InteractiveFly: GeneBrief

G protein β-subunit 13F: Biological Overview | References

The function of an organ relies on its form, which in turn depends on the individual shapes of the cells that create it and the interactions between them. Despite remarkable progress in the field of developmental biology, how cells collaborate to make a tissue remains an unsolved mystery. To investigate the mechanisms that determine organ structure, this work studied the cells that form the dorsal appendages (DAs) of the Drosophila eggshell. These cells consist of two differentially patterned subtypes: roof cells, which form the outward-facing roof of the lumen, and floor cells, which dive underneath the roof cells to seal off the floor of the tube. This paper presents three lines of evidence that reveal a further stratification of the DA-forming epithelium. Laser ablation of only a few cells in the anterior of the region causes a disproportionately severe shortening of the appendage. Genetic alteration through the twin peaks allele of tramtrack⁶⁹ (ttk^twk), a female-sterile mutation that leads to severely shortened DAs, causes no such shortening when removed from a majority of the DA-forming cells, but rather, produces short appendages only when removed from cells in the very anterior of the tube-forming tissue. Additionally it was shown that heterotrimeric G-protein function is required for DA morphogenesis. Like TTK⁶⁹, Gβ13F is not required in all DA-forming follicle cells but only in the floor and leading roof cells. The different phenotypes that result from removal of Gβ13F from each region demonstrate a striking division of function between different DA-forming cells. Gβ mutant floor cells are unable to control the width of the appendage while Gβ mutant leading roof cells fail to direct the elongation of the appendage and the convergent-extension of the roof-cell population (Boyle, 2010).

Much of the study of developmental biology has focused on understanding the mechanisms by which cells become selected to perform various roles in the formation of structures. Classically, this patterning involves changes in gene expression that often presage further changes in form and behavior. For example, during oogenesis in Drosophila, a uniform epithelium of follicle cells surrounds a cluster of 16 germline-derived cells composed of 15 nurse cells and one oocyte. By stage 10 (S10, of 14 stages) signaling between these cell types has established a polarized microtubule network within the oocyte and has also defined distinct domains within the follicular epithelium. Midway through S10 (at the onset of S10B), the follicle cells over the oocyte appear indistinguishable from each other at a morphological level, but have already become patterned into subregions destined for different functions. Most cells will secrete the main body of the eggshell while others will synthesize specialized structures involved in fertilization, gas exchange, or hatching (Boyle, 2010).

This study has investigated the link between the patterning and morphogenesis of the follicle cells that make the dorsal appendages (DAs), protrusions of chorion that aid in making oxygen accessible to the embryo. Once the cells destined to participate in DA formation have been determined—a process that culminates in S10B—these cells rearrange to form a tube during S11 and elongate that tube through S12 and S13, reaching the final product at S14. Notably, mitosis in follicle cells stops during S6, so this morphogenesis is achieved solely through cell shape change and movement. This cessation of cell division also places an upper limit on the time at which the genetic mosaics discussed in this paper were generated; thus, the expression of the gene removed will have ceased at least 13 h before the initiation of tube formation. The exception to this general rule involves the tramtrack^twk (ttk^twk) allele, in which a P element inserted into an upstream promoter disrupts expression only late in oogenesis. Since one of the proximal promoters (1b) is still functional early in oogenesis, TTK69 expression persists until S10B, making the ttk^twk allele useful in avoiding earlier tramtrack requirements (Boyle, 2010).

The patterning of the DA-forming cells starts with signaling by the EGF and DPP pathways, which leads to changes in the level at which follicle cells express the transcription factor Broad (BR). BR is initially expressed at a uniform, low level throughout the epithelium, but at S10B the cells destined to form the roof of the DA lumen express elevated BR. The single row of cells destined to dive underneath the roof to seal off the floor of the lumen (as well as adjacent cells that contribute to the anterior face of the eggshell) cease expressing BR altogether. Collectively the roof and floor cells form the DA primordium (Boyle, 2010).

While much about the mechanisms by which these cell types are patterned have been discovered, comparatively little is known about their relative functions in the process of DA morphogenesis. What forces do the roof and floor cells generate? Does anterior migration of the floor cells pull the roof cells forward or vice versa? Does convergent extension of the roof cells pull the floor, thereby determining the width of the tube, or does the inward migration of the floor drive convergent extension in the roof cells? Beyond even the roof vs. floor distinction, cells in different regions of the DA-forming epithelium must perform distinct tasks. For instance, among the roof cells lining the anterior of the primordium, the cells on the lateral side must swing toward the posterior in a hinge-closing movement while those at the hinge point must migrate anteriorly over the squamous follicle cells that lie over the nurse cells (the stretch cells) in order to elongate the tube. If cells form these different locations were removed or immobilized, how would the rest of the structure react? Two primary hypotheses are considered. First, the remaining cells may re-organize to take over the role of the missing cells no matter which cells are affected. Second, genetically altering or laser ablating cells at certain positions may cause the unrecoverable failure of a subset of the movements required for proper DA formation. Due to their obvious patterning differences, it was predicted that roof cells would not be able to assume the role of the affected floor cells. The possiblity was also considered that the roof and floor consisted of additional subregions, and it was asked exactly which subset of behaviors would be impacted when disrupting cells at various positions within the DA primordium (Boyle, 2010).

This paper addresses these questions using laser cell ablation and genetic mosaics. It was found that the DA primordium can be split not only into the two known regions (roof and floor) but that the roof population itself consists of two regions, which are referred to as the 'leading roof', those roof cells adjacent to the floor cells, and the 'trailing roof', the remainder of the roof-cell population. Of particular interest is the area surrounding the hinge point, as these cells lead the anterior migration. Ablation of a few cells near the hinge causes disproportionate defects in DA elongation, while ablation of cells in the posterior of the DA primordium causes only very minor elongation defects. Further, mosaics were generated of ttk^twk, a mutation in an upstream regulatory region that affects expression of the transcription factor TTK69 during S10B and later, and which causes a severe DA elongation defect. ttk^twk does not cause DA defects when removed from the majority of DA-forming cells, but only when clones occur in the anterior of the structure. Finally a role was demonstrated for heterotrimeric G-protein signaling in DA morphogenesis, and it was shown that Gβ13F is required for distinct behaviors in the floor and the leading roof, yet is dispensable from the remainder of the follicle cells (Boyle, 2010).

This study presents the strongest evidence to date that cells from distinct regions of the DA primordium are responsible for specific shape changes affecting the entire structure. Cells from the anterior 1/3 of the DA-forming region are responsible for elongation of the whole tube while the remaining cells are dispensable. Leading roof cells control convergent extension of the whole population, and floor cells control the lateral width of the lumen. Thus, it was shown that the DA-forming epithelium is more finely stratified than previously appreciated, both in terms of the roles that cells play in morphogenesis and the genes that they require to perform those roles. In doing so, it was demonstrated that heterotrimeric G-protein signaling is required for DA morphogenesis. A subset of the roof cells, defined as the leading roof, requires TTK69 and Gβ13F for DA elongation, yet those genes are not required in trailing roof cells. These results suggest that the leading roof cells, rather than the roof population as a whole, are responsible for driving DA elongation. Similarly, the different DA shapes that result when removing Gβ13F from the floor cells versus the roof suggest that the floor, and not the roof, controls lumen width (Boyle, 2010).

Laser ablation of the leading DA-forming cells severely disrupted tube elongation. Although dying cells could physically block more posterior cells, similar phenotypes occurred when disrupting leading cells by generating loss-of-function clones. Mosaic analysis also has potential drawbacks, including removal of gene function at earlier developmental periods (except when using specific alleles such as ttk^twk). Large clones are generated earlier in development, however, and such clones did not produce an effect unless specific cells were targeted, those same cells that exhibited a disproportionate effect upon laser ablation. Thus, these two distinct but complementary approaches reveal the importance of spatial position during DA morphogenesis (Boyle, 2010).

Clonal analysis of ttk^twk demonstrated that TTK69 is required during tube elongation, S12-S13, only in the anterior of the DA-forming region. This finding is particularly surprising for two reasons. One, earlier in oogenesis, at S10, TTK69 is required in all columnar follicle cells to pattern the epithelium; loss of function at that time produces cell shape changes in any cell that is mutated. Two, morphological analysis of ttk^twk cells revealed highly elongated yet apically constricted cell shapes throughout the DA-roof population. So how could removal of TTK69 (or Gβ13F) from the leading cells disrupt cell behaviors in other DA regions where these genes are still expressed? Two primary hypotheses are considered: TTK69, G proteins, or any such factor could be required specifically in those leading cells to relay a signal to the more posterior DA cells, which then respond by changing their shape and producing a correctly shaped DA. An alternative hypothesis is that these factors are required only to shape the leading roof and/or floor cells, which then pull or otherwise physically constrain the remainder of the cells into proper shape (Boyle, 2010).

Since the leading roof and floor cells outline the DA, moving them into proper position may be sufficient to shape the appendage if the remaining cells simply fill in the space in a lowest-energy fashion. It is also possible that TTK69 and Gβ13F act in all DA cells, but in a way that is only relevant to the leading roof and floor cells due to geographical constraints. For example, TTK69 and Gβ13F could regulate expression or activity of a heterophilic cell-cell adhesion molecule responsible for adhesion between roof cells and another cell type. If the function of this hypothetical adhesion protein were to diminish in some cells, it would not matter as long as those cells never come in contact with the cells expressing the protein's ligand. As the trailing roof cells contact only other roof cells along lateral surfaces (contacting the DA lumen at apical surfaces and extracellular matrix at basal surfaces), all would be well as long as proper function occurred in the leading roof that does contact other cell types (Boyle, 2010).

What role do G proteins play in DA morphogenesis and what molecular mechanisms underlie their genetic interaction with TTK69? The failure of Gβ13F^δ–96A mutant cells to cross the nurse-cell/oocyte boundary is suggestive. G-protein-coupled receptor signaling could be required in the DA-forming cells to receive a signal sent from the stretch -- or possibly some other anterior -- cells, a signal that attracts or orients them. Alternatively, the failure in convergent extension when Gβ13F is removed from the roof cells indicates that G proteins might be required to establish planar cell polarity at the boundary between roof and floor cells. This planar polarity could be required in turn not only for convergent extension of the roof, but also to orient the direction of roof-cell migration. Similar defects in the planar orientation of the floor cells could explain their disorganization and the consequent widening of the lumen (Boyle, 2010).

That Gγ1 is a suppressor of the ttk^twk phenotype suggests that TTK69 and heterotrimeric G-protein signaling downstream of the Gβγ subunit may be opposed in function, but the various G-protein mutants that were observed did not result in longer DAs, but in shorter ones. This result need not be inconsistent from a molecular perspective, however. In order for DA-forming cells to migrate toward the anterior of the egg chamber -- and in particular to cross over the nurse-cell/oocyte boundary -- they will require both the assembly and disassembly of adhesion complexes with their substrate, the stretch cells. If TTK69 and Gβ13F affect this expression in opposite directions, it would explain their opposition from a molecular perspective while still being required in the leading cells for anterior elongation of the DA tube (Boyle, 2010).

This hypothesis also explains the ttk^twk and Gβ13F phenotypes. ttk^twk results in highly elongated DA-roof cells stretched out along the path of their migration, consistent with an inability to disassemble adhesion complexes with the substrate along their migration path. By contrast, Gβ13F^δ1–96A mutant cells reach up to, but do not cross over, the stretch-cell-covered nurse cells, consistent with an inability to form such contacts (Boyle, 2010).

This study reveals that the boundaries of subpopulations are especially likely to behave in special ways due to their unique access to adjacent cell types. Parallels in other systems lead to the suggestion that this result is likely to be broadly applicable beyond DA morphogenesis. For instance, during Drosophila tracheal morphogenesis, a small number of cells at the tip of the branch directs the convergence of the cells that follow them. Similarly, during wound healing, cells along the leading edges form an actin purse-string and extend filopodia toward each other to facilitate the rapid closure of the gap. Like the leading roof cells, these cells at the edges are not fundamentally different from the cells that follow behind (both leading and trailing cells being, ultimately, a part of the wounded structure), yet their location causes them to perform an important function upon which all the cells behind them depend (Boyle, 2010).

In conclusion, this study has increased the resolution with which the process of DA morphogenesis can be understood, jointly in terms of the spatial organization of the cells that form the DA tubes, in terms of the functions of subpopulations in driving DA morphogenesis, and in terms of identifying new pathways that regulate tube elongation. Such a reductionist approach has proven highly valuable when studying other systems, such as gastrulation, where the development of modern understanding was dependent on breaking the complex movements into specific components such as convergent extension and spreading. Using the simpler system of DA morphogenesis, these intricate processes can be understood at a higher level of detail, thereby providing a model system that facilitates understanding morphogenesis from its most basic molecular components to the overarching macroscopic events (Boyle, 2010).

Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis

Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of myosin II. For instance, during Drosophila gastrulation, apical constriction and cell intercalation are mediated by medial-apical myosin II pulses that power deformations, and polarized accumulation of myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of myosin II activation and the ratchet-like myosin II dynamics. This study reports the function of a common pathway comprising the heterotrimeric G proteins Gα12/13 (Concertina), Gβ13F and Gγ1 in activating and polarizing myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog (Poor gastrulation, Pog & CG31660) was identified as being required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of myosin II and irreversible cell shape changes. It is proposed that GPCR and G proteins constitute a general pathway for controlling actomyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators (Kerridge, 2016).

