Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Dev Cell. 2008 Nov;15(5):704–713. doi: 10.1016/j.devcel.2008.10.001

Heterotrimeric G proteins regulate a non-canonical function of septate junction proteins to maintain cardiac integrity in Drosophila

Peng Yi 1,*, Aaron N Johnson 1,*, Zhe Han 2, Jiang Wu 1, Eric N Olson 1,†
PMCID: PMC2736786  NIHMSID: NIHMS79550  PMID: 19000835

Summary

The gene networks regulating heart morphology and cardiac integrity are largely unknown. We previously reported a role for the heterotrimeric G protein γ subunit 1 (Gγ1) in mediating cardial-pericardial cell adhesion in Drosophila. Here we show G-oα47A and Gβ13F cooperate with Gγ1 to maintain cardiac integrity. Cardial-pericardial cell adhesion also relies on the septate junction (SJ) proteins Neurexin-IV (Nrx-IV), Sinuous, Coracle, and Nervana2, and which together function in a common pathway with Gγ1. Furthermore, Gγ1 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 non-canonical functions to maintain cardiac integrity in Drosophila. Our findings unveil the components of a previously unrecognized network of genes that couple G-protein signaling with novel structural constituents of the heart.

Introduction

Cardiogenesis is a remarkably conserved process at both the morphological and the molecular level (Bodmer and Venkatesh, 1998; Olson, 2006). The Drosophila heart, or dorsal vessel, is a linear contractile tube formed by a layer of myoepithelial cells (cardial cells). Two rows of pericardial cells (PCs) flank the cardial cells (CCs), and carry out both structural and excretory functions (Cripps and Olson, 2002). The linear dorsal vessel in Drosophila is morphologically equivalent to the vertebrate heart prior to looping and chamber formation (Cripps and Olson, 2002; Zaffran and Frasch, 2002).

The gene regulatory networks directing heart cell fate specification have been extensively studied in Drosophila (Olson, 2006), however the molecular mechanisms regulating heart tube morphogenesis remain largely unknown. We previously performed a heart-specific genetic screen and identified a novel phenotype, broken hearted (bro), in which CCs lose adhesion to PCs (Yi et al., 2006). This screen identified HMG-CoA reductase (HMGCR) and downstream enzymes in the mevalonate pathway as key regulators of CC-PC adhesion. The mevalonate pathway ultimately mediates geranylgeranylation of the heterotrimeric G protein γ-subunit Gγ1, which is required to maintain CC-PC adhesion. In zebrafish, hmgcr1b mutants show abnormal heart morphology, defects in myocardial cell migration and pericardial edema, suggesting that the role of the mevalonate pathway in heart tube morphogenesis is evolutionarily conserved (D'Amico et al., 2007). In light of the similarities in cardiogenesis between insects and vertebrates, it is likely that heterotrimeric G proteins are also targets for the mevalonate pathway during heart formation in zebrafish.

Heterotrimeric G proteins regulate a multitude of developmental processes in metazoans by acting as intracellular effectors of G protein coupled receptors (GPCRs) (reviewed in Malbon, 2005). G proteins form heterotrimers with subunits designated as 〈, ®, and γ. In the basal state, the G protein 〈 subunit is bound to GDP and associates with the G®γ subunits; binding of 〈 to ®γ prevents all three subunits from interacting with downstream effectors (e.g. adenylyl cyclase). Ligand binding to a GPCR drives a conformational change in the G protein 〈 subunit that stimulates the release of GDP. The nucleotide-free 〈 subunit then binds GTP, which is present at a higher intracellular concentration than is GDP, and GTP binding decreases the affinity of the 〈 subunit for ®γ dimer and increases its affinity for downstream effectors. The 〈 subunit also possesses intrinsic GTPase activity that hydrolyzes the bound GTP and returns the 〈 subunit to the basal conformation. Regulators of G protein Signaling (RGS) proteins modulate 〈 subunit GTPase activity. In addition, GPCR-independent activation of heterotrimeric G protein complexes has been reported (Malbon, 2005). Heterotrimeric G proteins are represented by three large protein families and these proteins specifically interact with a number of GPCRs reviewed in (Albert and Robillard, 2002). The available G protein-receptor combinations, in addition to GPCR-independent G protein activation, create a robust signal transduction system with the potential to carry out a multitude of cellular functions.

Septate junctions (SJs) are spoke and ladder-like septa that connect adjacent plasma membranes and function as diffusion barriers in the epithelia and nervous system of insects (Tepass et al., 2001). The vertebrate nervous system has paranodal SJs, however insect epithelial SJs are functionally equivalent to chordate tight junctions (Hortsch and Margolis, 2003). Although SJs and vertebrate tight junctions have divergent morphologies and molecular components, the formation and function of both junctions requires members of the Claudin protein family, arguing SJs and tight junctions are indeed analogous (Wu et al., 2004). SJ-proteins (i.e. the set of proteins required for SJ formation and function) were also identified in a screen for genes controlling tracheal tube size, highlighting a function for SJ-proteins during organogenesis (Beitel and Krasnow, 2000). Interestingly, SJ-proteins act in multiple pathways to control tracheal tube size and the role of at least one SJ-protein, the Na,K-ATPase β-subunit Nervana2 (Nrv2), is independent of its role in regulating paracellular diffusion (Paul et al., 2003). Mechanistically, the secretion of extracellular matrix regulatory proteins, including Vermiform and Serpentine, into the tracheal lumen prevents tube overgrowth and requires the function of SJ-proteins (Wang et al., 2006; Wu et al., 2007). Thus, SJ-proteins fulfill both physiological and developmental functions and their regulation is indispensable for proper organ morphogenesis.

In the present study, we sought to identify both the mediators and the targets of Gγ1 signaling during embryonic cardiogenesis in Drosophila. We find that G-oα47A and Gβ13F are the Gα and Gβ subunits that function with Gγ1 to maintain CC-PC adhesion. Mutational analysis and overexpression studies indicate that cross regulation between the Gα and Gβγ subunits, in concert with the RGS protein Loco, ensures proper heart morphology. We also identify eight SJ-proteins that mediate CC-PC adhesion, including Neurexin-IV (Nrx-IV), Sinuous (Sinu), Coracle (Cora), and Nrv2. As SJs are absent from the embryonic heart, we conclude that these SJ-proteins perform novel functions in the mature dorsal vessel. By double mutant analysis, Nrx-IV, Sinu, Cora, and Nrv2 are shown to function in a common pathway with Gγ1, and proper subcellular localization of these four proteins in the dorsal vessel is Gγ1-dependent. In addition, Nrx-IV mediates CC-CC adhesion and ensures formation of the cardiac lumen. Our results show that heterotrimeric G protein signaling maintains cardiac integrity, in part, by regulating the activities of SJ-proteins.

