

RGS domains directly interact with G-protein alpha subunits, displaying a remarkable degree of specificity (De Vries, 1995; Berman, 1996; Druey, 1996; Hunt, 1996; Watson, 1996). If Loco indeed functions as a regulator of G-protein signalling, the presence of a G-protein would be anticipated in the lateral glial cells. The expression of Galphas, Galphai and Galphao RNAs was examined in the embryonic nerve cord: the Galphai subunit appears to be specifically expressed in the glial cells (Wolfgang, 1991). Further evidence for the interaction of Loco and Galphai was found in a yeast two-hybrid screen. A cDNA clone of Drosophila Galphai was used as ëbaití for interacting proteins. The Gai gene was fused in frame at its N terminus to a gene encoding a LexA DNA-binding domain. Yeast that express this fusion were transformed with a library carrying Drosophila cDNAs fused to a gene for a transcriptional activation domain. Clones that encoded putative Galphai-interacting proteins were identified by the ability of the transformed yeast colonies to express a LEU2 gene that contained LexA-dependent regulatory elements and the interaction was confirmed by reintroducing the putative positive clones into yeast that carried the LexA-Gai fusion. Six non-overlapping sets of interacting clones have been identified. Four non-identical loco clones have been recovered, with C-terminal fragments of various lengths fused to the lexA gene. The longest fragment begins at residue 443 of the predicted Loco c2 protein and includes the RGS domain; the shortest encodes only 199 amino acid residues, those that extend C-terminal from residue 977 of the predicted Loco c2 protein and includes the final 43 amino acids of the conserved region D closest to the C terminus. Thus LOCO appears to be an RGS domain protein specific for Galphai (Granderath, 1999).

Locomotion defects, together with Pins, regulates heterotrimeric G-protein signaling during Drosophila neuroblast asymmetric divisions

Heterotrimeric G proteins mediate asymmetric division of Drosophila neuroblasts. Free Gßgamma appears to be crucial for the generation of an asymmetric mitotic spindle and consequently daughter cells of distinct size. However, how Gßgamma is released from the inactive heterotrimer remains unclear. This study shows that Locomotion defects (Loco) interacts and colocalizes with Galphai and, through its GoLoco motif, acts as a guanine nucleotide dissociation inhibitor (GDI) for Galphai. Simultaneous removal of the two GoLoco motif proteins, Loco and Pins, results in defects that are essentially indistinguishable from those observed in Gß13F or Ggamma1 mutants, suggesting that Loco and Pins act synergistically to release free Gßgamma in neuroblasts. Furthermore, the RGS domain of Loco can also accelerate the GTPase activity of Galphai to regulate the equilibrium between the GDP- and the GTP-bound forms of Galphai. Thus, Loco can potentially regulate heterotrimeric G-protein signaling via two distinct modes of action during Drosophila neuroblast asymmetric divisions (Yu, 2005).

Heterotrimeric G proteins have been shown to be involved in controlling distinct microtubule-dependent processes in one-cell embryos of C. elegans. Gßgamma is important for correct centrosome migration around the nucleus and spindle orientation, while Galpha subunits, GOA-1 and GPA-16, are required for asymmetric spindle positioning. Recent studies have shown that the GoLoco-motif-containing proteins, GPR1/2, act as GDIs for GOA-1 and GPA-16 to translate polarity cues, mediated by the asymmetrically localized Par proteins, into asymmetric spindle positioning in the C. elegans zygote (Colombo, 2003; Gotta, 2003; Srinivasan, 2003). In Drosophila NBs, heterotrimeric G proteins Gß13F and Ggamma1 are required for the asymmetric localization/stability of the apical components and, hence, the formation of an asymmetric spindle (Yu, 2003b). This is likely to be achieved through the generation of free Gßgamma since depletion of Gßgamma function by overexpression of wild-type Galphai/Galphao or loss of Gß13F or Ggamma1 function can lead to the generation of a symmetric and centrally placed mitotic spindle, and NBs frequently divide to produce daughter cells of similar size (henceforth referred to as 'similarsized divisions,'). Thus, generation of free Gßgamma is crucial for NB asymmetric divisions. However, it is not clear whether Gßgamma mediates spindle geometry independently of the Galpha subunit(s) or alternatively by controlling the localization of Galpha subunit(s) and/or the GoLoco proteins. Pins has previously been shown to act as a GDI to facilitate the dissociation of Gßgamma from heterotrimers by binding to and stabilizing the GDP-bound form of Galphai (GDP-Galphai). However, paradoxically, loss of pins function does not produce the severe spindle defects seen in the Gß13F or Ggamma1 mutant NBs, suggesting that the absence of the Pins GDI activity does not prevent the generation of free Gßgamma. Similarly, loss of Galphai, while causing defects in spindle orientation and the localization of the basal proteins up to metaphase, like pins loss of function, also does not cause the severe spindle asymmetry defects seen in Gß13F or Ggamma1 mutant NBs; however, it remains possible that additional Galpha subunits may be involved in this process (Yu, 2005 and references therein).