During tissue morphogenesis, cells rearrange their contacts to invaginate, intercalate, delaminate or divide. During Drosophila gastrulation, invagination of the presumptive mesoderm in the ventral region of the embryo and of the posterior midgut requires apical cell constriction, a geometric deformation that occurs in different organisms. Elongation of the ventral–lateral ectoderm requires cell intercalation, a general topological deformation associated with junction remodelling. In the ectoderm, the so-called ‘vertical junctions’, oriented along the dorsal–ventral axis, shrink, followed by extension of new ‘horizontal’ junctions along the anterior–posterior axis. Despite differences in the cell deformations associated with intercalation and apical constriction, recent studies revealed that both processes require myosin II (MyoII) contractility. Cell shape changes rely on the pulsatile activity of MyoII in the apical–medial cortex, whereby MyoII undergoes cycles of assembly and disassembly allowing stepwise deformation1. Moreover, each step of deformation is stabilized and thereby retained, contributing to the irreversibility of tissue morphogenesis. In the mesoderm, each phase of apical area constriction mediated by MyoII pulses is followed by a phase of shape stabilization involving persistence of medial MyoII. In the ectoderm, medial–apical MyoII pulses flow anisotropically towards vertical junctions resulting in steps of shrinkage that are stabilized by a planar-polarized pool of junctional MyoII. This ratchet-like behaviour of MyoII is regulated by the Rho1–Rok pathway and requires quantitative control over MyoII activation. Low Rho1/Rok activity fails to form actomyosin networks, intermediate activation establishes MyoII pulsatility and high activation confers stability. The signalling mechanisms that cause stepwise activation of MyoII by Rho1 remain unknown. It is also unclear whether different pathways for Rho1 activation operate in the mesoderm and in the ectoderm as indeed Rho1 can be activated by numerous signalling mechanisms or whether a common pathway might exist (Kerridge, 2016).

Tissue-specific factors can result in polarized shape changes by signalling through cell surface receptors. For instance, in Drosophila ectoderm, pair rule genes encoding transcription factors control planar-polarized enrichment of MyoII through the combinatorial expression of the surface proteins Toll2, Toll6 and Toll8 in stripes. Likewise, in the mesoderm, Twist and Snail induce expression of Fog, a secreted ligand, and a G-protein-coupled receptor (GPCR) Mist (methuselah-like 1), which is reported to transduce Fog. The downstream G protein Gα_12/13 (known as Concertina (Cta) in Drosophila) is required for RhoGEF2 and thereby MyoII apical recruitment. As RhoGEF2 is a known GEF for Rho1, the requirement of Gα_12/13 for RhoGEF2 apical recruitment suggests that GPCRs and G-protein signalling mediate MyoII activation through the Rho1 pathway. These considerations prompted asking whether G-protein signalling directly controls the different regimes of MyoII dynamics (pulsatility and/or stability) in the mesoderm and planar polarized activation of Rho1 and MyoII in the ectoderm (Kerridge, 2016).

This study reports the function of the heterotrimeric G proteins Gα_12/13, Gβ13F and Gγ1 in activating and regulating MyoII dynamics both in the mesoderm and in the ectoderm. Receptor activation, through the GEF activity of the GPCR, converts Gα from an inactive GDP-bound state, in a complex with Gβγ, to an active GTP-bound state. This results in dissociation of Gβγ, enabling binding of both Gα–GTP and Gβγ to their respective effectors for signalling. This study found that Gα_12/13 and the Gβ13F/Gγ1 complex constitute distinct signalling modules, which regulate MyoII dynamics medial–apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog, was found to be required for cell shape changes associated with both mesoderm invagination and ectoderm elongation. During these morphogenetic events, Smog functions with other GPCRs, Mist in the mesoderm and an as yet unknown GPCR in the ectoderm, to activate the Rho1–Rok pathway. This results in stepwise activation of Rho1 and MyoII, ensuring irreversible cell shape changes (Kerridge, 2016).

First, this study reports that Gα_12/13 and Gβ13F/Gγ1 function as distinct signalling modules that control Rho1 and MyoII in different domains. Gα_12/13 activates medial–apical MyoII through its effector RhoGEF2 both in the ectoderm and the mesoderm. In mammals, p115–RhoGEF interacts directly with Gα12 suggesting that this may be a conserved signalling module. In contrast, Gβ13F/Gγ1 activates MyoII both at cell junctions and in the medial–apical domain. This modularity may provide distinct regulatory mechanisms for the activation of MyoII in different subcellular compartments owing to the existence of different molecular effectors of Gα–GTP and Gβγ. Second, stepwise activation of Rho1 by multiple GPCRs and their ligands determines the emergence of a pulsatile regime medial–apically, or stable activation. In the mesoderm, Smog and Mist GPCRs, together with high expression of their ligand Fog, ensure stabilization and rapid (<5 min) accumulation of MyoII ensuring apical constriction. In the ectoderm, low Fog expression and thus lower activation of Gα_12/13 and RhoGEF2 is responsible for intermediate medial–apical activation of MyoII and pulsatility. Indeed, Fog, constitutively active Gα_12/13QL and RhoGEF2 overexpression all lead to stable accumulation of MyoII instead of pulsation, similar to constitutively active RhoV14 (Kerridge, 2016).

Interestingly, the same receptor Smog controls MyoII activation in different subcellular domains during intercalation and apical constriction begging the question of how activation of Gα_12/13 and Gβγ is differentially achieved in the ectoderm and the mesoderm. The polarization of Smog activation is to some extent imparted by the ligand. Fog/Smog regulates medial–apical accumulation of MyoII in the two tissues: Fog induces medial Rho1 and Rok activation in the mesoderm and ectoderm and, when ectopically expressed in the ectoderm, it can increase Rho1 and Rok in the medial cortex. This argues that another mechanism results in junction-specific activation of Smog, Gβ13F/Gγ1, Rho1 and Rok in the ectoderm (Kerridge, 2016).

It is possible that an unknown ectoderm-specific ligand activates Smog specifically at junctions. Junctional localization of the Rho1 pathway by Smog may also be imparted by subcellular processing of Smog signalling, such as localization/activation of downstream effectors of Gα_12/13 and Gβγ. The recently identified Toll receptors required for MyoII planar-polarized activation may bias Smog signalling although the molecular mechanisms remain unclear. This could be through localization of RhoGEFs. In the mesoderm, the transmembrane protein T48 localizes RhoGEF2 apically through binding to its PDZ domain, and is required for apical MyoII activation in parallel with Smog, Gα_12/13 and Gβγ. Similarly, other GEFs may be required for junctional Rho1 activation by Smog (Kerridge, 2016).

What might be the advantage of having multiple GPCRs? Gastrulation sets the foundation for all other future processes in development and hence requires robustness. GPCRs with similar functions yet subtle differences such as ligand specificity may offer advantages compared with single ligand–receptor pairs. For instance, high cortical tension associated with mesoderm invagination may require multiple GPCRs activating parallel pathways to attain efficiency of the process. Moreover, multiple GPCRs may concede tissue-specific regulation of the common G-protein subcellular pathways. Finally, multiple GPCRs can allow stepwise activation of MyoII. Although activation by one GPCR is sufficient to induce pulsatility, more GPCRs are required to shift the actomyosin networks to more stable regimes (Kerridge, 2016).

The discovery that Smog and heterotrimeric G protein activate Rho1 and MyoII in two different morphogenetic processes provides a potentially general molecular framework for tissue mechanics. It is proposed that different developmental inputs tune a common GPCR/G-protein signalling pathway to direct specific patterns and levels of Rho1 activation. Quantitative control specifies the regime of MyoII activation through Rho1, namely pulsatility or stability of MyoII. Modular control defines the subcellular domains where MyoII accumulates (medial–apical or junctions) depending on molecular effectors. How developmental signals tune GPCR signalling will be important to decipher (Kerridge, 2016).

Distinct RhoGEFs Activate Apical and Junctional Contractility under Control of G Proteins during Epithelial Morphogenesis

Small RhoGTPases direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of actomyosin contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. This general problem was examined in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein-coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (Galpha and Gbetagamma) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized. However, the mechanisms responsible for polarizing Rho1 activity are unclear. This study reports that distinct guanine exchange factors (GEFs) activate Rho1 in these two cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1 but is recruited both medial-apically and at junctions by Galpha(12/13)-GTP, also called Concertina (Cta) in Drosophila. On the other hand, Dp114RhoGEF (Dp114), a newly characterized RhoGEF, is required for cell intercalation in the extending ectoderm, where it activates Rho1 specifically at junctions. Its localization is restricted to adherens junctions and is under Gbeta13F/Ggamma1 control. Furthermore, Gbeta13F/Ggamma1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. Finally, Dp114RhoGEF was absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results clarify the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia (Garcia De Las Bayonas, 2019).

Critical aspects of cell mechanics are governed by spatial-temporal control over Rho1 activity during Drosophila embryo morphogenesis. This work sheds new light on the mechanisms underlying polarized Rho1 activation during intercalation in the ectoderm. Rho1 activity was found to be driven by two complementary RhoGEFs under spatial control of distinct heterotrimeric G protein subunits. Notably, a regulatory module was uncovered specific for junctional Rho1 activation (Garcia De Las Bayonas, 2019).

Dp114RhoGEF was identified as a novel activator of junctional Rho1 in the extending ectoderm. Hence, two RhoGEFs, Dp114RhoGEF and RhoGEF2, coordinate independently the modular Rho signaling during tissue extension of the ectoderm. This has important implications, as it allows refinement of the nature of the interconnection between the two pools of Myo-II in this tissue. It has been shown previously that medial pulses of Myo-II flow toward and merge with the Myo-II pool at vertical junctions. However, to what extent these 'fusion' events contribute to junctional Myo-II was unclear. This study genetically uncoupled the regulation of both pools of Myo-II and showed that the loss of one pool does not compromise activation of Myo-II in the other. Indeed, junctional Myo-II levels and planar polarity are not affected in RhoGEF2 shRNA embryos or in RhoGEF2 germline clone where medial Myo-II is lost. This rules out the possibility of medial pulses being the main source of junctional Myo-II accumulation. Instead, it is concluded that actomyosin flow toward junctions contributes to junction shrinkage because it serves a distinct and direct mechanical function in junction remodeling rather than working by proxy by fueling junctional Myo-II (Garcia De Las Bayonas, 2019).

The division of labor in the molecular mechanisms of Rho1 activation in distinct cellular compartments lends itself to differential quantitative regulation. The activation kinetics of these different GEFs and nucleotide exchange catalytic efficiencies are likely to differentially impact Rho1 activity and therefore Myo-II activation at the junctional and medial-apical compartments. For example, RhoGEF2 mammalian orthologs, LARG and PDZ-RhoGEF, show a catalytic activity that is two orders of magnitude higher as compared with the Dp114RhoGEF orthologs subfamily. This may help to establish specific contractile regimes of actomyosin in given subcellular compartments. It is therefore important to tightly control RhoGEFs localization and activity to ensure a proper quantitative activation of the downstream GTPase (Garcia De Las Bayonas, 2019).

RhoGEF2 is a major regulator of medial-apical Rho1 activity during Drosophila gastrulation. Originally characterized in the invaginating mesoderm, it was found that RhoGEF2 also activates Rho1 medial-apical activity in the elongating ectoderm. There, RhoGEF2 localizes both medial-apically and at junctions where it is also planar polarized. Although RhoGEF2 and active Rho1 are both planar polarized at junctions, in RhoGEF2 mutants, junctional Rho1-GTP is not affected and ectopic recruitment of RhoGEF2 following expression of Gα12/13Q303L does not cause ectopic junctional Rho1-GTP accumulation. Thus, RhoGEF2 localization at the membrane is not strictly indicative of its activation status. Interestingly, Gα12/13/Cta is necessary for RhoGEF2 to translocate from microtubules plus ends to the plasma membrane where it signals. To date, experimental evidence favor a model whereby the binding of active Gα12/13/Cta to the RhoGEF in the vicinity of the cell membrane triggers its conformational change and stabilizes it in an open conformation able to bind to lipids via its PH domain and signal at the plasma membrane. There is no evidence that Gα12/13/Cta-GTP actively destabilizes RhoGEF2-EB1 interaction, but this is a formal possibility to be tested. Importantly, Gα12/13/Cta alone does not account for the restricted activation of Rho1 medial-apically (Garcia De Las Bayonas, 2019).

It is hypothesized that additional factors must regulate the spatial distribution of RhoGEF2 activity. In principle, RhoGEF2 signaling activity could either be specifically induced medial-apically independent of RhoGEF2 recruitment or RhoGEF2 could be inhibited at junctions and laterally. Sequestration of inactive RhoGEFs at cell junctions has been reported previously in mammalian cell cultures, suggesting that such mechanism could be evolutionary conserved. Phosphorylation can control the activity of the RH-RhoGEFs subfamily. Therefore, phosphorylation could promote activation or inhibition of RhoGEF2 activity in specific subcellular compartments in the ectoderm. RhoGEF2 is reported to be phosphorylated in the gastrulating embryo (Garcia De Las Bayonas, 2019).

