G protein oalpha 47A

DEVELOPMENTAL BIOLOGY

Embryonic

G-oalpha47A, as described by Yoon (1989), codes for two classes of cDNAs (class I and class II) have been extensively documented (de Sousa, 1989; Schmidt, 1989; Thambi, 1989, and Yoon, 1989). These transcripts arise from an alternative splicing in a unique gene and differ only in the ATG-containing first exon. Three developmentally regulated transcripts (3.4kb of maternal origin; 4.2kb and 6kb, zygotic in origin) have been revealed (Wolfgang, 1991). By using specific probes, it has been shown that the class I cDNA corresponds to the 6-kb mRNA appearing after 12h of development, whereas the class II cDNA corresponds to the 4.2-kb mRNA more abundant in earlier stages. Both cDNAs encode proteins of the same size (354 amino acids) that diverge only by 7 amino acids among their 21 N₂-terminal residues. It is not known if these amino acids exert different functions (Frémion, 1999). Despite the relatively high levels of maternal G-oalpha 47A mRNA the protein cannot be detected in ovaries. Just after the completion of germband retraction, elevated levels of both Gsalpha and G-oalpha 47A are first detected in the forming neuropil of the brain and ventral ganglion. This pattern persists for the duration of embryogenesis. The neuropil staining for both Gsalpha and G-oalpha 47A persists through adult life (Wolfgang, 1991).

By using the class II cDNA as a probe, a strong expression of G-oalpha47A has been observed in preblastoderm embryos due to the presence of the 3.4-kb maternal transcript. In early stage 11 embryos, the zygotic transcript can be detected for the first time in clusters of cells within 11 segments. The cells appear to be both cardial and visceral muscle progenitor cells, because based on G-oalpha47A staining patterns in later stage 12 embryos, the cells become integrated into the conspicuous monocellular layer of cardial and visceral mesoderm cells on each side of the embryo. This assumption is further supported by G-oalpha47A expression in embryonic tissues which are unambiguously constituted of such cells, and also by the pattern of expression in tinman (tin) loss of function mutants. In tin mutants, neither heart nor visceral muscles are formed and, correlatively, G-oalpha47A expression is completely abolished in the territories from which the precursor cells for these two tissues originate. From the middle of stage 11 onwards, neuroblasts of the central nervous system (CNS) become labeled and G-oalpha47A expression persists in the neurons of the CNS in later stages of embryogenesis. In a similar way, all the neurons of the peripheral nervous system (PNS) express the G-oalpha47A mRNA from stage 12 onwards, slightly before the onset of axonogenesis. Probes for either cDNA give identical spatial patterns of expression although class I transcripts are quantitatively less abundant and are expressed later than the class II transcripts. Antibodies directed against a COOH-terminal peptide whose sequence is conserved in the alpha subunit of all G_o proteins shows that the pattern of expression of the protein is superimposable on that of the mRNA during embryogenesis. However, probably due to the presence of the protein of maternal origin, these antibodies are poorly efficient in detecting a significant signal in the cardial cells as early as do mRNA probes (Frémion, 1999).

The alpha subunits of heterotrimeric G proteins are responsible for the coupling of receptors for a wide variety of stimuli to a number of intracellular effector systems. In the nervous system of vertebrates, high levels of a specific class of G protein (Go alpha) are expressed. The alpha subunit of Go serves as a substrate for modification by pertussis toxin (PTX). Drosophila heads contain high levels of a 40-kDa PTX substrate. Modification of this protein by PTX is modulated in a manner similar to that observed for vertebrate G proteins. The PTX substrate in Drosophila is also recognized specifically by antibodies raised against peptide sequences found specifically in vertebrate Go alpha. Vertebrate Go alpha probes were used to identify a Drosophila cDNA coding for a potential PTX substrate with high sequence identity (82%) to vertebrate Go alpha. An additional cDNA coding for a related Go alpha has also been isolated. The two cDNAs differ only in the 5'-untranslated and amino-terminal regions of the protein. This observation, in addition to Northern analysis, suggests that alternate splicing may generate a variety of Go alpha-like proteins in Drosophila. In situ hybridization of specific probes to tissue sections indicates that the mRNAs coding for Go alpha-like proteins in Drosophila are expressed primarily in neuronal cell bodies and, at lower levels, in the eyes (Thambi, 1989).

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, G-oalpha47A, hybridizes to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a fusion protein recognize a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies reveal expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. G-oalpha47A message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain (Schmidt, 1989).

In order to uncover the role of G proteins in the integrative functioning and development of the nervous system, a multidisciplinary study of the G proteins present in the fruit fly has been initiated. The distribution of 3 different G protein alpha-subunits in the adult Drosophila CNS is described as determined by immunocytochemical localization using affinity-purified antibodies generated to synthetic oligopeptide sequences unique to each alpha-subunit. Western blot analysis of membranes prepared from Drosophila heads indicates that antibodies specific for the Drosophila Go alpha and Gs alpha homologs recognize the appropriate protein species predicted by molecular cloning. The Gi alpha homolog could not be detected in head membranes by Western blotting, consistent with the negligible levels of expression observed for Gi alpha on Northern blots of head mRNA. However, a Drosophila Gi alpha fusion protein could be detected by these antibodies following expression in E. coli. Immunolocalization studies revealed that the Go alpha and Gs alpha homologs are expressed at highest levels in neuropils and at intermediate levels in the cortex of all brain and thoracic ganglion areas. Only the lamina contains low levels of these alpha-subunits in the CNS. Additionally, Gs alpha appears to be associated with the cell membranes of neuronal cell bodies, while Go alpha has a more diffuse distribution, suggesting its presence in the cytoplasm as well as cell membranes. In contrast to the wide distribution of Go alpha and Gs alpha, Gi alpha has a surprisingly restricted distribution in the CNS. It is present at high levels only in photoreceptor cell terminations, glomerulae of the antennal lobes, and the ocellar retina. Little or no Gi alpha is detected in other brain regions or in the thoracic ganglion. Gi alpha, then, appears to be uniquely associated with some primary sensory afferents and their terminations, suggesting the presence of specific receptor and/or effector systems which mediate the transmission of primary sensory information in Drosophila (Wolfgang, 1990).