This study shows that locomotion defects (loco), a gene previously shown to be required for glial cell differentiation and dorsal-ventral patterning, encodes a novel component of the NB apical complex that exhibits both guanine nucleotide dissociation inhibitor (GDI) and GTPase-activating protein (GAP) activities for Galphai. Loco interacts with GDP-Galphai through its GoLoco motif and forms a complex with Galphai in vivo. Loco colocalizes with Galphai and Pins at the apical cortex of NBs throughout mitosis and is required for the asymmetric localization/stabilization of Pins/Galphai. Analyses of various double-mutant NBs suggest that Loco, like Pins and Galphai, functions redundantly with the Baz/DaPKC pathway in regulating spindle geometry. Interestingly, loss of both loco and pins functions leads to similar-sized divisions in the majority of NBs, similar to that seen in either Gß13F or Ggamma1 mutants, suggesting that activation of Gßgamma is mediated in a redundant manner by both Loco and Pins. These data therefore provide functional support for the idea that the activation of heterotrimeric G-protein signaling through the generation of free Gßgamma, crucial for NB asymmetric divisions, can occur via a receptor-independent mechanism by using multiple GDIs that functionally overlap. Moreover, Loco can, through its RGS domain, also function as a GAP to regulate the balance between GDP-Galphai and GTP-Galphai. Hence, both the GDI and GAP functions of Loco are important for NBs to regulate the activities of Galphai and Gßgamma (Yu, 2005).

Previous studies have shown that heterotrimeric G-protein components play important roles in NB asymmetric divisions. This study considers the issues of how heterotrimeric G-protein activation might be mediated during NB asymmetric divisions and the roles that Gßgamma, GTP-Galphai, and GDP-Galphai play in this process. Loco is shown to be a novel asymmetrically localized component of the NB asymmetric division machinery that possesses both GDI and GAP activities for Galphai. Evidence is provided that indicates that the redundant GDI activities of Pins and Loco lead to the generation of free Gßgamma, which plays a crucial role for the formation of an asymmetric mitotic spindle and daughter cells of distinct size. Based on loss-of-function phenotype, Galphai appears to play a less important role than Gßgamma in this process; however, the proper balance between the levels of GTP- and GDP-bound forms of Galphai, which may be mediated, at least in part, by the GAP activity of Loco, is crucial for the asymmetric localization of Pins and Insc. It is important to note that there may exist additional Galpha subunit(s) that might functionally overlap with Galphai in the generation of an asymmetric spindle. Therefore the possibility that Gßgamma might mediate asymmetric spindle geometry by regulating the localization Galpha subunit(s) (and GoLoco proteins) cannot be excluded at this point (Yu, 2005).