Complementary to RhoGEF2, Dp114RhoGEF activates junctional Rho1 in the ectoderm. Dp114RhoGEF strictly localizes at junctions, providing a direct explanation for its junctional-specific effect. Gβ13F/G&gamma1 is also enriched at adherens junctions, where it controls Dp114RhoGEF junctional recruitment together with additional upstream regulators. Therefore, it is suggested that Gβ13F/Gγ1-dependent tuning of junctional Rho1 activation could be achieved through its ability to concentrate the GEF at junctions. Gβ/Gγ-dependent regulation of RhoGEFs has been described in mammals. One study proposes that mammalian p114RhoGEF may bind and be activated by Gβ1/Gγ2. Interestingly, recent work demonstrates that Gα12 can also recruit p114RhoGEF at cell junctions under mechanical stress in mammalian cell cultures where it promotes RhoA signaling. However, the region of mammalian p114RhoGEF that binds to Gα12 is absent in invertebrate RhoGEFs. How Gβ13F/Gγ1 controls Dp114RhoGEF at junctions in the Drosophila embryo remains an open question. A recent study reports that Dp114RhoGEF localizes at adherens junctions in the Drosophila ectoderm through multiple mechanisms, including interactions with Baz/Par3 and the Crumbs complex. Therefore, investigating a possible connection between Gβ13F/Gγ1 signaling and Baz/Crumbs should help decipher the mechanisms of Dp114RhoGEF localization (Garcia De Las Bayonas, 2019).

Importantly, neither Gβ13F/Gγ1 nor Dp114RhoGEF are themselves planar polarized at junctions. Hence, their distribution alone cannot explain polarized Rho1 activity at junctions. Strikingly, an increase in Gβ13F/Gγ1 dimers was found to hyperpolarize Rho1 activity and Myo-II at vertical junctions. Gβ13F/Gγ1 overexpression also leads to an overall increase in Dp114RhoGEF levels at junctions, although Dp114RhoGEF is not planar polarized in this condition. This indicates that recruitment at the plasma membrane and activation of Dp114RhoGEF are independently regulated, similar to RhoGEF2. In contrast, Dp114RhoGEF overexpression increases Myo-II at both transverse and vertical junctions, although a slightly stronger accumulation is observed at vertical junctions. Therefore, although Dp114RhoGEF junctional levels are increased in both experiments, only Gβ13F/Gγ1 overexpression leads to an increased planar polarization of Rho1-GTP and Myo-II at vertical junctions. This points to a key role for Gβ13F/Gγ1 subunits in the planar-polarization process associated with but independent from the sole recruitment of Dp114RhoGEF at junctions. In principle, Gβ13F/Gγ1 could bias junctional Rho1 signaling either by promoting its activation at vertical junctions or by inhibiting it at transverse junctions (e.g., RhoGAP polarized activation). Gβ13F/Gγ1 could also control active Rho1 distribution independent of its activation. For instance, a scaffolding protein binding to Rho1-GTP at junctions could be polarized by Gβ13F/Gγ1 to bias Rho1-GTP distribution downstream of its activation. Anillin, a Rho1-GTP anchor known to stabilize Rho1 signaling at cell junctions is a potential candidate in the ectoderm. Last, Toll receptors control Myo-II planar polarity in the ectoderm. Whether Gβ13F/Gγ1 and Tolls are part of the same signaling pathway is an important point yet to address in the future (Garcia De Las Bayonas, 2019).

Finally, this study sheds light on new regulatory differences underlying tissue invagination and tissue extension. This study found that Dp114RhoGEF localizes at junctions in the ectoderm, where it activates Rho1 and Myo-II. In contrast, maternally and zygotically supplied Dp114RhoGEF::GFP is not detected at junctions in the mesoderm. Little if any cytoplasmic signal is seen in this condition, suggesting that Dp114RhoGEF::GFP could be degraded in these cells. Thus, repression of Dp114RhoGEF protein in the mesoderm could be an important mechanism for cell apical constriction and proper tissue invagination. Of interest, Rho1 signaling is absent at junctions in the mesoderm. Therefore, it is tempting to suggest that the absence of Dp114RhoGEF at junction in the mesoderm accounts for cells' inability to activate Rho1 in this compartment. Importantly, the GPCR Smog and Gβ13F/Gγ1 subunits, found to control junctional Rho1 in the ectoderm, are common to both tissues. Dp114RhoGEF differential expression and/or subcellular localization could be a key element to bias signaling toward junctional compartment in the ectoderm (Garcia De Las Bayonas, 2019).

Cell contractility necessitates activation of the Rho1-Rock-MyoII core pathway. During epithelial morphogenesis, tissue- and cell-specific regulation of Rho1 signaling requires the diversification of Rho1 regulators, in particular RhoGEFs, as shown in this study, and RhoGAPs. Some of them are tissue specific with given subcellular localizations and activation mechanisms. The identification of signaling modules, namely Gα12/13-RhoGEF2 and Gβ13F/Gγ1-Dp114RhoGEF, provides a simple mechanistic framework for explaining how tissue-specific modulators control Rho1 activity in a given subcellular compartment in a given cell type. Therefore, it is suggested that the variation of (1) ligands, GPCRs, and associated heterotrimeric G proteins and (2) types of RhoGEFs and RhoGAPs as well as their combination, activation, and localization by respective co-factors underlies the context-specific control of Rho1 signaling during tissue morphogenesis. How developmental patterning signals ultimately control Rho regulators is an exciting area for future investigations (Garcia De Las Bayonas, 2019).

Competing activities of heterotrimeric G proteins in Drosophila wing maturation

Drosophila genome encodes six α-subunits of heterotrimeric G proteins. The α-subunit termed Gαs is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. This study shows that another α-subunit, Gαo, can specifically antagonize the Gαs activities by competing for the Gβ13F/Ggamma1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. These results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development (Katanayeva, 2010).

G protein-coupled receptors (GPCRs) represent the most populous receptor family in metazoans. Approximately 380 non-olfactory GPCRs are encoded by the human genome, corroborated by ca. 250 GPCRs in insect genomes, making 1%-1.5% of the total gene number dedicated to this receptor superfamily in invertebrates and mammals. GPCRs transmit their signals by activating heterotrimeric G protein complexes inside the cell. A heterotrimeric G protein consists of a GDP-bound α-subunit and a βα-heterodimer. Ligand-stimulated GPCR serves as a guanine nucleotide-exchange factor, activating the GDP-to-GTP exchange on the Gα-subunit. This leads to dissociation of the heterotrimeric complex into Gα-GTP and flγ, which transmit the signal further inside the cell (Katanayeva, 2010).

The β- and γ-subunit repertoire of the Drosophila genome is reduced as compared with that of mammals: only two Gγ and three Gβ genes are present in flies. Gγ30A and Gβ76C are components of the fly phototransduction cascade and are mostly expressed in the visual system. Gγ1 and Gβ13F have been implicated in the asymmetric cell divisions and gastrulation, while the function of Gβ5 is as yet unknown (Katanayeva, 2010).

Despite the fact that βγ can activate signal effectors, the main selectivity in GPCR coupling and effector activation is provided by the Gα-subunits. Sixteen genes for the α-subunits are present in the human genome, and six in Drosophila. All human Gαsubunit subgroups are represented in Drosophila: Gαi and Gαo belonging to the Gαi/o subgroup; Gαq belonging to the Gαq/11 subgroup; Gαs belonging to the Gαs subgroup, and concertina (cta) belonging to the Gα12/13 subgroup. Additionally, Drosophila genome encodes for Gαf which probably represents an insect-specific subfamily of Gαsubunits (Katanayeva, 2010).

Multiple functions have been allocated to different heterotrimeric G proteins in humans and flies. For example, in Drosophila development cta is a crucial gastrulation regulator, Gαo is important for the transduction of the Wnt/Frizzled signaling cascade, and Gαi controls asymmetric cell divisions during generation of the central and peripheral nervous system (the later in cooperation with Gαo. Gαq is the Drosophila phototransduction Gαsubunit, but probably has additional functions. Pleotropic effects arise from defects in Gαs function, while the function of Gαf has not yet been characterized (Katanayeva, 2010).

Among the developmental processes ascribed to the control by Gαs are the latest stages of Drosophila wing development. Newly hatched flies have soft and folded wings, which during the 1-2 hours post-eclosion expand and harden through intensive synthesis of components of the extracellular matrix. These processes are accompanied by epithelial-mesenchymal transition and apoptosis of the wing epithelial cells, producing a strong but mostly dead adult wing structure. Expression of the constitutively active form of Gαs leads to precocious cell death in the wing epidermis, which results in failure of the closure of the dorsal and ventral wing sheets and accumulation of the hemolymph inside the wing, producing wing blistering. Conversely, clonal elimination of Gαs leads to autonomous prevention of the cell death. Kimura (2004) has performed an extensive analysis of the signaling pathway controlling apoptosis at late stages of wing development. That study provided evidence suggesting that the hormone bursicon, synthesized in the head of post-eclosion Drosophila and secreted in the hemolymph, activates a GPCR rickets on wing epithelial cells, which signals through Gαs to activate the cAMP-PKA pathway, culminating at the induction of apoptosis. However, the identity and importance of the &βγ subunits in bursicon signaling, as well as possible involvement of other Ga proteins remained outside of their investigation. There also remain some uncertainties as to the phenotypic consequences of elimination of the bursicon-Gαs-PKA pathway in wings (Katanayeva, 2010).

This study describes a comprehensive functional analysis of the Drosophila heterotrimeric G protein proteome using loss-of-function and overexpression experiments. Loss of Gαs but not any other Gαsubunit leads to the failure of wing expansion after fly hatching. Gαo, but not another Gα, can compete with Gαs and thus antagonize its function. Finally, the Gβ13F and Gγ1 as the βγ subunits of the heterotrimeric Gs complex responding to the epithelial-mesenchymal transition and cell death-promoting signal (Katanayeva, 2010).

The soft folded wings of the young insect freshly hatched from the pupal case within 1-2 hours expand and harden, becoming a robust flight organ. This process is accompanied by epithelial-mesenchymal transition and cell death of the wing epithelial cells. Genetic dissection has revealed the function of the neurohormone bursicon and its wing epithelial receptor rickets in initiation of these processes. The GPCR rickets couples to the heterotrimeric G protein Gs; the Gαs-activated cAMP-PKA pathway culminates at the induction of apoptosis. However, the overall phenotypic consequences of the loss of the Gs signaling pathway in post-eclosion wings were unknown, as well as the nature of the Gβγ subunits of the heterotrimeric Gs complex responding to the bursicon-rickets signaling (Katanayeva, 2010).

This study consisted of an extensive analysis of the heterotrimeric G protein subunits in these post-eclosion stages of wing maturation. The whole-wing down-regulation of Gαs results in the failure of wing expansion, demonstrating that this change in the shape of the wing is the major morphological outcome of the bursicon-rickets-Gs signaling. The Gβ13F and Gγ1 subunits were also identified as the other two constituents of the heterotrimeric Gs complex, as downregulation of Gαs, Gβ13F, or Gγ1, but not any other Ga, Gβ, or Gγ subunits encoded by the Drosophila genome, each leads to the same folded wing phenotype (Katanayeva, 2010).

It was also shown that Gαo, but not any other Gαsubunit, can inhibit the wing expansion program through sequestration of the Gβ13F/Gγ1 heterodimer. The reason for the specificity of Gαo over other Gαsubunits in antagonizing the Gs signaling is unclear. It is unlikely that differences in expression levels of the tested Gαsubunits may account for the selective activity of Gαo. Indeed, most overexpression experiments were done with the X-chromosome-inserted MS1096-Gal4 driver, which results in markedly higher expression levels in males than heterozygous female flies, producing a more penetrant folded wing phenotype in males overexpressing Gαo. However, even in male flies overexpressing other Gαsubunits no instances of the folded wing phenotype could be seen. Furthermore, several independent insertions of the UAS-Ga transgenes were tested; while different Gαo transgenes all produced the folded wing phenotype upon overexpression, other Ga constructs remained ineffective (Katanayeva, 2010).

Similarly, the different Gαsubunits possess a similar affinity towards the interaction with the Gβγ heterodimer, not providing an explanation for a specific ability of Gαo to antagonize the Gs-mediated post-eclosion pathway. It is thus thus tempting to propose that a previously uncharacterized biochemical mechanism may allow for a specific antagonism physiologically existing between the Gs- and Go- mediated signaling pathways. As liberation of high amounts of GDP-loaded Gαo is predicted to be a consequence of activation of multiple Go-coupled GPCRs, and as Go is a heavily expressed G protein representing the major G protein species e.g. in the brain of flies and mammals, this specific ability of Gαo to antagonize the Gs-mediated signaling may have physiological implications in other tissues and organisms than Drosophila wing. However, it is added that these speculations are based on the analysis of the overexpression data and must be treated with caution when translating them into physiological situations (Katanayeva, 2010).

Only the GDP-loaded, but not the activated GTP-loaded form of Gαo is effective in antagonizing Gs. A proteomics analysis was performed of the Drosophila proteins which would discriminate between the two nucleotide forms of Gαo, and surprisingly few targets of this kind were revealed. While the chaperone Hsc70-3 and β1-tubulin preferentially interacted with the GTP-loaded Gαo, Gβ13F was found to specifically interact with Gαo-GDP. These data suggest that many Gαo-interaction partners do not discriminate between the two guanine forms of Gαo. These findings are in agreement with other experimental findings, as well as mathematical modeling predicting that high concentrations of free (monomeric) signaling-competent Gαo-GDP are produced upon activation of Go-coupled GPCRs (Katanayeva, 2010).