G proteins couple receptors for extracellular signals to several intracellular effector systems and play a key role in signaling transduction mechanisms. In particulate preparations of Drosophila melanogaster heads, only one substrate for pertussis toxin at 39-40 kd was detected. This substrate, which shows only one isoform when analysed by isoelectric focusing, was recognized by immunoblotting and immunoprecipitation techniques using a polyclonal antibody against the alpha subunit of the Go protein purified from bovine brain and can be thus considered as a Go-like protein. Antibodies obtained against a carboxy-terminal sequence of the alpha subunit of Go (but not of Gi1 or Gi2) and against an internal sequence shared by all the alpha subunits, were also able to cross-react with the alpha subunit of this protein in insects. The Go-like protein was studied in several D. melanogaster mutants, primarily in memory and learning mutants. In these mutants there is a sex-dependent enhancement in pertussis toxin-catalysed ADP-ribosylation with respect to the wild-type. This increase could be attributed in part to an increase in the alpha subunit of the Go-like protein, as revealed by immunoblotting with anti-Go alpha polyclonal antibody. This report constitutes the first evidence for the participation of a Go protein in learning and memory (Guillen, 1990).

In particulate preparations from Drosophila melanogaster embryos, only one substrate of 39,000-40,000 Da molecular weight could be ADP-ribosylated with pertussis toxin. This substrate reacts in immunoblotting and immunoprecipitation experiments with a polyclonal antibody directed against the carboxy-terminal sequence of the alpha subunit of the mammalian Go protein. The Drosophila Go alpha protein is present at all stages of embryonic development; however, its expression markedly increases after 10 h embryogenesis, a period of time during which there is an active development of axonal tracts. Immunolocalization on whole mount embryos has indicated that this protein is principally localized in the CNS and is mainly restricted to the neuropil without any labelling of the cell bodies. In contrast, all the axon tracts of the CNS appear to be highly labelled. The distribution of the Go alpha protein was also examined in several neurogenic mutants. The Go alpha protein expression is not altered in any of them but the pattern of labelling is disorganized as is the neuronal network. These results suggest a possible role for the Go protein during axonogenesis (Guillen, 1991).

Before the determination of cardiac precursors in the mesoderm, the overlying ectoderm is subdivided in segmentally repeated units partitioned into an anterior compartment (A-compartment) and a posterior compartment (P-compartment). Analysis of the expression of genes involved in the specification of mesodermal derivatives and other observations lend support to the idea that, after gastrulation, the mesoderm also becomes subdivided into segmentally repeated units, each of which consists of two separate domains. The domains that are located below the ectodermal P-compartments are subject to influences from the striped regulators eve and hh and have been termed 'P-domains' or 'eve-domains'. By contrast, the development of the metameric domains that are located below the A-compartments depends largely on the striped regulators wg and slp (sloppy-paired) and these domains have been termed 'A-domains' or 'slp-domains'. In this scheme, three basic groups of genes are at work to pattern the mesoderm either along the dorso-ventral axis (dpp and tinman) to specify the dorsal mesoderm, or along the anterior-posterior axis (wg and slp) to subdivide it into segmental units, or at defined positions to control tissue specification. For example, recent evidence suggests that wg, whose expression is restricted to striped domains in each of the A-compartments and which is required for a variety of inductive signaling events during embryonic development, is directly involved in heart formation: wg is necessary for further subdividing of the dorsal mesoderm and for specifying cardial cell fates. Elimination of the wg function shortly after gastrulation, at a time when tin becomes restricted to the dorsal mesoderm, results in the selective loss of heart progenitor cells with little effect on segmental patterning of the cuticle or other mesodermal derivatives. From these and other observations, a picture has emerged in which specification of precardiac and dorsal somatic muscle precursors requires intersections of the dorsal domains of dpp expression with the transverse stripes of the dorsal expression of wg (Frémion, 1999 and references).

However, the results presented in the Frémion (1999) study are merely consistent with some precursors of the cardial cells originating from the P-domains and subjected to the influence of hh signaling rather than that of wg. This hypothesis is somewhat difficult to verify because wg expression after gastrulation requires hh and vice versa, but, later in embryogenesis, the two signals become independent. Therefore, the expression of Tin in temperature-sensitive hh^ts2 mutant embryos submitted to a temperature shift ~5 h after egg laying (stage 10) was investigated. In these embryos, the wild-type expression of Tin in four cardial and four pericardial cells is expanded to the two cardial cells located in the P-domains of each segment. It is predicted that these two tin-expressing cells are the same as the two G-oalpha47A-expressing cells in this same domain (Frémion, 1999).