Heterotrimeric G proteins are classically known to transmit extracellular signals to targets within the cell through seven transmembrane, G-protein coupled receptors (GPCRs). Upon ligand binding, GPCR acts as a GEF to stimulate release of GDP from the Galpha subunit, which, in turn, is converted to the GTP-bound form. GTP-Galpha and Gßgamma dissociate and activate their respective effectors to initiate downstream signaling. G-protein signaling is attenuated through the hydrolysis of GTP to GDP by the GTPase activity of Galpha, which is accelerated by GAPs, which often contain an RGS domain. GDP-Galpha can reassociate with and inactivate Gßgamma (Yu, 2005).

Analyses of loss of function of Gß13F and Ggamma1 as well as gain of function of Galphai in NBs have provided compelling support for the view that free Gßgamma is required for the asymmetric localization/stability of both apical pathway components as well as the generation of asymmetric spindle and daughter cell size. Galphai is required primarily for the asymmetric localization of Pins and makes only a minor contribution in regulating spindle geometry and asymmetric daughter cell size. The mechanism by which heterotrimeric G-protein activation (generation of free Gßgamma) is mediated in NBs has been unclear. The fact that no G-protein-coupled receptors (GPCRs) have been implicated in NB asymmetric divisions, the apparent intrinsic polarity exhibited by cultured NBs, as well as the observed GDI activity associated with Pins have raised the possibility that heterotrimeric G-protein activation may occur via a receptor-independent mechanism since GoLoco-containing molecules like Pins should be able to generate free Gßgamma from the heterotrimeric complex by competing for binding to GDP-Galphai. However, loss of pins does not cause the majority of NBs to produce daughters of similar size and is therefore inconsistent with a failure to activate G-protein signaling (Yu, 2005).

This apparent contradiction is resolved by observations that indicate that receptor-independent activation of heterotrimeric G-protein signaling may be mediated through the GDI activities of both Pins and Loco. Like Pins, Loco can interact with GDP-Galphai through its GoLoco motif and form an in vivo complex with Galphai. In NBs, Loco colocalizes with Galphai and Pins at the apical cortex throughout mitosis. Removal of maternal and zygotic loco leads to delocalization of Pins/Galphai. Analysis of double mutants indicates that Loco functions redundantly with the Baz/DaPKC pathway with respect to the generation of differential daughter size. Simultaneous loss of both loco and pins results in phenotypic defects essentially indistinguishable from those seen in Gß13F or Ggamma1 loss-of-function NBs. These observations indicate that receptor-independent activation of heterotrimeric G proteins during Drosophila NB asymmetric division may be achieved through the actions of the two functionally redundant GDI activities of Pins and Loco (Yu, 2005).

In addition to its GDI activity, Loco also possesses an RGS domain that exhibits GAP activity for Galphai in vitro, suggesting that Loco can regulate Galphai via two distinct modes of action, both as a GDI and as a GAP. These studies suggest that Gßgamma, activated by the GDI activity of Pins and Loco, is crucial for NBs to produce daughters of unequal size, while the equilibrium between GDP-Galphai and GTP-Galphai, regulated, at least in part, by the GAP activity of Loco, is required for the localization of Insc/Pins/Loco at the apical cortex in NBs. When the equilibrium is shifted toward GTP-Galphai, that is, when GalphaiQ205L (the constitutively GTP-bound form) is expressed in the absence of endogenous wild-type Galphai, Pins becomes delocalized/destabilized because it requires binding to GDP-Galphai to localize to the cell cortex; however, the ability to generate an asymmetric spindle and unequal-size daughters is not compromised since Gßgamma function should not be compromised. Conversely, when the equilibrium is shifted toward GDP-Galphai, through the ectopic expression of GalphaiG204A (the constitutively GDP-bound form) in the absence of endogenous wild-type Galphai, free Gßgamma fails to be generated and defects similar to those seen in Gß13F or Ggamma1 loss of function result (Yu, 2005).