Gαo-mediated sequestration of Gβ13F/Gγ1 depletes the pool of the heterotrimeric Gs complexes. As only heterotrimeric Ga&βγ, but not monomeric Ga proteins can efficiently bind and be activated by their cognate GPCRs, overexpression of Gαo abrogates the rickets-Gs signaling. Phenotypic consequences of this abrogation are the failures of apoptosis and wing expansion. In contrast, expression of the constitutively activated form of Gαs induces premature cell death and wing blistering. This phenotype can be also induced by expression of cholera toxin, revealing that the ability of cholera toxin to specifically overactivate Gαs reported in mammalian systems is reproduced with Drosophila proteins. These data also confirm that not only exogenously overexpressed, but also the endogenous Gαs can induce the precocious cell death upon overactivation (Katanayeva, 2010).

However, prevention of apoptosis is not sufficient to produce the folded wing phenotype. Together with the observation that the constitutively active form of Gαs is ineffective in rescuing the wing expansion defects produced by Gαo overexpression, these data suggest that the Gαs-cAMP-PKA pathway culminating at apoptosis is not the sole signaling branch emanating from the bursicon-rickets GPCR activation. It is proposed that the second signaling branch initiated by the rickets-mediated dissociation of the heterotrimeric Gs complex is represented by the free Gββ subunits, signaling to epithelial-mesenchymal transition. Such a double signaling impact mediated by the two components of the heterotrimeric G protein complex leads to initiation of two cellular programs -- apoptosis and epithelial-mesenchymal transition -- which cumulatively result in wing expansion and solidification, producing the adult flight organ. This two-fold response of the Drosophila wing to the maturation signal, mediated by the two components of the heterotrimeric G protein complex activated by the single hormone-responsive GPCR, provides an elegant paradigm for the coordination of signaling and developmental programs (Katanayeva, 2010).

The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway

The Wnt/Frizzled signaling pathway plays crucial roles in animal development and is deregulated in many cases of carcinogenesis. Frizzled proteins initiating the intracellular signaling are typical G protein-coupled receptors and rely on the trimeric G protein Go for Wnt transduction in Drosophila. However, the mode of action of Go and its interplay with other transducers of the pathway such as Dishevelled and Axin remained unclear. This study shows that the alpha-subunit of Go directly acts on Axin, the multidomain protein playing a negative role in the Wnt signaling. G alpha o physically binds Axin and re-localizes it to the plasma membrane. Furthermore, G alpha o suppresses Axin's inhibitory action on the Wnt pathway in Drosophila wing development. The interaction of G alpha o with Axin critically depends on the RGS domain of the latter. Additionally, the betagamma-component of Go can directly bind and recruit Dishevelled from cytoplasm to the plasma membrane, where activated Dishevelled can act on the DIX domain of Axin. Thus, the two components of the trimeric Go protein mediate a double-direct and indirect-impact on different regions of Axin, which likely serves to ensure a robust inhibition of this protein and transduction of the Wnt signal (Egger-Adam, 2009).

This study has demonstrated that Gαo can physically bind the RGS domain of Axin and recruit it to the plasma membrane, the action likely leading to the destabilization of the Axin-based β-catenin destruction complex and propagation of the Wnt signal inside the cell. In support of this idea, this study has shown that Gαo can suppress the Wnt loss-of-function phenotypes induced by Axin over-expression in wing imaginal discs. This rescue critically depends on the presence of the RGS domain, reiterating the crucial role of this domain for the interaction with Gαo. While the GTP-bound form of Gαo is unable to change the phenotypes of the AxinΔRGS expression, the GDP-bound forms of Gαo even dramatically enhance these phenotypes. It is hypothesized that this enhancement is due to sequestration of the Gβγ heterodimer by the GDP-forms of Gαo. It was also shown that Gβγ can directly bind and recruit Dsh from the cytoplasm to the plasma membrane, thus possibly contributing to the propagation of the Wnt signal (Egger-Adam, 2009).

The RGS domain of Axin, responsible for the interaction with Gαo, is important for the full range of Axin activity in wing imaginal discs. Indeed, over-expression of the ΔRGS form of Axin only partially suppresses Wnt signaling in this tissue. The RGS domain of Axin is known to bind APC, another component of the β-catenin-destruction complex. The inability of AxinΔRGS to directly interact with APC is the likely reason for the reduced activity of this construct in Drosophila wings and in vertebrates. The Gαo and Gαq proteins were shown to dissociate the Axin-based destruction complexes in mammalian cells. It is proposed that in Drosophila, Gαo leads to a similar dissociation of the destruction complex through direct binding to the RGS domain of Axin, which recruits Axin to the plasma membrane and probably displaces APC from Axin (Egger-Adam, 2009).

In vitro, the purified RGS domain of Axin binds equally well both the GDP- and the GTP-loaded forms of Gαo. It also lacks the GTPase-activating protein (GAP) activity towards Gαo, typical for other RGS domains. These data agree with the absence of some of the conserved residues required for the GAP action in Axin RGS. Thus, biochemically Axin binds Gαo regardless of its nucleotide form. However, in vivo the GDP- and the GTP-loaded forms of Gαo behave differently towards Axin. Only Gαo[GTP] is capable of recruiting Axin-GFP to the plasma membrane in the salivary glands. Similarly, Gαo[GTP] is much more potent in rescuing the Axin full-length over-expression effects in wing imaginal discs and adult wings. This seeming contradiction is explained by the fact that in vivo the GDP-loaded forms of Gαo bind the βγ-subunits, recreating the trimeric Go complexes. Indeed, over-expressed, the wild-type Gαo was shown to compete with other Gα proteins for the βγ-subunits. Only the Gαo[GTP] form can stay free and thus exert its activities on Axin in full (Egger-Adam, 2009).

In contrast, the wild-type Gαo also possesses a capacity of over-activating the Wnt pathway in wing imaginal discs, and can to a certain degree rescue the phenotypes of Axin over-expression in this tissue. This contrasts with its inability to recruit Axin-GFP to the plasma membrane in salivary glands. These differences between the two tissues correlate with the degree of Wnt signal transduction. Indeed, the Wnt pathway is highly active in the wing imaginal discs, and Gαo can further enhance the pathway relying on the activity of Fz receptors. In contrast, in larval salivary glands the Wnt pathway is silent, which is illustrated by the cytoplasmic localization of Dsh in this tissue, expected to be plasma membrane localized when the pathway is on. It thus seems probable that in the salivary glands Gαo, forming trimeric Go complexes with Gβγ, fails to be further converted into the monomeric form due to the absence of the Wnt/Fz activity. In contrast, wing imaginal discs provide enough Wnt/Fz activity to activate endogenous as well as exogenous Go, which can then recruit Axin and thus propagate the signal (Egger-Adam, 2009).

The ability of the GDP-bound forms of Gαo to bind to the βγ-subunits is the likely reason for the aggravation of the AxinΔRGS phenotype induced by Gαo. This form, even upon conversion to the GTP-bound state by the action of the Wnt/Fz complexes, can no longer bind the RGS-lacking Axin and suppress Axin's negative action on the Wnt signal transduction. However, it can bind Gβγ. It is proposed that Gβγ plays, in addition to Gαo-GTP, a positive role in the Wnt signal transduction through its ability to bind and recruit Dsh to the plasma membrane. Over-expression of Gαo reduces the amounts of free Gβγ, reducing the efficiency of Dsh re-localization. It is proposed that when the endogenous full-length Axin is present, over-expression of Gαo has the overall stimulating effect on the Wnt signaling in wing discs due to increased generation of Gαo-GTP, which binds and antagonizes Axin. It is only in the artificial situation of over-expression of AxinΔRGS that the other, negative, effect of Gαo can be revealed. To prove that Gαo aggravates the AxinΔRGS phenotypes due to sequestration of Gβγ, the mutant Gαo[GDP] protein unable to charge with GTP but still capable to bind Gβγ was ested, and this form was found to be similar to Gαo in enhancing the AxinΔRGS phenotypes (Egger-Adam, 2009).

Direct experiments were performed testing the involvement of Gβγ in Wnt signaling. In accordance with predictions, down-regulation of Gβγ results in a clear reduction of the Wnt signaling in Drosophila wings and wing discs, affecting the short-range target genes of the Wnt pathway. As over-expression of Gβ alone leads to trapping Dsh in the cytoplasm, such over-expression also produces drastic dominant effects on Wnt signaling in wing discs. Unfortunately, it was not possible to confirm that Dsh was trapped in the cytoplasm of the epithelial cells of such discs due to the low resolution of the Dsh staining obtained in these thin columnar cells. Additionally, not only localization but also abundance of the components of the Wnt pathway are known to change in cells with high levels of Fz activation as part of the feedback regulation. Thus, interpretation of Dsh localization in wing imaginal discs upon perturbations of the Wnt pathway will be difficult. Instead, analysis of a tissue where the Wnt pathway is endogenously silent, such as salivary glands, allows analysis of the direct influence of the subunits of the trimeric Go complex on cellular localization of the components of the Wnt pathway. This analysis led to the identification of the plasma membrane re-localization of Axin by Gαo and of Dsh by Gβγ as such direct cellular responses. These primary responses are probably then utilized in the physiological context as the basis to build positive and negative feedbacks for the final outcome of Wnt signal propagation (Egger-Adam, 2009).

While the numerous data indicate that Gβγ is necessary for the proper activation of the Wnt pathway, probably through plasma membrane re-localization of Dsh, it was not possible to over-activate the Wnt pathway by over-expression of Gβ and Gγ together. Instead, the pathway was down-regulated, although to a weaker extent than that seen by over-expression of Gβ alone. This observation is not easy to reconcile with the other data. One possible explanation is that in the wing discs, unlike the salivary glands, co-overexpression of Gγ might be insufficient to attract the complete pool of Gβ to the plasma membrane, and significant amounts of Gβ may still remain cytoplasmic and retain Dsh. Along these lines, co-overexpression of Gγ shows a partial 'rescue' of the phenotypes induced by Gβ over-expression. Another possible explanation involves the notion of the negative feedback regulation in the Wnt cascade. Proteosomal degradation of Dsh during Wnt signal transduction has been demonstrated. A recent work has shown that targeted plasma membrane localization of Dsh by the Wnt activation or by the Gβγ subunits also destines it for the lysosomal degradation in vertebrate cells. Thus, the activity of Gβγ in the Wnt signaling may be multistep: the initial recruitment of Dsh from the cytosol may serve to activate the pathway, but the persistent membrane localization will lead to Dsh degradation. While Gβ RNAi targeting shows that the Gβγ complex is necessary for the proper Wnt signaling, activation of such a negative feedback loop may underlie the phenotypes observed upon the persistent over-expression of Gβγ. In this scenario, Gβγ will be added to the growing list of regulators of the Wnt pathway, which have both positive and negative activities in this signaling (Egger-Adam, 2009).

A model ia favored whereby Gβγ-induced plasma membrane re-localization of Dsh serves as an initial positive impact to activate the Wnt signal propagation. If this is correct, what may be the immediate consequences of the Gβγ-induced plasma membrane recruitment of Dsh? This scaffolding protein is known to become hyper-phosphorylated upon plasma membrane localization, which correlates with its activity in the Wnt signal transduction. Dsh is known to directly bind Axin through the DIX domain heterodimerization. Although a direct interaction of Gβγ with Axin's protein phosphatase 2A-binding region (N-terminal to the DIX domain) has recently been demonstrated in mammalian cells, no ability was found of Gβγ to re-localize or directly bind Drosophila Axin. Overall, the data and the above considerations lead to the proposal of the following model of the action of the trimeric Go protein in the Drosophila Wnt/Fz pathway (Egger-Adam, 2009).

The trimeric Go protein is a direct target of the activated Fz receptors. Wnt ligand binding to Fz activates the guanine nucleotide exchange activity of Fz towards Go. This in turn dissociates the trimeric Go complex into Gαo-GTP and Gβγ. It is proposed that both these components of the trimeric complex have the initial positive activity in Wnt signal propagation. Gαo-GTP directly binds to the RGS domain of Axin, recruiting Axin to the plasma membrane and dissociating the Axin-based β-catenin destruction complex. In contrast, Gβγ recruits and contributes to activation of Dsh, which then can bind the DIX domain of Axin and thus also promote dissociation of the destruction complex. These two branches of G protein–mediated signal propagation converge on the Axin complex to cooperatively ensure its efficient inhibition. Such a double effect on Axin emanating from the trimeric Go complex may serve to ensure a robust activation of the Wnt signaling (Egger-Adam, 2009).

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. Such neuroblasts were genetically created, and it was found that in embryos they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. These results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus (Kitajima, 2010).

This study investigated how the asymmetric mode of neuroblast division contributes to the specification and diversification of neuronal cell fate by generating neuroblasts that divide symmetrically. Combinations of dlg and Gβ13F mutants and of baz and Gβ13F mutants successfully generated neuroblasts that divide symmetrically with respect to both partition of determinants and daughter cell size during embryonic stages, allowing all progeny to differentiate into neurons that are often clonally homogeneous in temporal identity. At larval stages, dlg-Gβ13F neuroblasts generated overgrowing cell populations. Based on these results, the roles of asymmetric features of neuroblast division in the choice of self-renewal vs. differentiation and in cellular diversification are discussed (Kitajima, 2010).