Results

Gβ13F and Gγ1 operate in a single pathway to promote CC-PC adhesion

At the end of Drosophila embryogenesis, cardial and pericardial cells must adhere tightly to maintain the structural integrity of the dorsal vessel (Figure 1A). The bro phenotype is characterized by a failure in CC-PC adhesion and results in the loss of cardiac function and early larval lethality (Yi et al., 2006). The bro phenotype can be readily visualized as a perturbation in the ordered expression pattern of Hand-GFP in cardial and pericardial cells; mutations in Gγ1 induce a fully penetrant bro heart phenotype (Figures 1C). In addition, a subset of Gγ1 embryos (<3%) show a severe loss of CC-CC adhesion (data not shown).

Figure 1. Gβ13F and Gγ1 function in a common pathway to regulate CC-PC adhesion.

Figure 1

Open in a new tab

(A) Schematic drawings of a stage 17 embryonic heart (dorsal view and cross section). CCs, PCs and the lymph gland are indicated by green, red and yellow color, respectively. (B-F) Hand-GFP expression in the dorsal vessel of St17 embryos. In this and subsequent figures, embryos are oriented in a dorsal view with anterior to the left. Compared to WT (B), Gγ1N159 (C), Gβ13FΔ15 (D), and Gβ13Ff261 (E) mutant embryos show CC-PC cell adhesion defects (the bro mutant phenotype, arrows). The severity of the bro phenotype in Gβ13FΔ15; Gγ1N159 embryos (F) is comparable to that of either single mutant.

Since G proteins function as heterotrimers, we sought to identify the α, β, and γ subunits that mediate CC-PC adhesion in the embryonic dorsal vessel. The Drosophila genome encodes two Gγ subunits, Gγ1 and Gγ30A, and three Gβ subunits (Table S1). Gγ30A is known to mediate phototransduction in the adult eye (Schillo et al., 2004; Schulz et al., 1999); however, no lethal mutations have been reported for Gγ30A (Wilson et al., 2008). The two deficiencies uncovering Gγ30A, Df(2L)ED680 and Df(2L)N22-3, also uncover a known regulator of pericardial cell fate, numb, thus obscuring assessment of the bro phenotype. Based on the known role of Gγ30A in phototransduction and the lack of lethal mutations in the gene, we find it unlikely that Gγ30A functions during embryonic heart development.

Of the three Gβ subunits, two null mutations in Gβ13F induce the bro phenotype with full penetrance (Figures 1D,E), whereas deficiencies uncovering either Gβ5 or Gβ76C do not give rise to the bro phenotype (data not shown). To understand if Gβ13F and Gγ1 function in the same pathway, we performed double mutant analysis. Gβ13F; Gγ1 double mutant embryos show a bro heart phenotype comparable to that of either single mutant alone (Figure 1F), suggesting that Gβ13F and Gγ1 function in a common pathway during dorsal vessel development. Taken together, our genetic results identify Gγ1 and Gβ13F as the Gγ and Gβ subunits mediating CC-PC adhesion.

G-oα47A mediates CC-PC adhesion

Among the six Gα subunits encoded in the Drosophila genome (Table S1), only G-oα47A is known to be expressed in the embryonic heart (Fremion et al., 1999; Zaffran et al., 1995). In addition, embryos homozygous for Df(2R)47A, a deficiency uncovering G-oα47A, show a disrupted dorsal vessel and lack specific heart cell types (Fremion et al., 1999). To study the role of G-oα47A in CC-PC adhesion, we examined cardiac integrity in embryos homozygous for two mutations in G-oα47A; both mutations induce the bro phenotype with full penetrance (Figures 2A,B) and phenocopy Gβ13F and Gγ1 mutants. In addition, the G-oα47A mutation induces midline-positioning defects in a subset of embryos (A.N.J. and E.N.O. unpublished data). G-oα47A; Gγ1 double mutant embryos show a bro phenotype comparable to either single mutant alone (Figure 2C), indicating that G-oα47A and Gγ1 function in a common pathway to regulate cardiac integrity. We conclude that G-oα47A is the Gα subunit that functions with Gβ13F and Gγ1 to mediate CC-PC adhesion.

Figure 2. Regulated Gα signaling maintains CC-PC adhesion.

Figure 2

Open in a new tab

Hand-GFP expression in St17 embryos. Two null mutations G-oα47A007 (A) and G-oα47A0611 (B), induce a bro heart phenotype similar to that of Gβ13F and Gγ1 mutations. (C) The bro phenotype in Gγ1 G-oα47A embryos is comparable to that of either single mutant. (D) Embryos overexpressing G-oα47A in the heart, via Hand-Gal4, phenocopy G-oα47A mutant embryos. Over-expressing either WT (E) or constituitively active G-iα65A(Q205L) (F) in the heart induces the bro phenotype. (G) Embryos over-expressing inactive G-oα47A(G203T) in the heart do not show the bro phenotype. The bro phenotype can be rescued in Gγ1-/- G-oα47A+/- embryos (H), but not in Gγ1+/- G-oα47A-/- embryos (I). (J) The null mutation locoΔ13 induces the bro phenotype. Arrows denote the bro phenotype.

Regulation of G protein signaling during cardiogenesis

In the basal state, Gα and Gβγ associate in an inactive trimeric complex; in response to upstream signals, the Gα subunit dissociates from Gβγ. The Gα subunit then activates a set of downstream pathways distinct from that of Gβγ. Deletion of Gβγ can inactivate Gβγ-dependent pathways and concomitantly hyperactivate Gα pathways (Clapham and Neer, 1997). When we over-expressed G-oα47A in the heart using the Hand-Gal4 driver, we found the same bro phenotype observed in Gβ13F and Gγ1 mutants (Figure 2D), suggesting unregulated G protein signaling disrupts cardiac integrity.

The G-oα47A gain-of-function phenotype could result from either hyperactivation of Gα pathways or the depletion of available Gβ13F/Gγ1. To distinguish between these possibilities, we first over-expressed G-iα65A, a Gα known to couple to the same downstream signaling pathways as G-oα47A (Katanaev and Tomlinson, 2006). Over-expressing G-iα65A in the heart phenocopies embryos over-expressing G-oα47A (Figure 2E), however G-iα65A mutant embryos do not show the bro phenotype (data not shown). G-iα65A can therefore activate G-oα47A-dependent pathways in the dorsal vessel but G-iα65A is not required to maintain cardiac integrity.

Over-expressing a constitutively active form of G-iα65A that does not hydrolyze GTP and cannot bind Gβγ, induced the bro heart phenotype (Figure 2F). However, over-expressing a dominant negative G-oα47A, that constitutively binds Gβγ in both the GTP- and GDP-bound state (Hatley et al., 2003), did not cause the bro defect (Figure 2G). Thus, the G-oα47A gain-of-function phenotype is caused by hyperactivating G-oα47A downstream pathways rather than by sequestering Gβγ. We conclude that Gβγ prevents ectopic activation of G-oα47A and its downstream pathways in the embryonic heart and that an appropriate level of Gα signaling maintains cardiac integrity.