While the Loco-associated GAP activity can facilitate the conversion of GTP-Galphai to GDP-Galphai in NBs, how might the reverse reaction be catalyzed without invoking the involvement of a GPCR-associated GEF activity? A possible nonreceptor GEF that can fulfill this role may be the Drosophila homolog of the mammalian Ric-8A (Synembrin). Mammalian Ric-8A has been shown to act as a nonreceptor GEF for Galphao, Gq, and Galphai₁ subunits. Ric-8A is evolutionarily conserved from worm to mammals. More recent reports on C. elegans RIC-8 suggest that it functions as a GEF to regulate asymmetric divisions in the zygote for the Galpha subunits (GOA-1 and GPA-16). The fly homolog, DmRic-8, is indeed able to associate with Galphai and is involved in NB asymmetric divisions (Yu, 2005).

While receptor-independent activation of heterotrimeric G-protein signaling appears to be a mechanism conserved between fly and nematode, there are clear differences between the two systems. In the nematode zygote, previous studies have suggested that the Galpha subunits, GOA-1 and GPA-16, are required for generation of a net pulling force from the posterior cortex that leads to the displacement of the mitotic spindle toward the posterior cortex. Either (possibly both) of the GoLoco/GPR motif proteins, GPR1/2, which are enriched at the posterior pole of the zygote (Colombo, 2003; Gotta, 2003), can act as GDIs to asymmetrically activate heterotrimeric G-protein signaling. The Galpha subunits and GPR1/2 both appear to act downstream of the PAR proteins and their inactivation using RNAi results in identical spindle phenotypes that resemble those seen in par-2 mutants for which a reduction in cortical spindle forces have been directly demonstrated (Colombo, 2003; Gotta, 200). More recently, it has been reported that loss of ric-8 function also disrupts the movement of the posterior centrosome, suggesting that RIC-8 acts in the same pathway as GPR-1/2 to establish Galpha-dependent force generation, whereas loss of function of rgs-7, encoding a GAP protein for GOA-1, leads to overly vigorous posterior spindle rocking and more exaggerated size difference between two daughter cells, indicating that Galpha passes through the GTP-bound state during its activity cycle to regulate the force in one-cell-stage nematode embryos. In contrast, Gßgamma does not appear to regulate spindle displacement in the worm zygote (Yu, 2005).

For Drosophila NBs, spindle geometry and displacement appear to be regulated to a large extent through Gßgamma activation by the GoLoco proteins Loco and Pins. The spindle defects associated with loco/pins double loss-of-function NBs resemble those seen in the Gß13F and Ggamma1 mutants. However, it is clear that in Gß13F and Ggamma1 mutants there is a small degree of residual asymmetry in the size of the NB daughters; this residual size difference can be removed by the additional loss of baz function. There is no evidence implicating a major role for Galphai in spindle asymmetry since loss of Gi has relatively mild effects. However, the possibility that multiple Galpha subunits redundantly regulate NB spindle geometry cannot be ruled out (Yu, 2005).

Furthermore, in contrast to the C. elegans zygote where heterotrimeric G-protein signaling acts downstream of the PAR polarity cues, the precise hierarchical relationship between the heterotrimeric G proteins and the PAR proteins in Drosophila NBs is more complex. Some observations can be interpreted, at least formally, to suggest that free Gßgamma acts upstream of the apical components, since mutations in Gß13F and Ggamma1 cause delocalization of Pins/Loco/Galphai and affect the stability (intensity) of the Baz and DaPKC apical crescents. However, reduced levels of Baz and DaPKC can nevertheless asymmetrically localize and maintain residual levels of asymmetry despite the loss of free Gßgamma, suggesting that some aspects of NB asymmetry and PAR polarity cues act in parallel or upstream of heterotrimeric G proteins. This study provides evidence that in Drosophila NBs, both Loco and Pins contribute toward the generation of free Gßgamma and the asymmetric localization of Pins/Loco/Galphai depends not only on Gßgamma but also the right balance of GDP-Galphai and GTP-Galphai. It remains to be seen whether in NBs Gßgamma mediates the formation of an asymmetric spindle by regulating Galpha subunits (Yu, 2005).