Based on the observations, at embryonic stages, dlg-Gβ13F neuroblast divisions occur essentially without asymmetry in either daughter cell size or in the partition of the determinants from the first division. It is possible, however, that two daughter cells occasionally inherit different amounts of the determinants, leading to the generation of cell clusters expressing differential temporal identity genes in a single neuroblast progeny such as NB7-3. Such fluctuations in the partition of the determinants may occur stochastically because the apical/basal components are not tightly associated with cortex in dlg-Gβ13F neuroblasts (Kitajima, 2010).

All progenies of dlg-Gβ13F neuroblasts eventually differentiate in the embryonic stages. Which feature is then critical for the differentiation of all progeny; the asymmetric partition of determinants or of cell volume? On the one hand, the basal determinants are known to function in daughter cells' commitment to differentiation. On the other hand, all available results suggest that reduction in neuroblast cell size contributes to attenuation of cell cycle progression but not to the induction of differentiation. In the wild type, neuroblasts gradually reduce their size by budding off GMCs, and eventually enter the dormant state (Miranda+, Pros−, Elav−). In the Gβ13F single mutant, neuroblasts more rapidly lose volume by generating equal-sized daughters with the basal determinants normally segregating to one daughter, and remain in the same Miranda+, Pros−, Elav− state with the characteristic cell morphology of quiescent neuroblasts. This suggests that in the Gβ13F single mutant, neuroblasts also eventually enter the dormant state after the generation of a fewer number of GMCs. Thus, cell size reduction alone is not likely to cause neuronal differentiation of progenitors, but instead appears to cause them to remain in the undifferentiated state unless the basal determinants are present. This was confirmed in Gβ13F mutant neuroblasts at the larval stage (Kitajima, 2010).

What amount of the basal determinants is necessary to induce GMC fate? In Gβ13F mutants where neuroblast divisions give rise to daughters of equal size, a large daughter at the first division inherits most of the basal determinants and becomes differentiated into a GMC, indicating that a full amount of basal determinants can cause a daughter cell half the size of a newly born neuroblast to commit to the GMC fate. By contrast, neuroblasts undergoing symmetric divisions (dlg-Gβ13F mutants) appear to subsequently undergo at least two cell cycles and do not immediately commit to a GMC-like fate. This difference between embryonic Gβ13F mutant and dlg-Gβ13F mutant first daughters may mean that a half amount of the basal determinants is not sufficient to commit a daughter cell to the GMC fate. Alternatively, it has been argued that neuroblasts may express self-renewal factors that promote self-renewal and thereby proliferation and that asymmetrically segregate into the neuroblast. When dlg-Gβ13F neuroblasts undergo their first division symmetrically, those postulated factors and the basal determinants will be partitioned into both daughters and will counteract each other. This may cause a delayed commitment to the GMC fate, compared with the first GMC of Gβ13F mutant neuroblasts, which do not receive the self-renewal factors (Kitajima, 2010).

In the Drosophila CNS, the expression of the temporal identity genes changes sequentially in mother neuroblasts but is persistent in the sibling GMC progeny. Hence, the expression of such genes should also depend on the asymmetric mode of division. A significant difference was found in the expression of the temporal identity genes between normal neuroblast lineages and symmetrically dividing dlg-Gβ13F neuroblasts. In the latter, the neuroblast progeny frequently forms a clone of cells homogeneous for the expression of temporal identity genes, providing evidence for the importance of asymmetric division for the generation of neuronal diversity (Kitajima, 2010).

It has been shown that the first transition of temporal identity genes in embryonic neurogenesis, from Hb to Kr, requires cytokinesis, whereas the transition from Kr to Cas occurs without cell cycle progression. Symmetrically dividing neuroblasts pass through the initial transition from Hb to Kr in all lineages examined in this study (NB1-1, NB4-2, NB3-3 and NB7-3). A large proportion of neuroblast lineages appears to continue expressing the temporal genes in succession, but terminates earlier than normal, as revealed by their lack of Grh expression. Two observations suggest that the transition of temporal identity genes occurs sequentially (in the order of Hb to Kr to Pdm to Cas) in the majority of dlg-Gβ13F neuroblasts undergoing clonal expansion during early stages of neurogenesis; first, the size of Cas clusters is mainly 4 or 8 cells when Cas expression appears at 6–8 h AEL, suggesting that Cas expression starts in the clones that have already divided two or three times (some neuroblasts like NB3-3 start with Kr). Second, the cluster size of the clones become larger in the order of Hb, Kr, and Cas clusters at 8–10 h AEL (Kitajima, 2010).

The terminal temporal identity and the size of a particular neuroblast lineage are, however, not constant in dlg-Gβ13F mutants. Furthermore, a neuroblast progeny occasionally splits into two clusters with different temporal identities, as observed in the lineages of NB7-3. These observations suggest that stochastic processes are involved in the expression of temporal identity genes and cell cycle progression in the dlg-Gβ13F mutant neuroblast lineages (Kitajima, 2010).

Analysis of the relationship between clone size and clone homogeneity of NB7-3 reveals two characteristic features regarding dlg-Gβ13F neuroblast progenies. First, larger clones tend to be heterogeneous, containing both Kr-positive and Kr-negative cells (presumably Pdm-positive in their next identity), when compared to small-sized clones. Second, in heterogeneous clones, neurons with the same temporal identity form a cluster (Kr-positive and Kr-negative) and do not intermingle with each other, suggesting that cells with a different identity are also clonal instead being formed randomly during the expansion into a large heterogeneous clone. These observations regarding a single neuroblast lineage raise the possibility that a slight heterogeneity or difference created between sibling cells in early divisions become more pronounced in temporal identity in later stages as cells go through cell cycles. This and the remaining presence of a few Hb/Kr-double positive clones at late stages indicate that, in dlg-Gβ13F neuroblasts, cell cycle progression is not always linked to temporal identity progression as expected from looking at corresponding wild type lineages, although the progression of temporal identity is seen in this mutant (Kitajima, 2010).

Termination of temporal identity progression may depend on the amount of the basal determinants, including Prospero, given that the transition of temporal identity genes do not occur in wild type GMCs. Indeed, in dlg-Gβ13F neuroblasts, as cells divide, the size of the cell size is rapidly reduced to approach a GMC-like state. It is thus speculated that the progeny of symmetrically dividing neuroblasts eventually assume a GMC-like state, thereby terminating temporal identity gene progression prematurely (Kitajima, 2010).

A remarkable finding in this study is the opposite nature of the progeny of dlg-Gβ13F mutant neuroblasts in embryos and in larvae. When created at larval stages, dlg-Gβ13F mutant neuroblasts generate continuously proliferating progeny after reducing their cell size, in contrast to the embryonic situation. This difference would appear to reflect differences in the proliferation control of neuroblasts in the embryonic and larval stages. The function of the pros gene, which negatively regulates genes promoting cell cycle progression, appears to be pivotal because Pros functions as a tumor suppressor in larval brains but not in embryos. This difference in the effect of the loss of Pros has been attributed to the redundancy of Pros with Brat in embryos, while they are both necessary for normal larval lineages. pros mutant larval clones are thought to form tumors by the transformation of GMCs into proliferative cells, although proliferative dlg-Gβ13F mutant cells are likely derived from the transformation of neuroblasts into proliferative cells that undergo symmetric divisions (Kitajima, 2010).

Given that embryonic dlg-Gβ13F mutant neuroblasts appear to become GMC-like cells that inherit sufficient amounts of the basal determinants to differentiate, a simple explanation for the continuous proliferation of larval dlg-Gβ13F mutant cells is that the effective dosage of Pros (or other basal determinants) in those cells is insufficient to induce differentiation, unlike in their embryonic counterparts. Indeed, it was shown that elevation of Pros expression can induce proliferating cells in the dlg-Gβ13F mutant clones to exit the cell cycle and differentiate. It is of interest that, in interphase dlg-Gβ13F mutant cells of both embryonic and larval stages, Miranda is mainly cytoplasmic and Pros is largely nuclear, while during mitosis these proteins appear to form a cortical complex. There may be a larval mechanism by which neuroblasts reduce the nuclear entry of Pros in both wild-type and dlg-Gβ13F mutant cells. The ability of neuroblasts to prevent the nuclear import of Pros when it is overexpressed under the heatshock promoter was tested, and it was found that larval neuroblasts do not accumulate Pros protein in the nucleus at all under conditions in which embryonic neuroblasts show Pros nuclear accumulation. These results suggest that, compared with embryonic neuroblasts, larval neuroblasts have a strong ability to prevent nuclear accumulation of Pros (Kitajima, 2010).

A recent study has shown that cell cycle exit at the end of larval thoracic neurogenesis is programmed to reduce cell volume by symmetric divisions and nuclear localization of Pros; this is regarded as the mechanism terminating neuroblast division and allowing differentiation. As shown in larval Gβ13F mutant neuroblasts, the reduction of cell volume only limits the proliferative state or rate by idling or slowing the cell cycle progression, but does not induce differentiation. Furthermore, symmetric neuroblast divisions in the dlg-Gβ13F mutant resulted in reduction of cell volume and nuclear accumulation of Pros (although at a low level), but caused continuous proliferation of daughter cells. It may be that unlike the dlg-Gβ13F mutant, the level of nuclear Pros becomes high enough to terminate the cell cycle when wild-type neuroblasts stop division in the larval thorax. Alternatively, the progression of temporal identity in neuroblasts may induce additional mechanisms that cause neuroblasts to exit from the cell cycle into the differentiated state, as in the case for embryonic neuroblasts (Kitajima, 2010).

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 γ subunit 1 (Gγ1) has been shown to mediate cardial-pericardial cell adhesion in Drosophila. This study shows that G-oα47A and Gβ13F cooperate with Gγ1 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 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 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, 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, 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 α-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).

Drosophila Ric-8 regulates Gαi cortical localization to promote Gai-dependent planar orientation of the mitotic spindle during asymmetric cell division

Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of Galphai depends on Partner of Inscuteable (Pins). This study establishes that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Galphai, regulates cortical localization of the subunits Galphai and Gbeta13F. Ric-8 , Galphai and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis (David, 2005).

Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins; Ric-8 complexes with Pins through their mutual interactions with Galpha

Asymmetric division of Drosophila neuroblasts (NBs) and the C. elegans zygote uses polarity cues provided by the Par proteins, as well as heterotrimeric G-protein-signalling that is activated by a receptor-independent mechanism mediated by GoLoco/GPR motif proteins. Another key component of this non-canonical G-protein activation mechanism is a non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, RIC-8, which has recently been characterized in C. elegans and in mammals. The Drosophila Ric-8 homologue is required for asymmetric division of both NBs and pI cells. Ric-8 is necessary for membrane targeting of Galphai, Pins and Gbeta13F, presumably by regulating multiple Galpha subunit(s). Ric-8 forms an in vivo complex with Galphai and interacts preferentially with GDP-Galphai, which is consistent with Ric-8 acting as a GEF for Galphai. Ric-8 complexes with Pins through their mutual interactions with Galpha. Comparisons of the phenotypes of Galphai, Ric-8, Gbeta13F single and Ric-8;Gbeta13F double loss-of-function mutants indicate that, in NBs, Ric-8 positively regulates Galphai activity. In addition, Gbetagamma acts to restrict Galphai (and GoLoco proteins) to the apical cortex, where Galphai (and Pins) can mediate asymmetric spindle geometry (Wang, 2005).

In neuroblasts (NBs), two apically localized protein cassettes -- 1. Bazooka, Par3-DmPar6-DaPKC0 and 2. Galpha-Partner of Inscuteable [Pins, a GDP dissociation inhibitor (GDI) of Galpha], that are linked by Inscuteable (Insc) -- mediate all aspects of NB asymmetric division. These two conserved protein cassettes are spatially separated in pI cells of the sensory organ precursor (SOP) lineage: Pins-Galpha localizes to the anterior, whereas Baz-Par-6-DaPKC localizes to the posterior cortex. In both Drosophila and C. elegans asymmetry models, the activation of heterotrimeric G-protein signalling apparently occurs via a receptor-independent mechanism that is mediated by proteins containing GoLoco/GPR (G-protein regulatory) motifs with GDI activity (for example, Drosophila Pins and nematode GPR1/2), which can compete with Gbetagamma for GDP-Galpha. With respect to the spindle geometry of Drosophila NBs, Gbeta13FGgamma1 seems to have a more crucial role than Galpha and Pins in this process. By contrast, Galpha subunits, GOA-1 and GPA-16, and the GoLoco proteins GPR1/2 are essential in C. elegans, for the generation of a net posterior force that is necessary for asymmetric spindle positioning. Gbetagamma, in contrast, does not play a positive role in this process. More recently, RIC-8, a novel non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, has been shown to be required for asymmetric spindle positioning in the C. elegans zygote. This study characterizes the role of the Drosophila Ric-8 homologue in neural progenitor asymmetric division (Wang, 2005).