The observation that the cardial progenitor cells can be divided into two cell subpopulations is consistent with the situation in the mature heart tube in which two genetically distinct populations of cardial cells have been described (Bodmer, 1997). For example, tin as well as beta3-tubulin and several P-lacZ reporter genes from enhancer trap lines are expressed in only four cell pairs per segment among the six pairs present. In the same line, a D-mef2 enhancer element directs lacZ transcription in four cardial cell pairs per segment, consistent with a direct regulation by tin, which is expressed in these same cells (Gajewski, 1997). Interestingly, these two subpopulations of cardial cells reside, respectively, below the anterior and the posterior ectodermal parasegmental domains (Frémion, 1999). These different observations could mean that the two P-cardioblasts are specified by the inductive instruction of hh rather than by wg. However, it is not known whether the hh pathway provides a direct late cardiogenic signal or exerts its effect via suppressing the wg function in the posterior domain. This hypothesis is unlikely, since at that stage, reducing the function of hh in the epidermis does not lead to any visible effect on the wg signaling pathway. Indeed, wg expression in the dorsal epidermis is not expanded in a hh mutant. Therefore, it is predicted that hh might behave as a repressive signal for tin expression in the two P-cardioblasts. Cell heterogeneity in terms of gene expression could then be achieved along the anterior-posterior axis by an efficient cooperativity of wg and hh signals in the specification of the cardial cells (Frémion, 1999 and references).

Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).

Given the axon guidance defects in nerfin-1^null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1^null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1^null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).

Effects of Mutation or Deletion

In the nervous system of homozygous Df(2R)47A embryos, lacking the G-oalpha47A gene, longitudinal axons are often missing and important modifications are observed in the guidance and axonal growth of motoneurons. It should noted that the lola gene is also deleted in the Df(2R)47A deficiency and that mutations in this gene result in missing longitudinal axons of the CNS. Since the Df(2R)47A phenotype is slightly stronger (in terms of the phenotypic penetration) than that provoked by a loss of function of lola, this suggests a function per se for G_o in axonal growth or guidance. This was confirmed by the analysis of the G-oalpha47A⁰⁰⁷ mutation that fully complements mutations in the lola gene but continues to display a neuronal phenotype. In homozygous G-oalpha47A⁰⁰⁷ mutants, axons of the motoneurons are clearly misrouted. However, in contrast to the Df(2R)47A mutation, the longitudinal axons in the CNS are less frequently missing, but are often pinched (Frémion, 1999).

The formation of the dorsal vessel or heart in a Drosophila melanogaster embryo can be divided into three main steps: (1) the determination step allows individualization of heart precursor cells from the dorsal mesoderm. They are arranged in clusters of seven to nine cells, located in each of the eleven segments of the trunk. Preliminary observations suggest that the gene Notch could participate in the choice of fate that the cardioblasts and the pericardial cells will adopt within the cardiogenic region. In the same line, a new gene, whose expression, as revealed by a P-lacZ insertion, is initiated at gastrulation in the developing mesoderm and becomes restricted within the mesoderm to the myogenic lineages, could participate in the determination of the cardioblasts identity; (2) once the cardioblasts have separated from the dorsal mesoderm, they reorganize to form an epithelial monolayer. The gene coding for the alpha-subunit of the transduction protein Go, which is expressed in the cardioblasts shortly before this step, could be involved in this process. Indeed, mutants in the Go alpha gene are affected in the formation of the cardiac endothelium; and (3) the last step consists of the migration of the cardiac epithelium towards the dorsal midline of the embryo to form the dorsal vessel by apposition of the two layers of cardioblasts. An extracellular matrix component is specifically expressed at the surface of the dorsal vessel and could participate in the interaction between the dorsalmost ectodermal cells and the heart during this migration step (Zaffran, 1995).

Trimeric G protein-dependent Frizzled signaling in Drosophila

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. This study documents roles for the Galpha_o subunit (G_o) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. G_o is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (G_o-GTP) can signal when the receptor is removed. Thus, G_o is likely part of a trimeric G protein complex that directly tranduces Fz signals from the membrane to downstream components (Katanaev, 2005).

The evidence that Go transduces Wg signaling comes from the analysis of Go mutants, from overexpression studies, and from the epistasis experiments. These are addressed in the following discussion (Katanaev, 2005).

The inherent subviability of G_o clones prevented a frank assessment of their loss-of-function effects on Wg transduction: surviving cells likely carried perduring wild-type transcripts or protein. This offers a simple explanation for why not all G_o cells showed effects on Wg targets -- many cells still carried enough G_o function to transduce Wg. However, even given the lack of penetrance of the clones, there was a striking correspondence between G_o mutant clones and the loss of expression of Wg targets, thereby arguing that G_o gene function is critically required for Wg signal transduction (Katanaev, 2005).

Further evidence for the role of G_o in transducing Wg comes from the overexpression experiments. When G_o is overexpressed in the wing disc, clear upregulation of Wg targets is evident. If G_o achieves the upregulation of the target genes by hyperactivating the intracellular Wg transduction machinery, then abrogation of transduction downstream of G_o should nullify its effects. To this end, it was shown that the upregulation of Wg targets is arm and dsh dependent and is abolished by overexpression of sgg. Furthermore, G_o overexpression in embryos gives gain-of-function wg phenotypes that are arm dependent (Katanaev, 2005).

In arm and dsh clones (and fz, fz2 clones described below), residual Dll expression was sometimes found. This occurs in otherwise wild-type tissues and in both anterior and posterior domains of hh-Gal4; UAS-G_o wing discs and is most noticeable with dsh known for strong perdurance. However, arm and dsh clones in the regions of G_o overexpression lose Dll expression to a level comparable with clones in which G_o is not overexpressed. Thus, it is inferred that the upregulation of Wg targets induced by overexpression of G_o requires the Wg transduction pathway utilizing Dsh, Sgg, and Arm (Katanaev, 2005).