DEVELOPMENTAL BIOLOGY

Embryonic

Two enhancer trap lines, 3-109 and rC56, were selected based on their specific beta-Galactosidase expression in the lateral CNS glia. Both lines show identical beta-Galactosidase expression patterns and carry a P-element insertion at the cytological position 94B/C. In embryos carrying the rC56 enhancer trap insertion, first beta-Galactosidase expression can be detected in early stage 12 in cells which, based on their position, appear to be the progeny of the lateral glioblast. Interestingly, at this early stage these cells appear to be already different from surrounding cells. The anterior pair of progeny expresses elevated levels of beta-Galactosidase. As CNS development continues, these cells migrate medially and divide. By the end of embryogenesis, most glial cells except the midline glia express beta-Galactosidase. In addition, beta-Galactosidase expression can be detected in the dorsal leading edge cells in the lateral ectoderm (Granderath, 1999).

The mapping of exons by restriction analysis and genomic sequencing reveals two different loco variants differing in their 5' ends (transcripts c1 and c2). In situ hybridisation experiments with transcript-specific digoxigenin-labelled cDNA probes show that both LOCO RNA classes are expressed in the embryo. loco-c1 transcription is very weak and is detected only after prolonged incubation (6-12 hours) in the staining solution. Using a 200 bp loco-c1- specific probe, expression can be first detected in late stage 12 embryos. In stage 16 embryos, loco-c1 RNA is found in the dorsal leading edge cells in the lateral ectoderm, in the tracheal cells and in the lateral glial cells within the CNS. Except for loco expression in tracheal cells, this corresponds well with the beta-Galactosidase expression pattern observed for the two P-element insertions in the loco gene. loco-c2 transcripts are found only in scattered cells in the lateral ectoderm. Based on their position, these cells might correspond to PNS progenitor cells. No expression can be detected in the CNS (Granderath, 1999).

Effects of Mutation or Deletion

Two non-complementing lethal mutations, loco^delta13 and loco^delta293, were recovered using P-element excision. In loco^delta13, the proximal deletion breakpoint lies within the P-element leaving the lacZ gene intact. However, the relative level of beta-Galactosidase expression in different glial cells appears to be altered and, in loco^delta13 embryos, the longitudinal glial cells express only low levels of beta-Galactosidase. This indicates that glia-specific enhancer elements reside upstream of the rC56 enhancer trap insertion whereas a transcriptional activator acting specifically in the longitudinal glial cells must reside 3' of the rC56 insertion. The breakpoint in loco^delta13 was cloned and is at least 7 kb downstream of the loco gene. A small inversion as well as a deletion of about 2 kb of genomic sequence is associated with the loco^delta293 allele. Here, putative promotor sequences as well as the first exon are deleted. Both mutations are homozygous embryonic lethal. Additional loco alleles were obtained following EMS mutagenesis. These additional mutations are lethal in trans to both loco excision mutations at 29ƒC. Based on the complementation analyses, they were placed into an allelic series. At 25ƒC, loco M1 (but none of the other EMS-induced alleles) produces adult escapers when in trans to loco^delta133 or loco^delta293 . About 10% of the expected numbers of transheterozygous adults appear. They often fail to eclose from the opened puparium. Eclosed flies show a paralytic phenotype and drop into the food and die. If such flies are rescued from the food, they show a severe impairment of spontaneous locomotor activity and display a ëshakingí phenotype. Response to mechanical stimulation (e.g. after stimulation of thoracic bristles) is weak in loco^M1/loco^delta29 and undetectable in loco^M1/loco^delta13, indicating that D13 is a stronger allele than D293. Similar phenotypes, albeit with lower expressivity, are seen in flies heterozygous for loco^M1 and other EMS-induced alleles at lower temperatures. All adult escapers die after a maximum of 2 days (Granderath, 1999).

At least two transcripts of loco are expressed in oogenesis: (1) loco-c2 is observed in the anterior-dorsal follicle cells and is downstream of the epidermal growth factor receptor signaling pathway, initiated in the oocyte; (2) loco-c3 is a new transcript of loco that is expressed in the nurse cells from stage 6 onwards. Disrupting loco in follicle cells results in ventralized eggs, while disrupting loco in nurse cells results in short eggs, due to defective dumping of the nurse cell cytoplasm into the oocyte (Pathirana, 2002).