To further test this hypothesis, we generated Gγ1-/-; G-oα47A+/- embryos and found that reducing one copy of G-oα47A rescued the bro phenotype in 8.3 % of Gγ1 homozygous embryos (n=3/36, Figure 2H). Conversely, the bro phenotype is fully penetrant in Gγ1+/-; G-oα47A-/- embryos (n=27; Figure 2I). These experiments strengthen the conclusion that Gγ1 regulates G-oα47A in the dorsal vessel and demonstrate that G-oα47A is epistatic to Gγ1.

RGS proteins enhance Gα GTPase activity and function as negative regulators of Gα signaling (Dohlman and Thorner, 1997). Accordingly, mutations in the RGS protein loco induced the bro phenotype with full penetrance (Figure 2J). Therefore, G-oα47A, Gβ13F, Gγ1 and loco constitute a molecular pathway that mediates CC-PC adhesion, and maintenance of cardiac integrity requires appropriate regulation of Gα downstream pathways.

Nrx-IV mediates CC-PC adhesion and operates in a common pathway with Gγ1

One unmapped bro mutant in our collection, bro6, is Df(3L)Exel6116 that uncovers 25 genes. Embryos homozygous for Df(3L)Exel6116 display the bro phenotype with 100% penetrance (Figure 3B). To positionally clone the gene within this region that regulates CC-PC adhesion, we generated transheterozygotes for Df(3L)Exel6116 and four overlapping deficiencies within the region. This analysis identified a 55kb region, housing 11 genes, that contains the bro6 gene (Figure 3A). Among the 11 genes in this region, only Neurexin-IV (Nrx-IV) and Est-6 have reported lethal alleles, however a UAS-RhoGAP68FdsRNA line is available. Two Nrx-IV mutants showed a bro phenotype comparable to that of Df(3L)Exel6116 (Figures 3B,C,E), whereas neither the Est-6 mutant nor the RhoGAP68FdsRNA expressing embryos showed a heart phenotype (data not shown). Moreover, embryos transheterozygous for Nrx-IV/Df(3L)Exel6116 displayed a bro defect indistinguishable from that of embryos homozygous for Df(3L)Exel6116 (Figures 3C,F), confirming that Nrx-IV is the bro gene uncovered by Df(3L)Exel6116. In addition to the bro phenotype, Nrx-IV mutations occasionally induce mesoderm closure defects (Figure S1), which we conclude are secondary to the dorsal closure defects previously reported for Nrx-IV mutants (Baumgartner et al., 1996).

Figure 3. Cardiac integrity requires Nrx-IV.

Figure 3

Open in a new tab

(A) Mapping bro6 to the genomic region containing Nrx-IV. Transheterozygotes were generated for Df(3L)Exel6116 (bro6) and 4 overlapping deficiencies: Df(3L)vin5, Df(3L)vin4, Df(3L)BK9 and Df(3L)F10. A “+” indicates the transheterozgotes show the bro phenotype and a “-” indicates a normal heart. The critical region, identified by dashed lines, spans 11 genes. (B-G) Hand-GFP expression in St17 embryos. The bro phenotype is observed in embryos homozygous for Df(3L)Exel6116 (B), transheterozygous for Nrx-IVEY06647/Df(3L)Exel6116 (C) and Nrx-IVEP604/Df(3L)Exel6116 (D), and homozygous for Nrx-IVEY06647 (E), Nrx-IVEP604 (F), and Gγ1N159; Nrx-IVEY06647 (G). Arrows mark the bro phenotype. (H) A subset of Nrx-IVEY06647, Nrx-IVEP604, and Gγ1N159;Nrx-IVEY06647 embryos also show mesoderm closure defects. The penetrance of each mesoderm phenotype is shown. The frequency of mesoderm closure defects is slightly enhanced by Gγ1N159. n≥30 embryos per genotype.

Since heterotrimeric G proteins regulate SJ formation in the Drosophila brain-blood barrier (Schwabe et al., 2005), we tested whether Gγ1 acts in the same pathway as Nrx-IV during embryonic heart development. In embryos that complete mesoderm closure, the bro phenotype of Gγ1; Nrx-IV double mutants is comparable to that of either single mutant alone (Figure 3G). However, Gγ1; Nrx-IV embryos show a higher frequency of mesoderm closure defects than do Nrx-IV embryos (Figure 3H). These results indicate that Gγ1 and Nrx-IV operate in a common pathway to regulate CC-PC adhesion, yet act in separate pathways during dorsal closure.

SJ-proteins maintain cardiac integrity and support cardiac function

Since SJ formation requires the interdependent function of multiple SJ-proteins, we hypothesized that additional SJ-proteins maintain CC-PC adhesion. Indeed, the bro phenotype was fully penetrant in embryos homozygous for sinuous (sinu), coracle (cora), nervana2 (nrv2), and contactin (cont) mutations (Figure 4A-E). Similar to Nrx-IV mutants, a subset of cora mutants also showed mesoderm closure defects (Figure S1), presumably due to known dorsal closure defects in cora embryos (Lamb et al., 1998). The bro phenotype was also observed at low penetrance (20-30%) in embryos homozygous for mutations in Lachesin (Figure 4F), Gliotactin (Figure 4G), and Neuroglian (Figure 4H), which also encode SJ components. Since SJs themselves are absent from the embryonic heart (Rugendorff et al., 1994), we conclude that SJ-proteins fulfill a non-canonical function outside of SJs to maintain CC-PC adhesion.

Figure 4. SJ-proteins maintain cardiac integrity and support cardiac function.

Figure 4

Open in a new tab

Hand-GFP expression in St17 embryos. (A-E) The bro phenotype is observed in embryos homozygous for sinunwu7 (A1), cora14 (B1), nrv2ZCL1649 (C1), nrv2k13315 (D), and contex956 (E). The severity of the bro phenotype in Gγ1N159 sinunwu7 (A2), Gγ1N159 cora14 (B2), and Gγ1N159 nrv2ZCL1649 (C2) embryos is comparable to that of each single mutant. (F-H) The bro phenotype is observed at low penetrance (∼20%-30%) in embryos homozygous for LacBG01462 (F), Gli1 (G), and NrgG0488b (H). (I) Injection of 0.5μM dsRNA against prc induces the bro phenotype. Arrows denote the bro phenotype. (J) Mesoderm closure defects are apparent in a subset of SJ-protein mutants. The penetrance of each mesoderm phenotype is given. Gγ1N159 moderately enhances the frequency of mesoderm closure defects. n≥30 embryos per genotype. (K) Heart rate in St17 embryos is significantly reduced in Gγ1N159, locoΔ13, Nrx-IVEY06647, contex956 and sinunwu7 embryos compared to WT. n=3 for all genotypes.