Upon activation of serpentine receptors, GDP is exchanged for GTP on Galpha, and the complex dissociates, leaving Galpha-GTP and ßγ free to signal to downstream components. To test whether G_o-GTP is able to activate the transduction pathway, a form of G_o containing an inactive GTPase was overexpressed. Overexpression of G_o-GTP induces Wg targets, indicating that G_o-GTP is a positive transducer of the pathway and that one function of Fz activation is to catalyze the release of G_o-GTP. Any signaling role of the ßγ moiety remains to be investigated. Overexpression of the G_o-GDP mutant form did not produce any effect. This form has a low affinity for GTP and could be expected to have dominant-negative effects. However, this form may not be sufficiently inactive to allow any effects on Wg transduction (and the PCP pathway) to be detected (Katanaev, 2005).

The epistasis experiments provide two key indications that G_o represents an immediate transducer of Fz signaling. (1) Dsh (previously the highest element of the transduction cascade identified downstream of the receptors) is necessary for the effects of G_o overexpression. (2) Since serpentine receptors act as exchange factors for trimeric G proteins, the effects of overexpression of a wild-type form should require the presence of the exchange factor to load and subsequently reload GTP. Conversely, once loaded with GTP, the form lacking GTPase activity (G_o-GTP) will be a long-lived activated subunit. Thus, if Fz acts as the exchange factor for G_o, then it would be expected that wild-type G_o would require Fz for its overexpression effects but that the activated form would be significantly less dependent. This is what was observed: Wg signaling is significantly rescued in fz, fz2 cells concomitantly expressing G_o-GTP as compared to those expressing wild-type G_o (Katanaev, 2005).

Given that G_o functions in the Wg transduction pathway, given that its overexpression effects require Dsh, and given that its activated form is receptor independent, the simplest explanation is that G_o functions in a trimeric G protein complex that relays signals from Fz receptors. These data do not necessarily suggest that G_o is the exclusive transducer of Wg signals: other trimeric complexes may be involved, and non-G protein-mediated signaling may also occur (Katanaev, 2005).

In the wing, the key molecular events associated with PCP occur by 30 hr APF, when Fz becomes specifically localized to the distal membrane of the cell. The localization of Fz appears to require its own signaling, since, in dsh mutants, Fz localization does not occur. A similar effect occurs when Fz is overexpressed: Fz is no longer restricted to the distal membrane. Given this complexity, the following feature of G_o can be predicted if it indeed acts as a transducer of Fz signaling. (1) Loss of G_o activity should induce PCP phenotypes; (2) Fz localization should not occur correctly when G_o signaling is compromised. In regard to these two predictions, it has been shown that (1) reduction of G_o function or G_o overexpression induces clear PCP defects and (2) Fz localization is aberrant when G_o function is down- or up-regulated. Furthermore, it has been shown that G_o itself undergoes a striking asymmetric redistribution in a fz-dependent manner (Katanaev, 2005).

G_o clones can show nonautonomous polarity defects on their proximal side, whereas fz clones show effects on their distal sides. This may indicate that G_o relays a negative signal in PCP transduction. G_o localizes proximally in polarizing cells, as does Strabismus/van Gogh, which also shows proximal nonautonomous effects. Hence, the proximal nonautonomous effects of G_o may result from it functioning negatively in the PCP pathway, from it becoming localized proximally, or from some combination of the two. A further aspect of G_o clones is the inappropriate localization of Fz at the interface of mutant and wild-type cells. It is not clear if this protein is derived from the wild-type cells, the mutant cells, or both. But it implies that the cells are in communication, and again a similar phenomenon has been described for Strabismus/van Gogh clones that may relate to the nonautonomous effects (Katanaev, 2005).

Overexpression of either G_o or G_o-GTP causes PCP defects, suggesting that one function of Fz signaling in the PCP pathway is the generation of free G_o-GTP. However, given the difficulty in distinguishing gain-of-function from loss-of-function effects, it is not possible to say whether G_o-GTP acts positively (as in the Wg pathway) or negatively. Any role for the ß/gamma dimer in transducing PCP signals remains to be established. The secreted multiple wing hairs produced by overexpression of wild-type G_o or G_o-GTP show a marked difference: the effects of wild-type G_o require the presence of the receptor (Fz), whereas the activated form does not. As for the Wg pathway described above, the most likely explanation of this observation is that Fz functions as an exchange factor for G_o (Katanaev, 2005).

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 Gαi and Galphao, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. The data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. This study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core (Schwabe, 2005).

The Drosophila nerve cord is ensheathed by a thin single-layer epithelium, which in turn is surrounded by an acellular layer of extracellular matrix material. Ultrastructural analysis has revealed that septate junctions (SJs) between the epithelial cells are responsible for the insulation of the nerve cord. Fate-mapping studies have shown that the nerve cord is enveloped by glia expressing the glial-specific marker Repo, but to date there has been no direct proof that it is these surface glia that form intercellular SJs and thus the insulating sheath. Moreover, the time course for the formation of the sheath and of the SJ-mediated seal has not been established (Schwabe, 2005).

Several assays were developed to follow the morphogenesis of the surface glial sheath. Due to the onset of cuticle formation, immunohistochemistry becomes unreliable after 16 hr of development. Live imaging of GFP-tagged marker proteins was therefore used to visualize cell shapes, in particular the actin cytoskeleton marker GFP/RFP-Moesin and the SJ marker Neuroglian (Nrg)-GFP. Nrg-GFP expressed under its own promoter and RFP-Moesin driven by repo-Gal4 are colocalized in the same cells, establishing that the SJ-forming cells are repo positive and thus conclusively demonstrating the insulating function of the surface glia. To probe the permeability of the transcellular barrier, fluorescent dye was injected into the body cavity and dye penetration into the nerve cord was quantified by determining mean pixel intensity in sample sections (Schwabe, 2005).