Database searches of rat Ric-8A identified a putative Drosophila homologue, Ric-8 (CG15797, at cytological position 8D10 of the X chromosome), which shares ~31% amino-acid identity with rat Ric-8A. Ric-8 RNA is ubiquitously expressed with an abundant maternal component. In glutathione S-transferase (GST) pull-down assays, GST-Ric-8 interacts directly with Galpha in vitro. In co-immunoprecipitation experiments using embryonic extracts, Ric-8, similarly to Pins and Gbeta13F, interacts strongly with Galpha when GDP has been added in excess, but interacts poorly with Galpha in the presence of excess GTP-gammaS. This indicates that Ric-8 preferentially interacts with GDP-Galpha. These interactions are consistent with Ric-8 acting as a GEF for Galpha, similarly to its mammalian and nematode homologues (Wang, 2005).

To ascertain that the in vitro binding of Ric-8 with Galpha reflects an in vivo association, co-immunoprecipitation experiments were performed using embryonic extracts. Ric-8 was detected in immunocomplexes when precipitation was performed with anti-Galpha but not with the pre-immune control, indicating that Ric-8 complexes with Galpha in vivo. To further substantiate this interaction using a different approach, protein extracts from wild-type embryos were incubated with agarose beads coupled to bacterially expressed MBP-Galpha or MBP protein. Ric-8 was detected in the bound complex with MBP-Galpha but not MBP (Wang, 2005).

In Drosophila NBs, Galpha is present in at least two mutually exclusive complexes: a heterotrimeric complex with Gbeta13F, or with a GoLoco-containing protein, Pins, which acts as a GDI for, and can directly interact with Galphai. Conventional G-protein-coupled receptors (GPCRs) promote nucleotide exchange on the Galphai-Gbetagamma heterotrimeric complex, whereas the mammalian non-receptor GEF RIC-8A cannot act on the heterotrimer. To explore the molecular context in which Ric-8 might act on Galpha, whether Ric-8 can complex with Pins or Gbeta13F was examined in Drosophila using co-immunoprecipitation experiments with embryonic extracts. When precipitations were performed using anti-Ric-8, Pins was specifically detected in the immunocomplex; in precipitations using anti-Pins, Ric-8 was also specifically detected. No direct interaction was observed with Ric-8 and Pins in the in vitro binding assays, indicating that Ric-8 complexes with Pins through their mutual interactions with Galpha. To confirm these findings using a different approach, wild-type embryonic extracts were incubated with agarose beads coupled to bacterially expressed MBP-Ric-8 fusion protein. Pins but not Gbeta13F was found in the bound complex with MBP-Ric-8. Thus, Ric-8 preferentially binds to the GDP-Galpha-Pins complex, a similar finding to that seen in C. elegans embryos. This is in contrast to conventional GPCRs, which act on the heterotrimeric complex (Wang, 2005).

To determine the effects of ric-8 loss of function, several mutant alleles were isolated by imprecise excision of a P-element, EY05996. ric-8^P587 removes the entire coding region (-953 bp to +1853 bp; ric-8 transcriptional start is +1), whereas ric-8^P340 contains a larger deletion with unsequenced breakpoints. Both maternal and zygotic components were removed in the ric-8^P340 and ric-8^P587 germline clones (GLCs). These mutant embryos showed indistinguishable phenotypes, indicating that both are null alleles. Experiments were performed using embryos that were derived from ric-8^P587 GLCs (Wang, 2005).

Galpha shows punctated, cytosolic distribution in dividing and non-dividing NBs of ric-8 GLCs, in contrast to the apical cortical crescents seen in wild-type NBs. Pins also seemed to be cytosolic, which is consistent with findings that Galpha is required for the recruitment of Pins to the cortex. The issue of whether Gbeta13F is also required for membrane targeting of Galpha was examined using a newly generated anti-Galpha antibody, as it was unclear whether the reported inability to detect Galpha in Gbeta13F mutant NBs by immunofluorescence was due to low sensitivity of the previously available antibody. The specificity of this new antibody was demonstrated by the absence of immunoreactivity in Galphai mutant embryos or nota in both immunofluorescence and Western experiment. It was found that Galpha was uniformly localized on the cortex of Gbeta13F GLC NBs, with clearly reduced intensity compared with the wild type. Pins was also uniformly cortical in Gbeta13F GLC NBs, which indicates that the residual Galpha on the cortex is sufficient to recruit Pins. The localization of Galpha and Pins in blastoderm embryos that were derived from ric-8 and Gbeta13F GLCs lends further support to these findings. Strikingly, Galpha and Pins localized as punctated, cytosolic 'spots' in ric-8 GLC embryos, whereas in both wild-type and Gbeta13F GLC embryos, Galpha was membrane associated. Therefore, ric-8, but not Gbeta13F, is crucial for the membrane targeting of Galpha in NBs and other cell types (Wang, 2005).

In ric-8 GLC NBs, Insc was cytosolic. Baz and aPKC localized non-uniformly/asymmetrically on the cortex, but with reduced intensity and often as broader crescents, indicating that residual polarity cues remained. Mira crescents were often mislocalized in metaphase ric-8 NBs; mitotic domain 9 cells failed to re-orient their spindle by 90°, indicating that ric-8 is required for spindle re-orientation in cells of mitotic domain 9. These defects are similar to those seen in Galphai mutant NBs. Ric-8 is also required for the asymmetric division of pI cells. In ric-8 mutant metaphase pI cells, Galpha and Pins did not form the anterior cortical crescents. Similarly, in Galphai metaphase pI cells, the anterior crescent of Pins did not form. In both ric-8 and Galpha mutants, the Pon crescent was undetectable or significantly reduced. Nevertheless, Pon localized at the anterior cortex in anaphase pI cells of both mutants (Wang, 2005).

Antibodies specific for Ric-8 were generated against the amino-terminal (aa 1-150) or carboxy-terminal (aa 425-573) region of Ric-8. Ric-8 was localized to the cytoplasm of NBs throughout the cell cycle, even though Galpha was seen as an apical crescent in mitotic NBs. However, interestingly, Ric-8 was also observed as 'spot'-like structures at the apical cortex of metaphase NBs, partially colocalizing with the Galpha, indicating that their interaction might occur on the cytosolic face of the plasma membrane or in the cytoplasm. Similarly, in pI cells, Ric-8 was also cytosolic throughout the cell cycle (Wang, 2005).

ric-8 GLCs also exhibit abnormal gastrulation, in addition to defects in asymmetric divisions. Since gastrulation defects were also seen in Gbeta13F and Ggamma1 GLC embryos but not in Galphai embryos, the relationship was examined between ric-8 and Gbeta13F. During cellular blastoderm formation, Gbeta13F is delocalized from the cortex and is largely cytosolic in ric-8 GLC embryos, indicating that ric-8 is required for cortical localization of Gbeta13F during these early stages. Consistently, Gbeta13F is also largely cytosolic in NBs throughout the various stages of the cell cycle in stage-10 embryos derived from ric-8 GLCs. Given that Galphai loss of function alone does not disturb Gbeta13F localization and Gbeta13F does not complex with Ric-8, it was hypothesized that Ric-8 mediates the cortical localization of Gbeta13F through its regulation of another Galpha subunit. To further explore this possibility, it was asked whether Ric-8 can complex with Pins in embryos devoid of maternal and zygotic Galphai. If there was another Galpha subunit involved, it might allow Ric-8 to complex with Pins by interacting with both, even in the absence of Galpha. Indeed, Ric-8 complexes with Pins in the absence of Galpha. Given that Ric-8 does not display a direct interaction with Pins, these data indicate that an, as yet unidentified, Galpha subunit that is also regulated by Ric-8 may act (possibly in conjunction with Galpha) to mediate Gbeta13F cortical localization (Wang, 2005).

Gbeta13F protein levels in ric-8 GLCs are significantly reduced compared with wild-type embryos; Galpha and Pins levels remain unaffected. By contrast, Galpha protein levels in Gbeta13F GLCs are reduced, whereas Ric-8 levels do not change in Galpha or Gbeta13F GLCs. Gbetagamma might normally be in excess; therefore, despite the reduction in Gbetagamma levels in ric-8 mutants, sufficient cytosolic levels may remain to stabilize normal levels of Galpha. These data indicate that Ric-8 is required only for membrane targeting of Galpha but not its stability; Gbeta13F is required for the stability of Galpha but not for its membrane targeting. In addition, Ric-8 is involved in both membrane association and the stability of Gbeta13F, possibly by acting through another Galpha subunit (Wang, 2005).

The requirement of Ric-8 for cortical localization and stability of Gbeta13F prompted an examination of whether NB spindle geometry and difference in daughter-cell size are severely disrupted in ric-8 mutants, as shown for Gbeta13F GLCs. In telophase NBs of wild-type stage-10 embryos, the ratio of ganglion mother cell (GMC) and NB (GMC/NB) diameter never exceeded 0.8 (average ratio = 0.42. By contrast, a hallmark of Gbeta13F or Ggamma1 loss is the high frequency of divisions that generate daughters of approximately equal size. These cells are telophase NBs in which the GMC diameter/NB diameter ratio was 0.8 or more (for Gbeta13F NBs, 64% of divisions were similar sized with an average GMC/NB ratio of 0.82. The residual size asymmetry which remained was shown to be due to the reduced levels of asymmetrically localized Par proteins. However, a surprising observation was that, although cortical Gbeta13F localization was disrupted in ric-8 mutant NBs, only 16% of telophase NBs divided into two similar-sized daughter cells, similar to those observed in Galphai mutant NBs. Thus, ric-8 GLC NBs did not display a phenotype similar to that of Gbeta13F loss-of-function mutants. Further removal of Baz (by RNA interference) in ric-8 GLCs resulted in similar-sized division in 94% of NBs, indicating that partially localized Baz (Par proteins) can provide some asymmetry cues in ric-8 mutant NBs. Therefore, Ric-8 probably acts in the same pathway as Galpha to redundantly regulate the difference in daughter-cell size in the Baz pathway. It was shown previously that in Gbeta13F mutants, the number of abdominal Even-skipped positive lateral (EL) neurons in stage-15 embryos was severely decreased, presumably because a high frequency of similar-sized divisions rapidly reduces the cell volume of daughter NBs, resulting in early cessation of divisions. It was found that wild-type embryos produced an average of 9.0 EL neurons per abdominal hemisegment at stage 15; both ric-8 GLCs and Galphai mutants showed a similar reduction of EL neurons. By contrast, Gbeta13F GLC embryos showed a more marked reduction in the numbers of EL neurons. These data indicate that, with respect to both numbers of EL neurons and NB daughter-cell size asymmetry, ric-8 and Galpha mutants exhibit similar phenotypes that are less severe than those seen in Gbeta13F mutants (Wang, 2005).

Two alternative explanations are envisioned for why ric-8 and Gbeta13F mutants have different effects on the asymmetric size of the daughter cells. (1) The generation of functional Gbetagamma may occur even in the absence of ric-8 function, despite the majority of the molecules being cytosolic. (2) Alternatively, the severe phenotypes seen in Gbeta13F or G gamma1 mutant NBs may be an indirect consequence caused by the uniform cortical distribution of Galpha (and Pins); the failure of ric-8 GLC NBs to exhibit a marked decrease in asymmetric daughter size would be because Galpha and Pins are both cytosolic in ric-8 mutants and presumably inactive. To distinguish between these possibilities, ric-8, Gbeta13F double mutant GLC embryos were made in which both ric-8 and Gbeta13F would be completely removed. Interestingly, the double mutant GLC NBs exhibited phenotypes similar to those of ric-8 GLC NBs rather than Gbeta13F GLC NBs. In double GLC NBs, Galpha and Pins are cytosolic, whereas Baz localized non-uniformly/asymmetrically on the cortex. Only 24% of NBs divided into two similar-sized daughter cells. These observations indicate that the cytoplasmic Gbetagamma in ric-8 GLC NBs is non-functional and further suggests that the marked decrease in the difference in daughter-cell size of Gbeta13F GLC NBs is an indirect consequence of the uniform cortical localization of Galpha (and Pins) (Wang, 2005).

These data indicate that ric-8 mutants mediate asymmetric division of NBs and SOPs by regulating heterotrimeric G-protein localization. ric-8 acts at the top of a hierarchy for the sequential membrane/cortical localization of the apical proteins Galphai-Pins-Insc. The role of Ric-8 in membrane targeting of Galpha is novel. Interestingly, Ric-8 also promotes cortical localization of Gbeta13F in Drosophila. These data raise the possibility that this may be mediated indirectly by additional substrate(s) of Ric-8, which are presumably additional Galpha subunit(s). Rat Ric-8A interacts with multiple brain membrane Galpha subunits, including Galpha13, Galphao, Galphaq and Galpha1,2. It is therefore speculated that Ric-8 may control the localization and stability of Gbeta13F by regulating multiple Galpha subunits. Precedence for a role of Galpha in Gbetagamma membrane localization has been reported in mammalian cells (Wang, 2005).