We next tested, by double mutant analysis, whether heterotrimeric G proteins and SJ-proteins function in a common pathway. Heart morphology in Gγ1/Sinu, Gγ1/Cora, and Gγ1/Nrv2 double mutant embryos was comparable to that of each single mutant, except that Gγ1/Sinu and Gγ1/Cora double mutant embryos showed a higher frequency of mesoderm closure defects (Figures 4J, S1). We conclude that Gγ1, Sinu, Cora, and Nrv2 operate in a single pathway to regulate CC-PC adhesion, but Gγ1, Sinu and Cora function in separate pathways during dorsal closure.

The functional relationship between SJ-proteins and the ECM in tracheal tube size control (Wang et al., 2006) prompted us to ask if the ECM protein Pericardin (Prc) maintains CC-PC adhesion. Prc mediates the attachment of the dorsal mesoderm to the ectoderm and prc null mutants do not complete mesoderm closure (Chartier et al., 2002). Since hypomorphic prc alleles have not been identified, we investigated the requirement of prc for CC-PC adhesion by knocking down Prc expression with double-stranded RNA. As shown in Figure S1, injecting blastoderm embryos with 5μM prc dsRNA recapitulated the mesoderm closure phenotype prc null mutants (Chartier et al., 2002). However, injecting 0.5μM prc dsRNA induced the bro phenotype (Figure 4I). As a control, injecting white dsRNA did not affect heart morphology (data not shown). These findings support the conclusion that Prc functions, at least in part, to mediate CC-PC adhesion.

To understand the effect of the bro phenotype on cardiac function, we assessed heart rate in Stage 17 embryos homozygous for mutations in Gγ1 and loco, as well as the SJ components Nrx-IV, Cont, and sinu. We found that heart rate was dramatically reduced in Gγ1 and loco embryos and that the SJ mutants had a similar reduction in heart rate (Figure 4K). These results indicate that both heterotrimeric G protein signaling and SJ-proteins are indispensable for proper cardiac performance.

Correct localization of Nrx-IV in the dorsal vessel requires Gγ1

To further investigate the non-canonical, G protein-associated function of SJ-proteins in the embryonic heart, we characterized the expression of SJ-proteins in the dorsal mesoderm. Based on our genetic results, we predicted SJ-proteins would be expressed in the embryonic heart and that Gγ1 would regulate the expression or subcellular localization of SJ-proteins. By whole mount immunostaining, we found that Nrx-IV localizes to the cell membrane of both CCs and PCs (Figure 5A). In both Gγ1 and G-oα47A embryos, Nrx-IV does not correctly localize in CCs or PCs, and Nrx-IV expression is largely undetectable in cells that have lost PC-CC adhesion (Figure 5B,C; white arrows). In addition, Prc does not accumulate at wild type levels in either Gγ1 or G-oα47A mutants (Figure 5B,C). To further characterize Nrx-IV localization in the dorsal vessel, we also made transverse sections through wild type embryos and found that Nrx-IV localizes to all membrane domains of PC and CCs (Figure 5D). Thus, G protein-mediated localization of Nrx-IV and Prc is required to maintain cardiac integrity and the Nrx-IV expression pattern suggests it may promote cell-cell adhesion throughout the dorsal vessel.

Figure 5. Gγ1-dependent localization of Nrx-IV in the dorsal vessel maintains cardiac integrity.

Figure 5

Open in a new tab

(A-C) St16 WT, Gγ1N159, and Go-α47A0611 mutant embryos co-labeled with α-Prc, α-Nrx-IV, and Hoechst. (A) In WT embryos, Nrx-IV localizes to CC and PC membranes. (B,C) Nrx-IV does not correctly localize to CC and PC membranes in Gγ1N159 and Go-α47A0611 embryos, particularly in PCs that have lost adhesion with CCs (white arrows). Prc accumulation at the PC-CC boundary is also reduced in Gγ1N159 and Go-α47A0611 mutants. (D) Transverse section of a St17 WT embryo labeled with α-Nrx-IV and Hoechst. (D1) Low magnification view showing the entire embryo oriented with dorsal to the top. Red arrows indicate position of high magnification scans. (D2) High magnification. Nrx-IV localizes to all CC and PC membrane domains. Nrx-IV signal is highest at sites of CC-PC contact. (D3,4) DIC/Nrx-IV overlay shows Nrx-IV is indeed membrane localized. (E,F) Nrx-IV in situ hybridization in St16 embryos co-labeled with α-Mef2. (E) WT embryos express Nrx-IV in Mef2-positive CCs and in neighboring Mef2-negative PCs (F). Nrx-IV expression in Gγ1N159 embryos is comparable to WT, including the PCs that lose CC adhesion (white arrowheads). (G-J) Transverse sections of Nrx-IVEP604/+ and Nrx-IVEP604 homozygous embryos at St17. (G,H) Low magnification view, and accompanying schematics, depicts the position of CCs, PCs, and the cardiac lumen. The lumen is collapsed and CCs are often misaligned in Nrx-IVEP604 mutants. (I,J) High magnification scans reveal gaps (black arrows) between CC membranes in Nrx-IV embryos. (CC) cardial cell; (PC) pericardial cell; (EC) ectoderm.

The dramatic loss of Nrx-IV expression in Gγ1 embryos opened the possibility that Gγ1 signaling may transcriptionally regulate Nrx-IV. However, by in situ hybridization, we found that Nrx-IV expression in Gγ1 embryos is comparable to wild type embryos (Figure 5E,F), even in those PCs that have lost CC adhesion. Therefore, Gγ1 signaling provides post-transcriptional regulation of Nrx-IV in the embryonic heart.

The localization of Nrx-IV to the CC luminal membrane prompted us to ask if Nrx-IV also mediates CC-CC adhesion. By EM, we find that in Nrx-IV embryos, the heart lumen is collapsed (n=8/8; Figure 5G,H), the CCs are often misaligned, and the distance between adjoining CC membranes is increased (n=6/6; Figure 5I,J). The Nrx-IV CC phenotypes we observe are similar to those reported for Slit mutants, a known regulator of CC-CC adhesion (MacMullin and Jacobs, 2006). These results further demonstrate the essential role of Nrx-IV in maintaining not only PC-CC but also CC-CC adhesion.

Correct localization of the SJ-proteins Cora, Sinu, and Nrv1/2 in the dorsal vessel is Gγ1 dependent

Our genetic studies of Cora, Sinu, and Nrv2 suggested these SJ-proteins also carry out novel functions essential for CC-PC adhesion. Similar to Nrx-IV, both Cora and Sinu localize to CC and PC membranes (Figure 6A,B). Two Nrv isoforms, Nrv1 and Nrv2, are recognized by the Nrv antibody (Sun and Salvaterra, 1995), and Nrv1/2 also localize to CC and PC membranes (Figure 6C). Since Nrv1 and Nrv2 have non-overlapping subcellular localizations in other epithelial tissues (Paul et al., 2007), the expression pattern of the SJ specific isoform, Nrv2, may be more restricted than shown.