The surface glia are born in the ventrolateral neuroectoderm and migrate to the surface of the developing nerve cord, where they spread until they touch their neighbors (17 hr of development). The glia then join to form a contiguous sheet of square or trapezoidal cells, tiled to form three-cell corners. SJ material is visible as a thin contiguous belt by 18 hr but continues to accumulate until the end of embryogenesis. Similar to other secondary epithelia, the surface glia do not form a contiguous adherens-junction belt (zonula adherens), but only spotty, inconsistent adherens junctions were seen, as visualized by Armadillo-GFP (driven by own promoter). At 16 hr, the fluorescent dye freely penetrates into the nerve cord, but by 20 hr the nerve cord is completely sealed. The completion of the seal thus coincides with the onset of visible movements in the late embryo (Schwabe, 2005).

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

In a genome-wide screen for glial genes, using FAC sorting of GFP-labeled embryonic glia and Affymetrix microarray expression analysis, two novel GPCRs, Moody (CG4322) and Tre1 (CG3171) 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 (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).

The mevalonate pathway controls heart formation in Drosophila by isoprenylation of Gγ1

The early morphogenetic mechanisms involved in heart formation are evolutionarily conserved. A screen for genes that control Drosophila heart development revealed a cardiac defect in which pericardial and cardial cells dissociate, which causes loss of cardiac function and embryonic lethality. This phenotype resulted from mutations in the genes encoding HMG-CoA reductase, downstream enzymes in the mevalonate pathway, and G protein Gγ1, which is geranylgeranylated, thus representing an end point of isoprenoid biosynthesis. These findings reveal a cardial cell-autonomous requirement of Gγ1 geranylgeranylation for heart formation and suggest the involvement of the mevalonate pathway in congenital heart disease (Yi, 2006).

Mutations in genes controlling heart development frequently cause fatal cardiac malformations, the most common type of birth defect in humans. Because many of the mechanisms involved in heart development are evolutionarily conserved, the fruit fly Drosophila represents a powerful model for genetically dissecting this complex developmental process. The Drosophila heart, or dorsal vessel, which pumps bloodlike cells through an open circulatory system, is composed of parallel rows of contractile cardial cells (cardioblasts) tightly attached to pericardial cells; the latter perform supportive and secretory functions (Yi, 2006).

A P-element genetic screen was performed for Drosophila mutants with heart defecusing transgenic flies harboring a green fluorescent protein (GFP) transgene under control of the Hand enhancer, which is specific for cardial cells, pericardial cells, and the lymph gland—a hematopoietic organ in fruit flies. The Hand-GFP transgene allows visualization of the developing heart at single-cell resolution. Among a collection of mutants with cardiac abnormalities, a heart defect was observed in which pericardial cells dissociated from cardioblasts in the dorsal vessel at the end of embryogenesis. This phenotype was termed 'broken hearted' (bro). Five such mutants of different genetic loci are described in this study. In contrast to the wild-type dorsal vessel in which the pericardial cells are intimately associated with cardioblasts, in each of these mutants, the relative positions of pericardial cells and cardioblasts changed with each heartbeat (Yi, 2006).

The P element in the bro1 locus [l(3)01152] is located in the first exon of the hydroxymethyl-glutaryl (HMG)–coenzyme A (CoA) reductase gene (HMGCR), which is expressed in the dorsal vessel and the gonadal mesoderm, where it is required for migration of primordial germ cells (Van Doren, 1998). Mutants trans-heterozygous for HMGCR⁰¹¹⁵² and a deficiency line Df(3R)Exel9013, in which the HMGCR gene is deleted, or two EMS mutants, HMGCR^clb26.31 and HMGCR^clb11.54, showed similar, but more severe, cardiac defects than homozygous HMGCR⁰¹¹⁵² mutants. Expression of HMGCR in the heart, with the use of a Hand-GAL4 driver and a UAS-HMGCR transgene, rescued the cardiac defects in the HMGCR⁰¹¹⁵² mutant (Yi, 2006).

HMGCR controls a rate-limiting step in the conversion of HMG-CoA into mevalonate, a precursor for the synthesis of cholesterol and isoprene derivatives that modify the C termini of proteins containing a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid). In contrast to mammalian cells, Drosophila does not use the mevalonate pathway to synthesize cholesterol. Injection of embryos at the syncytial blastoderm stage with 0.1 µM mevinolin, a statin drug that lowers cholesterol level by inhibiting HMGCR activity, caused cardiac defects at stage 17 similar to those of the HMGCR mutants (Yi, 2006).

To investigate whether either of the two major isoprenoids, farnesyl pyrophosphate (farnesyl-PP) and geranylgeranyl pyrophosphate (geranylgeranyl-PP), might be required for heart formation, mutants were examined in the genes encoding geranylgeranyl pyrophosphate synthase (GGPPS) and geranylgeranyl transferase type I ß subunit (ßGGT-I), which act downstream of HMGCR and are required for the biosynthesis of geranylgeranyl-PP or transfer of geranylgeranyl-PP to protein, to find out whether they also cause cardiac defects. Indeed, GGPPS (also called qm) mutant embryos showed 100% penetrance for the bro phenotype, just as HMGCR mutants did, and at least 30% of the ßGGT-I mutants displayed the same phenotype. In contrast, two deficiency lines [Df(2L)Exel6010 or Df(3R)Exel6269] deleting either the farnesyl transferase α (CG2976) or ß (CG17565) subunit did not display similar cardiac defects. These findings suggested that the cardiac defects of HMGCR mutant embryos resulted from a failure of geranylgeranylation of a target substrate protein required for the adhesion between cardioblasts and pericardial cells (Yi, 2006).