This analyses of ric-8, Galphai, Gbeta and ric-8;Gbeta mutants support the view that, in NBs, cortically localized Galpha mediates asymmetric spindle geometry and asymmetric daughter-cell size, which is positively regulated by Ric-8, and that an important role of Gbetagamma is to restrict Galpha from the basal cortex. In the absence of Gbetagamma, the GoLoco/Galpha complex expands from its normal apical localization, becomes uniformly cortical and can largely override the residual polarity cues that are provided by the asymmetrically localized, but drastically reduced levels of, Par proteins to greatly reduce spindle asymmetry and the difference in daughter size. The residual asymmetry that is present in the absence of Gbeta13F is lost following further removal of Par function. The negative regulation of Galphai by Gbeta13F in Drosophila NBs is similar to that in the C. elegans zygote, in which excess Galpha activity was observed following loss of function of Gbeta or Ggamma. The findings that ric-8 mutants are genetically epistatic to Gbeta mutants, both with respect to Galphai-Pins localization and to spindle geometry, are different from those reported in C. elegans embryos, in which inactivation of Gbetagamma alleviates the requirement for RIC-8 in asymmetric division. This indicates that different mechanisms of heterotrimeric G-protein regulation are present in the asymmetric division of nematode embryos and Drosophila NBs. These findings are consistent with a model in which Ric-8 has a crucial role in Galpha activity by localizing the GoLoco/Galpha complex onto the cortex and/or generating GTP-Galpha as a GEF to mediate spindle geometry. Ric-8 also regulates the cortical localization and activity of Gbeta, possibly through its regulation of multiple Galpha subunits; Gbeta acts to restrict Galpha localization only to the apical cortex. Galpha subunits that are asymmetrically localized at the apical cortex, in conjunction with Par proteins, mediate asymmetric spindle geometry and differences in daughter-cell size (Wang, 2005).

Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division

Drosophila neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. This study identified a Drosophila mutant in the Ggamma1 subunit of heterotrimeric G protein, which produces Ggamma1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gß13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gß13F to the membrane, indicating an essential role of cortical G1-Gß13F signaling in asymmetric divisions. In Ggamma1 and Gß13F mutant NBs, Pins-Galphai, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gßgamma and other mutants indicates a predominant role of Partner of Inscuteable-Gi in spindle orientation. It is thus suggested that the two apical signaling pathways have overlapping but different roles in asymmetric NB division (Izumi, 2004).

Because the Gß13F-Ggamma1 complex, which distributes uniformly in the cortex, functions in asymmetric organization of the spindle, differential activation or inactivation of Gßgamma signaling must occur in the apical-basal direction. Two apical signaling pathways are implicated in the apical-basal difference in spindle development in a redundant fashion. What is the relationship between the apical signals and the Gßgamma signal? Spindle size is reduced by an increase in the amount of Gßgamma, but a lack of Gßgamma results in formation of a large, symmetric spindle. These findings raise the possibility that spindle development is suppressed by the Gßgamma signal, which is repressed by the presence of an apical complex on the apical side in the wild-type cells, resulting in a large apical and small basal spindle. This model suggests that the apical complex acts upstream of the Gßgamma signal. In contrast, elimination of Gß13F affects the localization of the apical components: Pins becomes uniformly distributed and Galphai becomes undetectable. In addition, Ggamma1^N159 and Gß13F mutations appear to destabilize the localization of the components in the Baz-DaPKC pathway, as judged by the reduced staining by their antibodies (although this may be an indirect consequence of the mislocalization of Pins-Galphai). The Gßgamma signal is thus required for normal distribution of the components of both apical pathways, consistent with the idea that the apical pathways acts downstream of the Gßgamma signal in regulating spindle asymmetry. Tests for epistasis between the apical pathways and the Gßgamma signal are needed to clarify their relationship in the regulation of spindle organization (Izumi, 2004).

The effects of Ggamma1^N159 and Gß13F mutations on cell-size asymmetry are remarkable but different from those in double mutants in which both apical pathways are disrupted simultaneously, where daughter cell sizes are completely equal. The cell-size ratio of GMCs to their sibling NBs shows a broad distribution: from 0.6 to 1 in the Ggamma1 (and Gß13F) mutants. This residual asymmetry in daughter cell size is due to Baz-DaPKC activity. The components of this pathway indeed distribute asymmetrically in Ggamma1 (and Gß13F) mutant NBs in which Pins-Galphai activity is no longer asymmetric (Pins is uniformly distributed and Galphai is absent) (Izumi, 2004).

Why does this polarized Baz-DaPKC activity cause less asymmetry in daughter cell size in spite of the redundant function of the Baz-DaPKC pathway and Pins-Galphai? Antibody staining for Baz, DaPKC, and DmPar-6 suggests that their levels and their polarized distribution are weakened in Ggamma1 (and Gß13F) mutants. A possible explanation is that low levels of polarized Baz-DaPKC activity confer only low levels of asymmetry to the daughter cell size in the absence of polarized Pins-Galphai. Thus, the degree of cell-size asymmetry resulting from NB divisions may depend on the dosage of the components of one apical pathway when the other is absent or uniformly distributed. In contrast, Miranda localization does not appear to be severely impaired in Ggamma1^N159 and Gß13F mutants until late embryonic stages, indicating that the polarized Baz-DaPKC activity in these mutants is sufficient to localize Miranda. Therefore, full asymmetry in daughter cell size may require relatively higher levels of Baz-DaPKC activity than does polarized distribution of cell-fate determinants does (Izumi, 2004).

In Ggamma1^N159 and Gß13F mutants, Insc has a distribution different from the other components of the Baz-DaPKC pathway. In most of these mutant NBs, Insc distributes broadly to both the cytoplasm and the cortex in a slightly asymmetric way, but Baz, DaPKC, and DmPar-6 localize asymmetrically in the cortex. The cytoplasmic distribution of Insc is also slightly asymmetric in pins mutant NBs. It is not known whether cytoplasmic Insc is functional. Interestingly, Insc distribution often appears to correlate better with the asymmetry in daughter cell size than do the other components of the Baz-DaPKC pathway in Ggamma1^N159 and Gß13F mutants: in most telophase NBs that are cleaving into two equal daughters, DaPKC and DmPar-6 are excluded from the daughter GMC, but Insc tends to distribute evenly to both daughter cells. This occurs also in pins mutants, in which ~15% of NBs divide equally but most NBs divide unequally. In pins NBs cleaving equally, Insc is found equally in the cytoplasm of both daughter cells, but DaPKC and DmPar-6 remain in the newly forming NB; in unequally dividing NBs, all three components are found preferably on the NB side. These observations raise the intriguing possibility that Insc has more important roles in the generation of spindle asymmetry than do the other components of the Baz-DaPKC pathway. Because the absence of Baz results in mislocalization of Insc and vice versa, it is technically difficult to discriminate Insc-specific from Baz-specific functions. It may be Insc or some unknown Insc-associating effectors, rather than Baz, that functions in parallel with Pins-Galphai in the establishment of cell-size asymmetry (Izumi, 2004).

The question of whether the two apical pathways have redundant functions in aspects of NB division other than cell-size asymmetry has been elusive. In this paper, examination of Ggamma1^N159 and Gß13F mutant NBs, as well as those overexpressing baz, suggests that the asymmetric localization of Miranda depends solely on polarized Baz activity and not on Pins-Galphai function. Miranda always distributes on the cortical side, opposite the distribution of Baz in these mutants and in the wild-type. This also occurs for sensory precursor cells in the peripheral nervous system: in sensory precursor cell division Insc is not expressed, and Pins and Baz distribute on cortical sides opposite to each other, unlike in NBs; however, both Miranda and Numb localize to the cortex opposite Baz, as seen in NBs (Izumi, 2004).

Phosphorylation of the Lethal (2) giant larvae protein by DaPKC directs the localization of cell-fate determinants to the basal cell cortex. When baz is overexpressed in NBs, ectopically distributed Baz excludes Miranda from the Baz region and DaPKC colocalizes with the ectopic Baz. In contrast, a decrease in Baz activity in the wild-type results in cytoplasmic localization of DaPKC and uniform cortical distribution of Miranda. All these findings suggest that the Baz-directed localization of DaPKC excludes Miranda from the apical cortex via Lethal (2) giant larvae phosphorylation. In the absence of Baz, Miranda is eventually concentrated to the budding GMC during telophase by unknown mechanisms, a phenomenon called 'telophase rescue'. This phenomenon did not occur by depleting both baz activity and Gßgamma signaling, suggesting that telophase rescue involves Gßgamma signaling or asymmetric Pins-Galphai localization (Izumi, 2004).

The absence of any single component of the apical complex has the same effect on spindle orientation during NB division, which is normally perpendicular to the apical-basal axis. Thus, proper orientation of the spindle has been thought to require all the apical components. However, observations on epithelial cells and mitotic domain 9 cells indicated that the spindle always points to the location of Pins when Pins is localized in the cell. This alignment of the spindle toward Pins occurs irrespective of the localization of the Baz-pathway components. For instance, wild-type epithelial cells divide parallel (Pins direction) but not perpendicular to the apical-basal axis (Baz direction); so do most epithelial cells and mitotic domain 9 cells in Gß13F and Ggamma1 mutants. Therefore, the Pins-Galphai pathway, rather than the Baz-DaPKC complex, is likely to play a dominant role in controlling spindle orientation (Izumi, 2004).

In most NBs in pins, Gß13F, and Ggamma1 mutants, the spindle is oriented in the direction of Baz localization and therefore follows the localization of the cell-fate determinants. This coincidence results in the determinants' virtually normal segregation to one daughter cell despite the random orientation of division. Thus, only when Pins-Galphai are absent or uniformly distributed in NBs, polar Baz activity appears to be capable of directing spindle orientation. Alternatively, the mitotic spindle may position the Baz-DaPKC complex over one spindle pole (Izumi, 2004).

In the NB in which the Baz-DaPKC pathway is depleted, Pins-Galphai can still localize asymmetrically and orient the spindle. Interestingly, the Pins crescent forms in random orientations in this situation, leading to random spindle orientation. This fact suggests that the Baz-DaPKC complex or its combination with Pins-Galphai is necessary to orient the Pins-Galphai crescent in the apical direction of the NB, raising an intriguing possibility that there are unknown mechanisms by which formation of the apical complex occurs on the apical side. This postulated mechanism may involve interactions with neighboring epithelial cells (Izumi, 2004).

What is the molecular mechanism by which Pins-Galphai orient the spindle? It is interesting to assume that Pins has the ability to attract the spindle pole. This idea is consistent with previous evidence; although epithelial cells do not normally express Insc, its ectopic expression in these cells recruits Pins-Galphai to the apical cortex and reorients the mitotic spindle in the apical-basal direction. The C. elegans homologues of Pins, GPR-1/GPR-2, interact with Galphai/Galphao and a coiled-coil protein, LIN-5, which is required for GPR-1/GPR-2 localization. All these molecules are indeed involved in the regulation of forces attracting spindles during early cleavages. Although Lin-5 has no obvious homologue in other species, functional homologues may regulate Pins localization and/or the connection between the spindle pole and Pins in Drosophila. Furthermore, the C. elegans gene ric-8, which interacts genetically with a Galphao gene, is also required for embryonic spindle positioning. Its homologue in mammals acts as a guanine nucleotide exchange factor for Galphao, Galphaq, and Galphai. An analysis of the Drosophila RIC-8 homologue may give insight into the mechanisms by which Pins-Galphai regulate spindle orientation (Izumi, 2004).

Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions

Cell division often generates unequally sized daughter cells by off-center cleavages, which are due to either displacement of mitotic spindles or their asymmetry. Drosophila neuroblasts predominantly use the latter mechanism to divide into a large apical neuroblast and a small basal ganglion mother cell (GMC), where the neural fate determinants segregate. Apically localized components regulate both the spindle asymmetry and the localization of the determinants. This study shows that asymmetric spindle formation depends on signaling mediated by the G beta subunit of heterotrimeric G proteins. Gβ13F distributes throughout the neuroblast cortex. Its lack induces a large symmetric spindle and causes division into nearly equal-sized cells with normal segregation of the determinants. In contrast, elevated Gβ13F activity generates a small spindle, suggesting that this factor suppresses spindle development. Depletion of the apical components also results in the formation of a small symmetric spindle at metaphase. Therefore, the apical components and Gβ13F affect the mitotic spindle shape oppositely. It is proposed that differential activation of Gβ signaling biases spindle development within neuroblasts and thereby causes asymmetric spindles. Furthermore, the multiple equal cleavages of Gβ mutant neuroblasts accompany neural defects; this finding suggests indispensable roles of eccentric division in assuring the stem cell properties of neuroblasts (Fuse, 2003).

In the first division of C. elegans eggs, eccentric cleavage occurs due to the displacement of the symmetric spindle, which is pulled asymmetrically by astral microtubules. This process depends on Gα, but not on Gβ. In contrast, the unequal-sized divisions of Drosophila neuroblasts are predominantly promoted by the asymmetric organization of the mitotic spindle, which requires biased microtubule development along the apical-basal axis. This study has shown an essential role of Gβ13F in forming asymmetric spindles in neuroblasts. The elimination of Gβ13F activity enhances spindle development, but its elevation inhibits spindle growth. These findings suggest that Gβ signaling acts to suppress microtubule development. In comparison, simultaneous disruption of the two apical pathways appears to reduce the size of mitotic spindles; this finding suggests that these signals normally act to enhance spindle development. Therefore, Gβ and the two apical signals likely exert opposite effects on microtubule development. These observations led to a simple model in which Gβ signaling is active on the basal cortex to suppress spindle growth but is inhibited by the apical signals on the apical side. In an alternative model, the apical signals enhance spindle growth, and Gβ13F acts to exclude this activity of the apical complex from the basal side. Currently, both models equally explain the data obtained in this study and suggest that Gβ signaling confers the basal character to the cell cortex. This differential Gβ signaling ultimately induces biased spindle development, which results in the asymmetric spindle. For better understanding of the mechanisms that regulate spindle asymmetry, it would be necessary to assess where Gβ13F is active in neuroblasts and to elucidate how it relates to the apical signals (Fuse, 2003).