Figure 6. Gγ1 dependent localization of SJ-proteins in the dorsal vessel.

Figure 6

Open in a new tab

St16 embryos labeled for Cora (A,D), Sinu (B,E), and Nrv (C,F) co-stained with α-Mef2 or Hoechst. In WT embryos (A-C), Cora, Sinu, and Nrv1/2 are detected in all CC and PC membrane domains. Cora and Sinu prominently localize to sites of PC-CC contact (arrowhead) and along the CC luminal domain (arrow); localization of both proteins along the membrane joining ipsilateral CCs is less pronounced. (D) In CCs of Gγ1N159 mutants, Cora fails to localize to the luminal domain and localization to sites of PC-CC contact is often interrupted. (E) Sinu localizes to the correct CC membrane domains in Gγ1N159 mutants, although the punctate nature of Sinu localization is compromised. Correct localization of Cora and Sinu in PCs is also lost in Gγ1N159 embryos. (F) In Gγ1N159 embryos Nrv1/2 localization is diffuse, particularly in misaligned CCs (open arrowhead). (CC) cardial cell (PC) pericardial cell

In Gγ1 mutant embryos, subcellular localization of Cora to the CC luminal membrane is lost and localization to sites of CC-PC adhesion is often disrupted (Figure 6D). CC localization of Sinu in Gγ1 mutants is largely unaffected, however the punctate organization of Sinu along the cell membrane is compromised (Figure 6E). In addition, proper subcellular localization of Cora and Sinu in PCs is disrupted in Gγ1 embryos. Accordingly, Nrv1/2 fails to restrict to CC and PC membranes in Gγ1 embryos (Figure 6F). Thus, correct subcellular localization of Cora, Sinu, and perhaps Nrv2 in the dorsal vessel requires Gγ1 signaling.

Gγ1 does not regulate CC polarity

Since heterotrimeric G proteins activate the planar cell polarity pathway downstream of Frizzled receptors (Katanaev et al., 2005), we asked if these G proteins regulate cardial cell polarity such that SJ-protein mislocalization could be a secondary effect to polarity loss. However, by immunostaining, heterotrimeric G proteins mutants do not show mislocalization of the CC polarity markers α-spectrin or FasIII (Figure S2). We conclude that CC polarity is not regulated by heterotrimeric G protein signaling and that this signaling pathway specifically regulates the localization of SJ proteins in CCs.

Discussion

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 bro mutant (Nrx-IV) and characterizing additional candidate genes, we discovered a non-canonical role for SJ-proteins in mediating CC-PC and CC-CC adhesion outside SJs. We found four SJ-proteins, Nrx-IV, Sinu, Cora, and Nrv2, that operate in a common pathway with Gγ1 to maintain cardiac integrity and that require Gγ1 for proper subcellular localization in the heart. Mechanistically, the presence of SJ-proteins in both CCs and PCs suggest that these proteins act in trans to maintain cell-cell adhesion in the dorsal vessel (Figure 7). We favor a model in which the extracellular domain of Nrx-IV engages in heterophilic interactions with SJ-proteins such as Neuroglian or Contactin (reviewed in Hortsch and Margolis, 2003), and that these interactions would be stabilized by ECM proteins such as Prc. Alternatively, the SJ proteins may directly interact with ECM proteins to provide a structural basis for cardiac integrity.

Figure 7. A model for G protein function in the dorsal vessel.

Figure 7

Open in a new tab

Heterotrimeric G proteins mediate SJ-protein membrane localization in CCs and PCs. To promote CC-PC adhesion, SJ-proteins on the basal membrane of CCs likely interact in trans with SJ-proteins along the juxtaposed PC membrane. Alternatively, SJ-proteins may regulate the function of, or directly interact with, ECM proteins such as Prc. Similar mechanisms are likely to ensure CC-CC adhesion and cardiac lumen formation. In the absence of heterotrimeric G proteins, SJ-proteins are mislocalized and CC-PC and CC-CC adhesion is lost. For simplicity, SJ-proteins are only shown in membrane domains participating in CC-PC/CC-CC adhesion.

GPCR function and cardiac integrity

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 et al., 2005). Although loco mutant embryos show the bro heart phenotype (Figure 2F), moody mutations do not induce a heart phenotype (data not shown). 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 (data not shown). Since our screen did not identify a GPCR that maintains cardiac integrity, we conclude 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.

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 (reviewed in Knust, 2001). During neuroblast cell division, heterotrimeric G proteins are activated by the GTPase exchange factor (GEF) Ric-8, but not by GPCRs (Afshar et al., 2004; David et al., 2005; Wang et al., 2005). However, the lethal mutation ric-8G0397 does not induce the bro phenotype (data not shown).

Gα signaling in the dorsal vessel is distinct from other tissues

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 (Schwabe et al., 2005). A similar relationship exists among heterotrimeric G proteins during asymmetric cell division in neuroblasts (Schaefer et al., 2001; Yu et al., 2003). On the other hand, sequestering Gβγ in the dorsal vessel has no effect on cardiac integrity (Figure 2F) while hyperactivating G-oα47A in the embryonic heart induces the bro phenotype (Figures 2D). We conclude 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.

G protein signaling and SJ proteins

G protein signaling regulates SJ formation in Drosophila and tight junction formation in mammalian cells. Even though SJ are analogous to vertebrate tight junctions, it is striking that G protein signaling components co-localize with both SJ and tight junction proteins (Denker et al., 1996; Saha et al., 2001; Schwabe et al., 2005). In addition, Gαs interacts with the tight junction protein ZO-1 throughout junction formation, suggesting that Gα subunits physically regulate tight junction assembly (Denker et al., 1996). Thus, septate/tight junction proteins appear to be direct targets of G proteins in both flies and vertebrates.

Although the embryonic heart lacks SJs, our 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. We propose that an appropriate level of Gα signaling mediates SJ-protein localization whereas loss or hyperactivation Gα signaling mislocalizes SJ-proteins leading to a loss in cardiac integrity.

Cell adhesion during Drosophila heart morphogenesis

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 et al. (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. Our studies suggest that a third CC membrane domain, which we refer 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.

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. We do observe the type-2 phenotype 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.

The guidance ligand Slit regulates multiple aspects of cardiogenesis in Drosophila, and mutations in slit induce type-1, type-2, and likely type-3 phenotypes (MacMullin and Jacobs, 2006; Qian et al., 2005). In addition, slit mutant embryos show mesoderm migration and CC polarity defects (Qian et al., 2005), however these defects are genetically separable from cardiac integrity defects (MacMullin and Jacobs, 2006). Slit signals through the Robo receptors and mutations in genes encoding downstream components of the Robo signaling pathway do not dominantly enhance slit mutations. On the other hand, 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 (MacMullin and Jacobs, 2006). 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.