Analysis of another bro mutant (bro4) suggested that the G protein γ subunit 1 (Gγ1), which contains a C-terminal CAAX motif, is the substrate of this geranylgeranylation modification required for heart formation. The P element in the bro4 locus l(2)k08017 is inserted into the splice donor site after the first exon of the Gγ1 gene. Gγ1 expression level was reduced by more than 50% in homozygous l(2)k08017 embryos, which suggested that l(2)k08017 is a hypomorphic mutant allele of the Gγ1 gene. Mutants trans-heterozygous for the l(2)k08017 insertion and a deficiency that deletes the Gγ1 gene [Df(2R)H3E1] or for Df(2R)H3E1 and a Gγ1 null allele showed the same cardiac defects as the homozygous l(2)k08017 embryos. Double mutants of the hypomorphic HMGCR and Gγ1 alleles showed a more severe cardiac defect than either single mutant. A fifth bro mutation was mapped to the Sar1 gene, which encodes a guanosine triphosphatase that controls budding of vesicles overlaid with coat protein complex II (COPII) from the endoplasmic reticulum (ER) to the Golgi network (Yi, 2006).

The developmental onset of cardiac defects was identical in the HMGCR, Gγ1, GGPPS/qm, ßGGT-I, and Sar1 mutants. Cardioblasts and pericardial cells were properly specified and aligned until stage 16. However, at stage 17, pericardial cells began to dissociate from the dorsal vessel. These observations suggest that these genes are required to maintain cardiac integrity. The phenotypes of the different mutants were also comparable, except for the two HMGCR EMS mutants or the HMGCR⁰¹¹⁵²/Df(3R)Exel9013 mutant, which was more severe and showed distortion of the shape of the dorsal vessel (Yi, 2006).

The final C-terminal residues of all G protein γ subunits contain a CAAX motif in which the variable amino acid X determines the type of lipid modification: If X is serine, methionine, alanine, or glutamine, the cysteine is modified by farnesylation, whereas if X is leucine or valine, it is modified by geranylgeranylation. Using an in vitro prenylation assay, it was found that Drosophila Gγ1 protein, which contains a CAAX motif of Cys-Thr-Val-Leu (CTVL), was modified by geranylgeranylation, but not by farnesylation, in agreement with the requirement of GGPPS/qm and ßGGT-I for cardiac development (Yi, 2006).

To determine directly if geranylgeranylation of Gγ1 is essential for heart development, whether wild-type and mutant forms of Gγ1 protein could rescue the cardiac defect of the Gγ1 mutant was tested. Targeted expression of wild-type Gγ1 in the heart was sufficient to rescue the cardiac defects of Gγ1 mutants, whereas mutant forms of Gγ1, in which geranylgeranylation was abolished by either a substitution of Ser for Cys⁶⁷ (Gγ1-C67S) in the CAAX box or a deletion of the CAAX box (Gγ1-δCAAX), failed to rescue the cardiac defects in Gγ1 mutants. It is concluded that geranylgeranylation of the CAAX motif of Gγ1 is required for its normal activity during Drosophila heart formation (Yi, 2006).

Lipid modification of the CAAX motif facilitates the association of proteins with membranes. To further explore how geranylgeranylation of Gγ1 affects its biological function, the subcellular localization of the Gγ1 protein was examined in Drosophila S2R⁺ cells. Wild-type Gγ1 protein was always excluded from the nucleus in S2R⁺ cells, whereas the two mutant forms of Gγ1, which were not geranylgeranylated, were located throughout the cytoplasm and nucleus. Because Gγ1 is a small protein and can enter the nucleus freely, the specific localization of wild-type Gγ1 protein to the cytoplasm likely reflects its interaction with membranous structures, which requires modification by geranylgeranylation (Yi, 2006).

In S2R⁺ cells treated with three HMGCR inhibitors (atorvastatin, mevinolin, and simvastatin), as well as HMGCR double-stranded RNA, the wild-type Gγ1 protein displayed the same abnormal subcellular distribution as the two mutant forms of Gγ1. These findings suggest that abnormal subcellular localization of Gγ1 accounts for the cardiac defects in the mevalonate pathway mutants and Gγ1 mutants. Gα has also been shown to be required at an earlier stage of heart development for proper alignment of cardioblasts (Fremion, 1999), which is distinct from the function of Gγ1 revealed here (Yi, 2006).

Cardiac defects of HMGCR or Gγ1 mutants could be completely rescued by targeted expression of UAS-HMGCR and UAS-Gγ1 transgenes, respectively, using a Hand-GAL4 driver, which directs expression in both cardioblasts and pericardial cells, or a Mef2-GAL4 driver, which is expressed in cardioblasts but not in pericardial cells. In contrast, targeted expression of HMGCR or Gγ1 using Dot-GAL4, which drives expression only in pericardial cells, failed to rescue the cardiac defects in either mutant. These results demonstrate that HMGCR and Gγ1 function specifically in cardioblasts to adhere with pericardial cells and exclude the possibility that the bro cardiac phenotype arises secondarily from general metabolic abnormalities (Yi, 2006).

HMGCR and downstream enzymes in the biochemical pathway leading to the synthesis of geranylgeranyl-PP are specifically required in cardioblasts to modify Gγ1. It is proposed that geranylgeranylation, which is required for the proper intracellular localization of Gγ1, is in turn required for generating a signal for pericardial cells to adhere to cardioblasts throughout heart formation. Indeed, Gßγ has been shown to control Golgi apparatus organization and vesicle formation during exocytosis in mammalian cells. The finding that a mutation in Sar1 causes the same cardiac phenotype as the Gγ1 mutation further supports the possibility that this collection of mutations perturbs the secretion of a factor required for maintenance of cardiac integrity. Inhibition of this pathway with statins results in cardiac defects similar to those resulting from mutations in HMGCR and downstream genes required for isoprenoid biosynthesis, which raises the possibility that congenital heart defects reportedly associated with the use of statins, which are contraindicated during pregnancy, may reflect perturbation in a similar developmental pathway (Yi, 2006).