The observed egg phenotypes laid by loco³⁷¹ homozygous females suggest a role for loco in DV axis formation of the egg. This data was corroborated by heatshock induced anti- sense-loco experiments. However, the mutant analysis was not straightforward. Although molecular studies clearly showed that mutants had been generated in the loco gene and there were no other P-elements in the stocks, the complementation analysis did not show a more severe phenotype when newly generated mutants were crossed to the existing deficiencies in the region. loco is a large gene with several differently spliced forms, which would suggest a complex mechanism of gene regulation. Preliminary analysis with antibodies has also shown there are different isoforms of the protein present at distinct developmental stages suggesting different roles for different protein isoforms. As a negative regulator of G-protein levels, the types of protein expressed would be critical in maintaining equilibrium in signaling systems. All these factors could contribute to the observed behavior of loco³⁷¹ which has a small insertion and retains rearranged parts of the P-element in the loco gene. This would be likely to interfere with expression of some transcripts but not others. This misregulation would lead to unusual complementation analysis, since having two copies of a gene that is expressed in the wrong cells or at the wrong time, or as the wrong isoform is likely to be more developmentally disruptive than a single copy. This would explain why homozygotes are mostly lethal and hemizygotes are not (Pathirana, 2002).

Heatshock antisense results were crucial for interpreting how the different mutant phenotypes were generated. The different heatshock lines generate transcripts in different cell types. This allowed loco to be disrupted separately in the germ-line and follicle cells. loco-c3 is expressed in the nurse cells and, when disrupted, results in a dumpless phenotype resulting in smaller than normal eggs being laid. When loco-c2 is disrupted in the anterior-dorsal follicle cells, this results in a range of dorsal defects in the eggs. The anterior-dorsal egg defects vary from dorsal appendages, which are fused at the base, to appendages fused along the whole length, to completely absent appendages. These results suggest a role for G-protein signaling in DV pattern formation in oogenesis (Pathirana, 2002).

The role of RGS proteins is to negatively regulate G-protein signaling. It seems likely that G-protein signaling has a role in DV axis formation. In the absence of loco there is a loss of DV polarity in the egg and embryo. This phenotype is similar to the one induced by Gurken mutants and suggests that in the absence of loco repression EGFR signaling occurs. Since RGS genes negatively regulate G-protein signaling, one can assume that in wild type ovaries G-protein signaling inhibits EGFR signalling but the presence of loco prevents this inhibition in anterior-dorsal follicle cells. This adds to the complexity of regulation of the EGFR pathway and suggests that the tight regulation or modulation of EGFR is critical for the correct sequence of morphological events to occur in the specification of follicle cell fates along the DV axis with time (Pathirana, 2002).