The cellular function of SJ proteins in the dorsal vessel

SJ-proteins are functionally interdependent and localization of Sinu to SJs requires Nrx-IV, Cora, and Nrv2 (Wu et al., 2004), while Nrx-IV, Cora, Cont, and Nrg are equally interdependent for localization to SJs (Baumgartner et al., 1996; Faivre-Sarrailh et al., 2004). In addition, both Nrv2 and Nrx-IV are transmembrane proteins, and the extracellular domain of Nrv2 at least is required for SJ function (Baumgartner et al., 1996; Paul et al., 2007). Since every SJ-protein mutant we 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 Prc, to maintain cardiac integrity. A search of the Drosophila protein interaction map reveals an interaction between Prc and Sinu, supporting the latter possibility. Alternatively, SJ-proteins could be required for the formation or function of adherens junctions in the dorsal vessel.

broken hearted genes are evolutionally conserved

All of the bro genes have close vertebrate orthologs (Table S2). Since the function of mevalonate pathway genes in heart development is conserved from Drosophila to vertebrates (D'Amico et al., 2007; Edison and Muenke, 2005; Yi et al., 2006), we speculate 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 (Zolk et al., 2000). On the other hand, Sinu is a member of the Claudin protein family and even though this protein family is rather divergent (Wu et al., 2004), vertebrate Claudin-1 is required for normal heart looping in the chick (Simard et al., 2006). In addition, Claudin-5 localizes to the lateral membrane of cardiomyocytes and is associated with human cardiomyopathy (Sanford et al., 2005). Lastly, mutations in the prc ortholog, collagen alpha-1(IV), cause vascular defects in mice and humans (Gould et al., 2005). Taken together, our studies raise the possibility that heterotrimeric G proteins and tight junction proteins ensure proper vertebrate cardiovascular morphogenesis.

Experimental Procedures

Drosophila strains

The following fly stocks were used: Hand-GFP (Han et al., 2006), Gγ1N159 (Izumi et al., 2004); Gβ13FΔ15 and Gβ13Ff261 (Fuse et al., 2003); G-oα47A007 (Fremion et al., 1999); G-oα47A0611 (Katanaev et al., 2005); locoΔ13 (Granderath et al., 1999); Contex956 (Faivre-Sarrailh et al., 2004); sinunwu7 (Wu et al., 2004); Gγ1k08017, Nrx-IVEY06647, Nrx-IVEP604, nrv2k13315, nrv2ZCL1649, cora14, Gli1, LacBG01462, NrgG0488b, Df(3L)Exel6116, Df(3L)vin5, Df(3L)vin4, Df(3L)BK9 and Df(3L)F10 (Bloomington Stock Center). Overexpression studies used the following fly lines: Hand-GAL4 (Han et al., 2006); UAS-G-oα47A and UAS-G-oα47A(G203T) (Katanaev et al., 2005); UAS-G-iα65A and UAS-G-iα65A(Q205L) (Schaefer et al., 2001).

Double-stranded RNA injection

dsRNA synthesis and injection was performed as described in (Kennerdell and Carthew, 1998). dsRNAs were generated for prc and white. Hand-GFP blastoderm embryos were injected and assessed for cardiac phenotypes. Primers used to generate the templates for in vitro transcription reactions are available upon request.

Immunohistochemistry

Immunostaining of Drosophila embryos was performed as described (Yi et al., 2006), except that embryos were heat fixed as described (Wu et al., 2004) for α-Sinu immunostaining. The following primary antibodies were used: α-Nrx-IV (gift from H. Bellen), α-Sinu (gift from G. Beitel), α-Mef2 (gift from B. Paterson), α-Cora (gift from R. Fehon), α-Prc, α-Nrv, α-FasIII, and α-alpha-Spectrin (Developmental Studies Hybridoma Bank). Primary antibodies were detected with Alexa488 and Alexa633 conjugated secondary antibodies (Molecular Probes, Carlsbad, CA) except α-Nrx-IV, α-Sinu, and α-Cora which were detected with Tyramide Signal Amplification (TSA; Molecular Probes). Zenon647 (Molecular Probes) was used to detect α-Mef2 for the Cora/Mef2 double label. To image Hand-GFP, St17 embryos were dechorionated and mounted in halocarbon oil. Images were obtained with a Zeiss LSM510-Meta confocal microscope.

In situ hybridization

in situ hybridization was performed as described in (Johnson et al., 2007) except that TSA was used to detect labeled RNA probes. Nrx-IV probes were generated from the DGRC clone RE18634.

Electron Microscopy

Embryos were collected in grape agar plates for 30 min and aged to St17. Homozygous mutant embryos were identified by the absence of GFP expressed from balancer chromosomes. Heterozygous embryos were used as control. Embryos were dechorionated and manually devitellinated in 4% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer, post-fixed in 1% osmium tetroxide, and embedded in Epon–Spurr resin. Sections were stained with uranyl acetate and lead citrate. Images were gathered on a Philips CM-100 transmission electron microscope.

Heart rate quantification

St17 WT or homozygous mutants embryos bearing the hand-GFP transgene were collected and manually dechorionated. Heart rate was counted under a fluorescent microscope. For each genotype, three embryos were selected and counted. For each embryo, heart rate was measured for 2 minutes.