HMGCR has also been shown to be required for recruitment of primordial germ cells (PGCs) to the gonad in Drosophila, but the protein target(s) of the mevalonate pathway that mediate this process have not been identified. Perhaps Gγ1 functions in the gonad mesoderm to guide PGC migration. It is speculated that lipid modifications mediated by the mevalonate pathway contribute to directed cell migration and subsequent cell-cell adhesion in diverse cell types. Given the conservation of cardiac developmental control mechanisms, it will be of interest to investigate the potential involvement of the mevalonate pathway in mammalian heart development and congenital heart disease (Yi, 2006).

Heterotrimeric G proteins regulate a noncanonical function of septate junction proteins to maintain cardiac integrity in Drosophila

The gene networks regulating heart morphology and cardiac integrity are largely unknown. The heterotrimeric G protein gamma subunit 1 (Ggamma1) has been shown to mediate cardial-pericardial cell adhesion in Drosophila. This study shows that G-oalpha47A and Gbeta13F cooperate with Ggamma1 to maintain cardiac integrity. Cardial-pericardial cell (CC-PC) adhesion also relies on the septate junction (SJ) proteins Neurexin-IV (Nrx-IV), Sinuous, ">Coracle, and Nervana2, which together function in a common pathway with Ggamma1. Furthermore, Ggamma1 signaling is required for proper SJ protein localization, and loss of at least one SJ protein, Nrx-IV, induces cardiac lumen collapse. These results are surprising because the embryonic heart lacks SJs and suggest that SJ proteins perform noncanonical functions to maintain cardiac integrity in Drosophila. These findings unveil the components of a previously unrecognized network of genes that couple G protein signaling with structural constituents of the heart (Yi, 2008).

The results of this study show that the heterotrimeric G proteins G-oα47A, Gβ13F, and Gγ1 function together to maintain CC-PC adhesion during the late stage of heart formation in Drosophila. By mapping a new broken hearted (bro) mutant (Nrx-IV) and characterizing additional candidate genes, a noncanonical role was discovered for SJ proteins in mediating CC-PC and CC-CC adhesion outside SJs. Four SJ proteins, Nrx-IV, Sinu, Cora, and Nrv2, operate in a common pathway with Gγ1 to maintain cardiac integrity; these proteins require Gγ1 for proper subcellular localization in the heart. Mechanistically, the presence of SJ proteins in both CCs and PCs suggests that these proteins act in trans to maintain cell-cell adhesion in the dorsal vessel. A model is favored in which the extracellular domain of Nrx-IV engages in heterophilic interactions with SJ-proteins such as Neuroglian or Contactin, and that these interactions would be stabilized by ECM proteins such as Pericardin (Prc). Alternatively, the SJ proteins may directly interact with ECM proteins to provide a structural basis for cardiac integrity (Yi, 2008).

Heterotrimeric G proteins G-oα47A/G-iα65A, Gβ13F, and Gγ1 function with the GPCR moody and the RGS protein loco to regulate SJ formation in the Drosophila brain-blood barrier (Schwabe, 2005). Although loco mutant embryos show the bro heart phenotype, moody mutations do not induce a heart phenotype. A search of the Drosophila protein interaction map reveals that the GPCR CG32447 interacts with both the SJ protein Sinu and the RGS Kermit. Kermit also interacts with Loco, suggesting that the CG32447 GPCR participates in the control of cardiac integrity. However, a deficiency uncovering CG32447 does not induce the bro phenotype. Since the screen for bro mutants, visualized as a perturbation in the ordered expression pattern of Hand-GFP in cardial and pericardial cells, did not identify a GPCR that maintains cardiac integrity, it is concluded that the GPCR regulating cardiac integrity is either pleiotropic, with an early embryonic function that precludes its identification as a regulator of cardiac integrity, or is redundant to a second GPCR in the dorsal vessel (Yi, 2008).

Alternatively, cardiac integrity may be regulated by a GPCR-independent mechanism. In neuroblasts, G-iα65A, Gβ13F, Gγ1, and loco regulate mitotic spindle orientation, protein localization, and ultimately asymmetric cell division via a GPCR-independent signaling pathway. During neuroblast cell division, heterotrimeric G proteins are activated by the GTPase exchange factor (GEF) Ric-8, but not by GPCRs (see David, 2005). However, the lethal mutation ric-8^G0397 does not induce the bro phenotype (Yi, 2008).

During blood-brain barrier formation, sequestering Gβγ or hyperactivating G-oα47A signaling in glial cells leads to SJ defects, whereas hyperactivating G-iα65A signaling does not affect SJ function. A similar relationship exists among heterotrimeric G proteins during asymmetric cell division in neuroblasts. In contrast, sequestering Gβγ in the dorsal vessel has no effect on cardiac integrity, while hyperactivating G-oα47A in the embryonic heart induces the bro phenotype. It is concluded that the bro phenotype in Gβ13F or Gγ1 mutants is caused by misregulation of G-oα47A signaling. This is in sharp contrast to the G proteins regulating blood-brain barrier formation and asymmetric cell division where Gβγ dimers activate a set of downstream effectors distinct from that of G-oα47A signals (Yi, 2008).

G protein signaling regulates SJ formation in Drosophila and tight junction formation in mammalian cells. Even though SJs are analogous to vertebrate tight junctions, it is striking that G protein signaling components colocalize with both SJ and tight junction proteins. In addition, Gα_s interacts with the tight junction protein ZO-1 throughout junction formation, suggesting that Gα subunits physically regulate tight junction assembly. Thus, septate/tight junction proteins appear to be direct targets of G proteins in both flies and vertebrates (Yi, 2008 and references therein).