Distinct roles of Galphai and Gbeta13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions

The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprised of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Galphai. In addition, Gß13F is also required, however, the role of Galphai and the hierarchical relationship between the G protein subunits and apical components are not well defined. This study describes the isolation of Galphai mutants and shows that Galphai and Gß13F play distinct roles. Galphai is required for Pins to localize to the cortex, and the effects of loss of Galphai or pins are highly similar, supporting the idea that Pins/Galphai act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gß13F appears to regulate the asymmetric localization/stability of all apical components, and GßF loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, these results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts (Yu, 2003).

This study reports the isolation and analysis of loss of function mutations in Galpha and show that the loss of Galpha and Gß13F have distinct effects on NB asymmetric cell divisions. Galphai is required for Pins cortical association and asymmetric localization; loss of Galphai causes Pins to localize to the cytosol, and mutant NBs exhibit phenotypes that are highly similar to those seen in pins mutants. Analyses of double mutant combinations confirm Galphai RNAi results showing that Pins/Galphai and Baz/DaPKC/Insc act in an redundant fashion to mediate the formations of an asymmetric mitotic spindle and the generation of NB daughters of unequal size. Importantly, these analyses also revealed a new aspect of apical complex function: that the two apical pathways also act redundantly to suppress the formation of astral microtubules from the basal centrosome of NBs. In contrast, Gß13F appears to act upstream of the apical components and is required for their asymmetric localization/stability. The defects associated with NBs lacking Gß13F function are highly similar to those seen when the function of both apical pathways have been compromised. In addition, it was shown that high level overexpression of two different Galpha subunits, which can bind/complex to Gß13F, results in similar phenotypes seen in Gß13F mutant NBs, suggesting that it is the depletion of free Gß13F, which is responsible for the mutant phenotypes (Yu, 2003).

Pins and Galphai apical localization are mutually dependent. In pins NBs, Galphai is evenly distributed to the NB cortex, and in Galpha mutant NBs, Pins localizes to the cytosol. Pins asymmetric localization to the apical cortex of the NBs is a two-step process: Pins needs to be targeted to the cortex first: this requires the COOH-terminal Goloco motifs that can bind Galphai before Galphai can be recruited to the apical cortex in a process which requires the Galphai NH₂-terminal TPR that can interact with Insc. The current results therefore suggest that Pins cortical targeting is most likely mediated by Galphai, which not only binds Pins, but also is able to localize to the plasma membrane through lipid modifications (Yu, 2003).

However, in Gß13F mutant NBs, although the levels of Pins are drastically reduced, the residual Pins is localized both to the cytosol and to the cell cortex. This poses a problem since in the Gß13F mutant NBs not only is Gß13F absent but Galphai also is undetectable with an anti-Galphai antibody. One possible explanation is that although Galphai is undetectable, there is still some Galphai remaining in the Gß13F NBs: this may account for the low level residual uniform cortical distribution of Pins. Alternatively, the possibility cannot be ruled out that the cortical Pins in Gß13F NBs is due to some unknown molecule that can recruit Pins to cortex in the absence of both Galphai and Gß13F (Yu, 2003).

The analysis of Gß13F function is complicated by the fact that in the Gß13F mutant NBs, Galphai levels are also down-regulated presumably due to the instability of the protein in the absence of Gß13F. Although loss of either Galpha or Gß13F causes aberrations in localization of the basal components and orientation of the mitotic spindle, it is clear that at least some of the defects associated with the loss of Gß13F cannot be attributable solely to the depletion of Galphai. In the great majority of Galphai mutant NBs, DaPKC and Baz still localize asymmetrically to a subset of the cell cortex. And consistent with the proposal that spindle geometry and the size asymmetry of the NB daughters are mediated by two redundant apical pathways, Pins/Galphai and Baz/DaPKC, the great majority (79%) of the Galpha mutant NBs generate an asymmetric mitotic spindle and divide to produce unequal size daughters. In contrast, in Gß13F NBs not only do Pins/Galphai always fail to become asymmetrically localized but the majority of mutant NBs (71%) also fail to asymmetrically localize Baz/DaPKC; consequently ~65% of NBs fail to generate an asymmetric mitotic spindle and divide to produce equal size daughters. Therefore, at least formally, Gß13F acts upstream of the two apical pathways (Yu, 2003).

It is believed that the major reason for the phenotypes associated with loss of Gß13F function is due to the disruption of Gßgamma signaling. Overexpression of Galphai will cause a high frequency of equal size divisions. In addition, overexpression of Galphao, a Galpha subunit that interacts with Gß13F but is not itself required for asymmetric divisions in wt NBs, will also mimic the Gß13F loss of function phenotype. For both overexpression of Galphai and Galphao, the frequency of equal size divisions is significantly higher than that seen in Gß13F loss of function. This difference may be due to the existence of other Gß subunits which might also function in NB asymmetric divisions. Three Gß genes have been identified by the Drosophila genome project, and although one of these genes, concertina, appears not to be involved in the process, it is possible that overexpression of Galpha molecules may deplete not only Gß13F but also Gß76C. This possibility could be addressed by the analysis of double mutants of Gß genes. Nevertheless, these observations are consistent with the view that the depletion of free Gßgamma, and not Galphai, is the major cause for the symmetric divisions seen in Gß13F mutant NBs. Hence, although previous analysis of Gß13F loss of function did not report any effects on NB daughter size, the current data are consistent with the notion that Gß13F plays a major role in mediating the distinct size of NB daughter cells (Yu, 2003).

The apical centrosome associates with prominent astral microtubules, whereas the basal centrosome connects to few if any astral microtubules in wt NBs and in mutants in which one of the two apical pathways is compromised. In contrast, in NBs that lack both apical pathways a symmetric mitotic apparatus is established that features extensive arrays of astral microtubules at both centrosomes. Therefore, either of the two apical pathways appears sufficient to prevent formation of basal astral microtubules. It is not clear how this might be accomplished at a mechanistic level. However, one might speculate that there exists an asymmetrically localized molecule, which can act to promote the formation of astral microtubules. When either of the apical pathways is functional, this molecule is asymmetrically localized and promotes the formation of astral microtubules only over the centrosome it overlies. However, when both apical pathways are mutated, or when Gß13F is mutated or when all apical components become uniformly cortical, e.g., when Galphai is overexpressed, then the hypothetical molecule becomes uniformly cortical and can promote the formation of astral microtubules over both centrosomes. This type of model can readily explain why either loss or uniform cortical localization of both apical pathways leads to symmetric astral microtubule formation over both centrosomes (Yu, 2003).

In summary, the results demonstrate that for NB asymmetric divisions Galphai and Gß13F play distinct roles. Galphai and Pins are members of one of the two apical pathways and Baz/DaPKC/Insc forms the other. Loss of Galphai function results in defects in NB asymmetry that are essentially indistinguishable from those seen in pins mutants. Gß13F (Gßgamma) functions upstream of both Pins/Galphai and Baz/DaPKC/Insc pathways to mediate their stability and/or asymmetric localization (and function). Without Gß13F, the function of both apical pathways are attenuated; Galphai levels are dramatically reduced and Pins/Galphai pathway is defective; in addition, the asymmetric localization of members of the Baz/DaPKC/Insc pathway is often defective. Consequently, loss of Gß13F function yields phenotypes that are similar to those seen when both apical pathways are disrupted by mutations (Yu, 2003).

Cloning of a Drosophila melanogaster guanine nucleotide regulatory protein β-subunit gene and characterization of its expression during development

A Drosophila gene encoding a protein with greater than 80% sequence identity to the beta subunits of mammalian guanine nucleotide-binding regulatory proteins (G proteins) has been cloned. The gene, which was mapped to 13F on the X chromosome by in situ hybridization, was cloned from a Drosophila genomic library by using a bovine transducin beta-subunit cDNA probe. Genomic DNA blot hybridization analysis indicated that there is a single Drosophila G-protein beta-subunit gene. Multiple transcripts were detected throughout development; in adult flies the mRNA is expressed at higher levels in heads than in bodies. The proposed coding region is uninterrupted by introns, but there is evidence for differential mRNA splicing in the 5' nontranslated region (Yarfitz, 1988).

Silencing of odorant receptor genes by G protein betagamma signaling ensures the expression of one odorant receptor per olfactory sensory neuron

Olfactory sensory neurons express just one out of a possible approximately 1,000 odorant receptor genes, reflecting an exquisite mode of gene regulation. In one model, once an odorant receptor is chosen for expression, other receptor genes are suppressed by a negative feedback mechanism, ensuring a stable functional identity of the sensory neuron for the lifetime of the cell. The signal transduction mechanism subserving odorant receptor gene silencing remains obscure, however. This study demonstrates in the zebrafish that odorant receptor gene silencing is dependent on receptor activity. Moreover, signaling through G protein βγ subunits was demonstrated to be both necessary and sufficient to suppress the expression of odorant receptor genes and likely acts through histone methylation to maintain the silenced odorant receptor genes in transcriptionally inactive heterochromatin. These results link receptor activity with the epigenetic mechanisms responsible for ensuring the expression of one odorant receptor per olfactory sensory neuron (Ferreira, 2014).

Search PubMed for articles about Drosophila Gbeta13f

Boyle, M. J. et al. (2010). Division of labor: Subsets of dorsal-appendage-forming cells control the shape of the entire tube. Dev. Biol. 346: 68-79. PubMed ID: 20659448

David, N. B., Martin, C. A., Segalen, M., Rosenfeld, F., Schweisguth, F. and Bellaiche, Y. (2005). Drosophila Ric-8 regulates Gαi cortical localization to promote Gai-dependent planar orientation of the mitotic spindle during asymmetric cell division. Nat. Cell Biol. 7(11): 1083-1090. PubMed ID: 16228010

Egger-Adam, D. and Katanaev, V. L. (2009). The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway. Dev. Dyn. 239(1): 168-83. PubMed ID: 19705439

Ferreira, T., Wilson, S. R., Choi, Y. G., Risso, D., Dudoit, S., Speed, T. P. and Ngai, J. (2014). Silencing of odorant receptor genes by G protein betagamma signaling ensures the expression of one odorant receptor per olfactory sensory neuron. Neuron 81: 847-859. PubMed ID: 24559675

Fuse, N., Hisata, K., Katzen, A. L. and Matsuzaki, F. (2003). Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions. Curr. Biol. 13: 947-954. PubMed ID: 12781133

Garcia De Las Bayonas, A., Philippe, J. M., Lellouch, A. C. and Lecuit, T. (2019). Distinct RhoGEFs Activate Apical and Junctional Contractility under Control of G Proteins during Epithelial Morphogenesis. Curr Biol 29(20): 3370-3385. PubMed ID: 31522942

Izumi, Y., et al. (2004). Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division. J. Cell Biol. 164(5): 729-38. PubMed ID: 14981094

Kitajima, A., Fuse, N., Isshiki, T. and Matsuzaki, F. (2010). Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development. Dev. Biol. 347(1): 9-23. PubMed ID: 20599889

Katanaev, V. L., Ponzielli, R., Semeriva, M. and Tomlinson, A. (2005). Trimeric G protein-dependent frizzled signaling in Drosophila. Cell 120: 111-122. PubMed ID: 15652486

Katanayeva, N., Kopein, D., Portmann, R., Hess, D. and Katanaev, V. L. (2010). Competing activities of heterotrimeric G proteins in Drosophila wing maturation. PLoS One. 5(8): e12331. PubMed ID: 20808795

Kerridge, S., Munjal, A., Philippe, J. M., Jha, A., de Las Bayonas, A. G., Saurin, A. J. and Lecuit, T. (2016). Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis. Nat Cell Biol 18(3): 261-70. PubMed ID: 26780298

Kimura, K., Kodama, A., Hayasaka, Y. and Ohta, T. (2004). Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster. Development 131: 1597-1606. PubMed ID: 14998927

Wang, H., Ng, K. H., Qian, H., Siderovski, D. P., Chia, W. and Yu, F. (2005). Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins. Nat. Cell Biol. 7(11): 1091-8. 16228012

Yarfitz, S., Provost, N. M. and Hurley, J. B. (1988). Cloning of a Drosophila melanogaster guanine nucleotide regulatory protein β-subunit gene and characterization of its expression during development. Proc. Natl. Acad. Sci. 85: 7134-7138. PubMed ID: 3140235

Yi, P., Johnson, A. N., Han, Z., Wu, J. and Olson, E. N. (2008). Heterotrimeric G proteins regulate a noncanonical function of septate junction proteins to maintain cardiac integrity in Drosophila. Dev. Cell 15(5): 704-13. PubMed ID: 19000835

Yu, F., Cai, Y., Kaushik, R., Yang, X. and Chia, W. (2003). Distinct roles of Galphai and Gbeta13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions. J. Cell Biol. 162(4): 623-633. PubMed ID: 12925708

date revised: 25 March 2014

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