Supplementary Material

01

Acknowledgments

We thank F. Matsuzaki, M. Semeriva, A. Tomlinson, U. Gaul, M. Bhat, G. Beitel, and the Bloomington Stock Center for providing the fly stocks. We thank H. Bellen, G. Beitel, B. Paterson, R. Fehon, and the Developmental Studies Hybridoma Bank for antibodies. We are grateful to the UTSW Molecular Pathology Core for embryo sectioning and Mark Hatley for critical reading of the manuscript. E.N.O. was supported by grants from the NIH, the Donald W. Reynolds Foundation and the Robert A. Welch Foundation. ANJ was supported by NRSA fellowship F32GM083530.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Afshar K, Willard FS, Colombo K, Johnston CA, McCudden CR, Siderovski DP, Gonczy P. RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos. Cell. 2004;119:219–230. doi: 10.1016/j.cell.2004.09.026. [DOI] [PubMed] [Google Scholar]
  2. Albert PR, Robillard L. G protein specificity: traffic direction required. Cell Signal. 2002;14:407–418. doi: 10.1016/s0898-6568(01)00259-5. [DOI] [PubMed] [Google Scholar]
  3. Baumgartner S, Littleton JT, Broadie K, Bhat MA, Harbecke R, Lengyel JA, Chiquet-Ehrismann R, Prokop A, Bellen HJ. A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell. 1996;87:1059–1068. doi: 10.1016/s0092-8674(00)81800-0. [DOI] [PubMed] [Google Scholar]
  4. Beitel GJ, Krasnow MA. Genetic control of epithelial tube size in the Drosophila tracheal system. Development. 2000;127:3271–3282. doi: 10.1242/dev.127.15.3271. [DOI] [PubMed] [Google Scholar]
  5. Bodmer R, Venkatesh TV. Heart development in Drosophila and vertebrates: conservation of molecular mechanisms. Dev Genet. 1998;22:181–186. doi: 10.1002/(SICI)1520-6408(1998)22:3<181::AID-DVG1>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  6. Chartier A, Zaffran S, Astier M, Semeriva M, Gratecos D. Pericardin, a Drosophila type IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure. Development. 2002;129:3241–3253. doi: 10.1242/dev.129.13.3241. [DOI] [PubMed] [Google Scholar]
  7. Clapham DE, Neer EJ. G protein beta gamma subunits. Annu Rev Pharmacol Toxicol. 1997;37:167–203. doi: 10.1146/annurev.pharmtox.37.1.167. [DOI] [PubMed] [Google Scholar]
  8. Cripps RM, Olson EN. Control of cardiac development by an evolutionarily conserved transcriptional network. Dev Biol. 2002;246:14–28. doi: 10.1006/dbio.2002.0666. [DOI] [PubMed] [Google Scholar]
  9. D'Amico L, Scott IC, Jungblut B, Stainier DY. A mutation in zebrafish hmgcr1b reveals a role for isoprenoids in vertebrate heart-tube formation. Curr Biol. 2007;17:252–259. doi: 10.1016/j.cub.2006.12.023. [DOI] [PubMed] [Google Scholar]
  10. David NB, Martin CA, Segalen M, Rosenfeld F, Schweisguth F, Bellaiche Y. Drosophila Ric-8 regulates Galphai cortical localization to promote Galphai-dependent planar orientation of the mitotic spindle during asymmetric cell division. Nat Cell Biol. 2005;7:1083–1090. doi: 10.1038/ncb1319. [DOI] [PubMed] [Google Scholar]
  11. Denker BM, Saha C, Khawaja S, Nigam SK. Involvement of a heterotrimeric G protein alpha subunit in tight junction biogenesis. J Biol Chem. 1996;271:25750–25753. doi: 10.1074/jbc.271.42.25750. [DOI] [PubMed] [Google Scholar]
  12. Dohlman HG, Thorner J. RGS proteins and signaling by heterotrimeric G proteins. J Biol Chem. 1997;272:3871–3874. doi: 10.1074/jbc.272.7.3871. [DOI] [PubMed] [Google Scholar]
  13. Edison RJ, Muenke M. Gestational exposure to lovastatin followed by cardiac malformation misclassified as holoprosencephaly. N Engl J Med. 2005;352:2759. doi: 10.1056/NEJM200506303522622. [DOI] [PubMed] [Google Scholar]
  14. Faivre-Sarrailh C, Banerjee S, Li J, Hortsch M, Laval M, Bhat MA. Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function. Development. 2004;131:4931–4942. doi: 10.1242/dev.01372. [DOI] [PubMed] [Google Scholar]
  15. Fremion F, Astier M, Zaffran S, Guillen A, Homburger V, Semeriva M. The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila. J Cell Biol. 1999;145:1063–1076. doi: 10.1083/jcb.145.5.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fuse N, Hisata K, Katzen AL, Matsuzaki F. Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions. Curr Biol. 2003;13:947–954. doi: 10.1016/s0960-9822(03)00334-8. [DOI] [PubMed] [Google Scholar]
  17. Giot L, Bader JS, Brouwer C, Chaudhuri A, Kuang B, Li Y, Hao YL, Ooi CE, Godwin B, Vitols E, et al. A protein interaction map of Drosophila melanogaster. Science. 2003;302:1727–1736. doi: 10.1126/science.1090289. [DOI] [PubMed] [Google Scholar]
  18. Gould DB, Phalan FC, Breedveld GJ, van Mil SE, Smith RS, Schimenti JC, Aguglia U, van der Knaap MS, Heutink P, John SW. Mutations in Col4a1 cause perinatal cerebral hemorrhage and porencephaly. Science. 2005;308:1167–1171. doi: 10.1126/science.1109418. [DOI] [PubMed] [Google Scholar]
  19. Granderath S, Stollewerk A, Greig S, Goodman CS, O'Kane CJ, Klambt C. loco encodes an RGS protein required for Drosophila glial differentiation. Development. 1999;126:1781–1791. doi: 10.1242/dev.126.8.1781. [DOI] [PubMed] [Google Scholar]
  20. Han Z, Yi P, Li X, Olson EN. Hand, an evolutionarily conserved bHLH transcription factor required for Drosophila cardiogenesis and hematopoiesis. Development. 2006;133:1175–1182. doi: 10.1242/dev.02285. [DOI] [PubMed] [Google Scholar]
  21. Hatley ME, Lockless SW, Gibson SK, Gilman AG, Ranganathan R. Allosteric determinants in guanine nucleotide-binding proteins. PNAS. 2003;24:14445–50. doi: 10.1073/pnas.1835919100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hortsch M, Margolis B. Septate and paranodal junctions: kissing cousins. Trends Cell Biol. 2003;13:557–61. doi: 10.1016/j.tcb.2003.09.004. [DOI] [PubMed] [Google Scholar]
  23. Izumi Y, Ohta N, Itoh-Furuya A, Fuse N, Matsuzaki F. Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division. J Cell Biol. 2004;164:729–738. doi: 10.1083/jcb.200309162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Johnson AN, Burnett LA, Sellin J, Paululat A, Newfeld SJ. Defective decapentaplegic signaling results in heart overgrowth and reduced cardiac output in Drosophila. Genetics. 2007;176:1609–1624. doi: 10.1534/genetics.107.073569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Katanaev VL, Ponzielli R, Semeriva M, Tomlinson A. Trimeric G protein-dependent frizzled signaling in Drosophila. Cell. 2005;120:111–122. doi: 10.1016/j.cell.2004.11.014. [DOI] [PubMed] [Google Scholar]
  26. Katanaev VL, Tomlinson A. Dual roles for the trimeric G protein Go in asymmetric cell division in Drosophila. Proc Natl Acad Sci U S A. 2006;103:6524–6529. doi: 10.1073/pnas.0601853103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kennerdell JR, Carthew RW. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell. 1998;95:1017–1026. doi: 10.1016/s0092-8674(00)81725-0. [DOI] [PubMed] [Google Scholar]