Although the embryonic heart lacks SJs, the current results are consistent with the idea that SJ proteins are direct targets of G proteins in the dorsal vessel. G protein mutants phenocopy SJ-protein mutants and G proteins operate in a common pathway with SJ proteins to maintain cardiac integrity. In addition, proper localization of SJ proteins in the embryonic heart requires G protein signaling, and G proteins regulate at least one SJ protein at the posttranscriptional level. Finally, loss of G-oα47A signaling (G-oα47A mutants) and hyperactivation of G-oα47A signaling (overexpressing G-oα47A) both result in the bro phenotype; thus Gα signaling is localized to specific foci in cells of the dorsal vessel. It is proposed that an appropriate level of Gα signaling mediates SJ-protein localization, whereas loss or hyperactivation of Gα signaling mislocalizes SJ proteins leading to a loss in cardiac integrity (Yi, 2008).

Cell-cell adhesion plays an essential role during organ morphogenesis. In the Drosophila heart, cell-cell adhesion along three distinct CC membrane domains is required to maintain cardiac integrity. Medioni (2008) provide a detailed description of two CC domains participating in cell-cell adhesion: the adherent domain, positioned immediately dorsal and ventral to the cardiac lumen, promotes cell-cell adhesion between CCs on opposing sides of the heart, and the basal-lateral adherent domain, positioned along the lateral CC membrane, promotes cell-cell adhesion between neighboring CCs on one side of the heart. These studies suggest that a third CC membrane domain, referred to as the pericardial adherent domain, is positioned opposite to the luminal domain and promotes PC-CC adhesion. The loss of cell-cell adhesion along each of the three CC domains gives rise to a unique phenotype: luminal collapse (referred to hereafter as type-1), breaks between neighboring cardial cells (type-2), and loss of PC-CC adhesion (type-3), respectively. The unique nature of these three phenotypes can provide insight into the molecular pathways regulating cardiac integrity (Yi, 2008).

Loss of heterotrimeric G proteins or SJ proteins induces the type-3 (bro) phenotype, and mutations in at least one SJ-protein gene, Nrx-IV, leads to the type-1 phenotype. In addition, Sinu, Cora, and Nrv2 localize to the luminal and perhaps the adherent domains, suggesting that loss of these proteins will also cause the type 1 phenotype. The type 2 phenotype is observed in a subset of Gγ1 embryos, but not in any other heterotrimeric G protein or SJ-protein mutants. Thus, the pathways regulating cell-cell adhesion along the CC basal-lateral membrane may be distinct from those identified in this study (Yi, 2008).

The guidance ligand Slit has been shown to regulate multiple aspects of cardiogenesis in Drosophila, and mutations in slit induce luminal collapse (referred to hereafter as type-1), breaks between neighboring cardial cells (type-2), and likely loss of PC-CC adhesion (type-3) phenotypes. In addition, slit mutant embryos show mesoderm migration and CC polarity defects, however these defects are genetically separable from cardiac integrity defects. Slit signals through the Robo receptors and mutations in genes encoding downstream components of the Robo signaling pathway do not dominantly enhance slit mutations. In contrast, mutations in genes encoding integrins or integrin ligands, such as scab, mys, and Lan-A, dominantly enhance slit mutations and transheterozygous embryos show the type-2 phenotype. This study suggests that Slit activates two pathways during cardiogenesis: one pathway utilizes typical Robo signaling to regulate mesoderm migration and CC polarity while a second pathway uses atypical, or Robo-independent, signaling to regulate cell adhesion between neighboring CCs and likely between opposing CCs to promote lumen formation. Although the role of Slit in regulating PC-CC adhesion has not been studied in detail, one possibility is that Slit signals through G-oα47A/Gβ13F/Gγ1 to regulate CC-CC and even PC-CC adhesion (Yi, 2008).

SJ proteins are functionally interdependent and localization of Sinu to SJs requires Nrx-IV, Cora, and Nrv2 (Wu, 2004), while Nrx-IV, Cora, Cont, and Nrg are equally interdependent for localization to SJs. In addition, both Nrv2 and Nrx-IV are transmembrane proteins, and the extracellular domain of Nrv2 at least is required for SJ function. Since every SJ-protein mutant examined showed PC-CC adhesion defects, SJ proteins likely form interdependent complexes in PCs and CCs. The extracellular domains of SJ proteins may act in trans, either through direct interactions with SJ proteins along opposing membranes or through indirect interactions with ECM proteins such as Pericardin, to maintain cardiac integrity. A search of the Drosophila protein interaction map reveals an interaction between Pericardin and Sinu, supporting the latter possibility. Alternatively, SJ proteins could be required for the formation or function of adherens junctions in the dorsal vessel (Yi, 2008).

All of the bro genes have close vertebrate orthologs. Since the function of mevalonate pathway genes in heart development is conserved from Drosophila to vertebrates (D'Amico, 2007; Edison, 2005; Yi, 2006), it is speculated that G protein-mediated regulation of SJ proteins is also evolutionarily conserved. To date, the role of heterotrimeric G proteins in regulating vertebrate heart development has not been identified, but heterotrimeric G proteins do play a role in heart disease. In contrast, Sinu is a member of the Claudin protein family and even though this protein family is rather divergent (Wu, 2004), vertebrate Claudin-1 is required for normal heart looping in the chick. In addition, Claudin-5 localizes to the lateral membrane of cardiomyocytes and is associated with human cardiomyopathy. Lastly, mutations in the prc ortholog, collagen alpha-1(IV), cause vascular defects in mice and humans. Taken together, these studies raise the possibility that heterotrimeric G proteins and tight junction proteins ensure proper vertebrate cardiovascular morphogenesis (Yi, 2008).

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