How does loco fit into the existing EGF pathway? loco is downstream of Egfr/torpedo in the follicle cells and appears to be activated both at high and moderate levels of torpedo activation. loco is downstream of pointed, a target of Egf signaling. loco has been identified in a screen for genes downstream of pointed in specific subsets of cells in the central nervous system (CNS). pointed P1 and P2 are expressed in the anterior-dorsal follicle cells in oogenesis. The expression of pointed in oogenesis is dynamic, expression first being observed in the germarium, then later at stage 8, downstream of torpedo, in the posterior follicle cells and again at stage 10, also downstream of torpedo, in anterior-dorsal follicle cells. Since pointed is observed in anterior-dorsal follicle cells in a similar pattern to loco at stage 10, it is possible that loco is downstream of pointed at this particular stage of oogenesis. The relationship between loco and pointed was analyzed in these cells. Using a pointed 1/UAS sense fly line, expression of pointed was driven in all the follicle cells using a T155 GAL4 driver. In situ hybridization to RNA in the GAL4/UAS-pointed ovaries, using a pointed probe, clearly shows high levels of pointed expression in all the follicle cells that cover the oocyte at stage 10. The expression of loco-c2 was examined in egg chambers where pointed was being ectopically expressed. A normal spatial distribution was seen in anterior-dorsal follicle cells at stage 10, though levels of expression were somewhat reduced. This reduction in expression is not uniform over the anterior-dorsal region, with the anterior-most follicle cells maintaining their normal level of expression. At later stages the expression pattern is slightly different from wild type, since there is a patch of cells expressing loco in the dorsal position that has not migrated as far anteriorly as would be expected at this stage. This is due to overexpression of pointed in oogenesis resulting in failure to make dorsal appendages. This suggests that cells normally expressing loco do not migrate as far as in wild type egg chambers. This experiment shows that there is not a simple relationship between loco and pointed. Ectopic pointed expression does disrupt the normal loco-c2 expression pattern at stage 10, with its level of expression dropping in the anterior-dorsal follicle cells, except in the anterior-most follicle cells. This indicates that loco-c2 is downstream of pointed, although not directly (Pathirana, 2002).

The DV defects in twist expression observed in the embryos of eggs laid by fly line loco³⁷¹ help clarify the role of loco in oogenesis, showing that disruption of maternal loco in oogenesis results in the disruption of native twist expression, a marker for ventral cell fate, and clear DV defects are observed in the embryos that fail to hatch, as many fail to form normal guts. The range of patterning defects observed are related to the disruption of native twist expression. This suggests that maternally expressed loco is required for normal embryonic development. It cannot be distinguished if the failure of embryos to develop properly and the observed defects in twist expression in the early embryo result from the mutant follicle cells affecting the developing egg chamber and oocyte or if the maternal contribution of loco from the nurse cells deposited in the egg is being needed for zygotic development of the D/V axis. However, the twist expression observed only in the activation of the embryo does suggest that disruption of loco in anterior-dorsal follicle cells may affect embryonic patterning. Dorsal follicle cell identity is determined as the follicle cells migrate posteriorly over the egg chamber, coming into contact with the localized gurken signal over the oocyte nucleus. If EGFR signaling is disrupted in follicle cells during the course of this migration, an embryo that has abnormal D/V patterning along only part of its AP axis would result (Pathirana, 2002).

The small egg phenotype observed in both the mutant lines and heatshock antisense lines indicates that loco is required for cytoplasmic dumping from the nurse cells to the oocyte. Several mutants have been identified that are involved in this process, and the phenotypes can be attributed to two defective processes. The first is the failure of the ring canals to grow, restricting the flow of cytoplasm from the nurse cells to the oocyte. The second is a change in the actin cytoskeleton, with the nurse cells failing to centralize the nurse cell nuclei. When this happens the nuclei block the ring canals, preventing dumping. Which process requires loco during nurse cell dumping remains to be determined (Pathirana, 2002).

Thus, two roles for the loco gene in oogenesis have been identified; cytoplasmic dumping and DV axis formation. Goalpha mRNA is present in oocyte and nurse cells and Gsalpha protein in follicle cells; Gialpha mRNA is present in nurse cells and the oocyte, with Gialpha protein present in stage 10 anterior-dorsal follicle cells and the oocyte. The expression patterns of the different Galpha subunits correlate with the expression patterns of the loco isoforms, further suggesting a role for G-protein signaling in nurse cell dumping and a link between G-protein signaling and the EGFR pathway in follicle cells (Pathirana, 2002).

GPCR signaling is required for blood-brain barrier formation in Drosophila

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 Galphai 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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) 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 PNS 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 (Schwab, 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 (Schwab, 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 (Schwab, 2005).

Protein expression and distribution of the GPCR signaling components were examined in greater detail in third-instar larval nerve cords. By this stage, the surface glia have doubled in size and show robust protein expression of GPCR signaling and SJ components (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 (Schwab, 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 made at the light- and electron-microscopic levels (Schwab, 2005).

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date revised: 15 April 2007

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