  28. Knust E. G protein signaling and asymmetric cell division. Cell. 2001;107:125–128. doi: 10.1016/s0092-8674(01)00534-7. [DOI] [PubMed] [Google Scholar]
  29. Lamb RS, Ward RE, Schweizer L, Fehon RG. Drosophila coracle, a member of the protein 4.1 superfamily, has essential structural functions in the septate junctions and developmental functions in embryonic and adult epithelial cells. Mol Biol Cell. 1998;9:3505–3519. doi: 10.1091/mbc.9.12.3505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. MacMullin A, Jacobs JR. Slit coordinates cardiac morphogenesis in Drosophila. Dev Biol. 2006;293:154–164. doi: 10.1016/j.ydbio.2006.01.027. [DOI] [PubMed] [Google Scholar]
  31. Malbon CC. G proteins in development. Nat Rev Mol Cell Biol. 2005;6:689–701. doi: 10.1038/nrm1716. [DOI] [PubMed] [Google Scholar]
  32. Medioni C, Martine A, Zmojdzian M, Jagla K, Semeriva M. Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation. J Cell Biol. 2008;182:249–61. doi: 10.1083/jcb.200801100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Olson EN. Gene regulatory networks in the evolution and development of the heart. Science. 2006;313:1922–1927. doi: 10.1126/science.1132292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Paul SM, Palladino MJ, Beitel GJ. A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control. Development. 2007;134:147–155. doi: 10.1242/dev.02710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Paul SM, Ternet M, Salvaterra PM, Beitel GJ. The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system. Development. 2003;130:4963–4974. doi: 10.1242/dev.00691. [DOI] [PubMed] [Google Scholar]
  36. Qian L, Liu J, Bodmer R. Slit and Robo control cardiac cell polarity and morphogenesis. Curr Biol. 2005;15:2271–2278. doi: 10.1016/j.cub.2005.10.037. [DOI] [PubMed] [Google Scholar]
  37. Rugendorff A, Younossi-Hartenstein A, Hartenstein V. Embryonic origin and differentiation of the Drosophila heart. Roux's Arch Dev Biol. 1994;203:266–280. doi: 10.1007/BF00360522. [DOI] [PubMed] [Google Scholar]
  38. Saha C, Nigam SK, Denker BM. Expanding role of G proteins in tight junction regulation: Galpha(s) stimulates TJ assembly. Biochem Biophys Res Commun. 2001;285:250–256. doi: 10.1006/bbrc.2001.5154. [DOI] [PubMed] [Google Scholar]
  39. Sanford JL, Edwards JD, Mays TA, Gong B, Merriam AP, Rafael-Fortney JA. Claudin-5 localizes to the lateral membranes of cardiomyocytes and is altered in utrophin/dystrophin-deficient cardiomyopathic mice. J Mol Cell Cardiol. 2005;38:323–332. doi: 10.1016/j.yjmcc.2004.11.025. [DOI] [PubMed] [Google Scholar]
  40. Schaefer M, Petronczki M, Dorner D, Forte M, Knoblich JA. Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system. Cell. 2001;107:183–194. doi: 10.1016/s0092-8674(01)00521-9. [DOI] [PubMed] [Google Scholar]
  41. Schillo S, Belusic G, Hartmann K, Franz C, Kuhl B, Brenner-Weiss G, Paulsen R, Huber A. Targeted mutagenesis of the farnesylation site of Drosophila Ggammae disrupts membrane association of the G protein betagamma complex and affects the light sensitivity of the visual system. J Biol Chem. 2004;279:36309–36316. doi: 10.1074/jbc.M404611200. [DOI] [PubMed] [Google Scholar]
  42. Schulz S, Huber A, Schwab K, Paulsen R. A novel Ggamma isolated from Drosophila constitutes a visual G protein gamma subunit of the fly compound eye. J Biol Chem. 1999;274:37605–37610. doi: 10.1074/jbc.274.53.37605. [DOI] [PubMed] [Google Scholar]
  43. Schwabe T, Bainton RJ, Fetter RD, Heberlein U, Gaul U. GPCR signaling is required for blood-brain barrier formation in drosophila. Cell. 2005;123:133–144. doi: 10.1016/j.cell.2005.08.037. [DOI] [PubMed] [Google Scholar]
  44. Simard A, Di Pietro E, Young CR, Plaza S, Ryan AK. Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail. Mech Dev. 2006;123:210–227. doi: 10.1016/j.mod.2005.12.004. [DOI] [PubMed] [Google Scholar]
  45. Sun B, Salvaterra PM. Characterization of nervana, a Drosophila melanogaster neuron-specific glycoprotein antigen recognized by anti-horseradish peroxidase antibodies. J Neurochem. 1995;65:434–443. doi: 10.1046/j.1471-4159.1995.65010434.x. [DOI] [PubMed] [Google Scholar]
  46. Tepass U, Tanentzapf G, Ward R, Fehon R. Epithelial cell polarity and cell junctions in Drosophila. Annu Rev Genet. 2001;35:747–784. doi: 10.1146/annurev.genet.35.102401.091415. [DOI] [PubMed] [Google Scholar]
  47. Wang H, Ng KH, Qian H, Siderovski DP, Chia W, Yu F. Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins. Nat Cell Biol. 2005;7:1091–1098. doi: 10.1038/ncb1317. [DOI] [PubMed] [Google Scholar]
  48. Wang S, Jayaram SA, Hemphala J, Senti KA, Tsarouhas V, Jin H, Samakovlis C. Septate-junction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea. Curr Biol. 2006;16:180–185. doi: 10.1016/j.cub.2005.11.074. [DOI] [PubMed] [Google Scholar]
  49. Wilson RJ, Goodman JL, Strelets VB, Consortium tF. FlyBase: integration and improvements to query tools. Nucleic Acids Research. 2008;36 doi: 10.1093/nar/gkm1930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wu VM, Schulte J, Hirschi A, Tepass U, Beitel GJ. Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control. J Cell Biol. 2004;164:313–323. doi: 10.1083/jcb.200309134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wu VM, Yu MH, Paik R, Banerjee S, Liang Z, Paul SM, Bhat MA, Beitel GJ. Drosophila Varicose, a member of a new subgroup of basolateral MAGUKs, is required for septate junctions and tracheal morphogenesis. Development. 2007;134:999–1009. doi: 10.1242/dev.02785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yi P, Han Z, Li X, Olson EN. The mevalonate pathway controls heart formation in Drosophila by isoprenylation of Ggamma1. Science. 2006;313:1301–1303. doi: 10.1126/science.1127704. [DOI] [PubMed] [Google Scholar]
  53. Yu F, Cai Y, Kaushik R, Yang X, Chia W. Distinct roles of Galphai and Gbeta13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions. J Cell Biol. 2003;162:623–633. doi: 10.1083/jcb.200303174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zaffran S, Astier M, Gratecos D, Guillen A, Semeriva M. Cellular interactions during heart morphogenesis in the Drosophila embryo. Biol Cell. 1995;84:13–24. doi: 10.1016/0248-4900(96)81314-1. [DOI] [PubMed] [Google Scholar]
  55. Zaffran S, Frasch M. Early signals in cardiac development. Circ Res. 2002;91:457–469. doi: 10.1161/01.res.0000034152.74523.a8. [DOI] [PubMed] [Google Scholar]
  56. Zolk O, Kouchi I, Schnabel P, Bohm M. Heterotrimeric G proteins in heart disease. Can J Physiol Pharmacol. 2000;78:187–198. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS79550-supplement-01.pdf (87KB, pdf)

RESOURCES