BIOLOGICAL OVERVIEW

Gastrulation, the morphologenic process that creates mesoderm and endoderm from a uniform blastula early in development, involves multiple cell movements in Drosophila, unlike vertebrate gastrulation in which ingressing cells move through a single blastopore. During early stages of gastrulation in Drosophila, two populations of cells invaginate. At the posterior pole of the embryo, the posterior midgut invagination brings endodermal precursors into the interior. The ventral furrow forms along the ventral surface of the central body region of the embryo and internalizes the mesoderm precursors. Both these morphogenic movements are driven by individual cell shape changes within the invagination primordia. Both invaginations are initiated by apical constrictions that transform columnar cells to a wedge- or cone-like shape. As the cells constrict their apices (the outer surface of the cell), the apical plasma membrane folds into many small protrusions, or blebs, rather than decreasing in surface area. This observation suggests that the force that drives apical constriction is generated below the cell membrane. Since cytoplasmic myosin accumulates at the apical ends of cells in the ventral furrow and posterior midgut primordia at the start of cell shape changes, contraction of a cortical actin-myosin network may mediate apical constriction (Costa, 1994 and references).

How are the cell shape changes coordinated during gastrulation so that all cells in the gastrulating primordia behave synchronously? Two genes have been identified that are required specifically for the ventral furrow and the posterior midgut invaginations: the maternal gene concertina and the zygotic gene folded gastrulation (fog) do not seem to be essential for the mechanical aspects of constriction of the apical cytoplasm, but rather are necessary for coordination of cell shape changes throughout the invaginating primordia. Concertina is a G-protein alpha subunit, suggesting a role for cell-cell signaling in invagination (Parks, 1991). It is suggested Fog functions as a secreted signal that activates the G-protein alpha subunit encoded by concertina in neighboring cells, and thus a secreted signal ensures the rapid, orderly progression of constriction initiations from the middle of invagination primordia out toward the margins. Apparently Fog acts through an unidentified G-protein coupled receptor, that is, a 7 pass transmembrane serpentine receptor (Costa, 1994).

Additional components in this pathway have been identified. DRhoGEF2, a guanine nucleotide exchange factor for Rho1 mediates specific cell shape changes in response to the extracellular ligand, Folded gastrulation. fog was expressed ectopically from a huckebein promoter, which is normally active in a subset of cells at the anterior and posterior ends of the embryo. In all the hkb-fog expressing embryos a characteristic transient depression in the dorsal head region can be seen. The surfaces of cells in this depression exhibit membrane blebbing and constrictions closely resembling those normally seen in cells along the ventral furrow in wild-type embryos. In addition, the nuclei of these cells have migrated from an apical to a basal position. In the absence of DRhoGEF2, ectopic Fog expression fails to induce any detectable cell shape changes, despite equivalent levels of fog transgene expression. Together, these results establish a Rho-mediated signaling pathway that is essential for the major morphogenetic events in Drosophila gastrulation (Barrett, 1997).

The central domain of DRhoGEF2 contains a likely phorbol ester-response motif. The homologous domain in Protein kinase C mediates kinase activation in response to diacylglycerol, which is generate by phospholipase C (PLC). Thus is it possible that the GEF activity of DRhoGEF2 is responsive to diacylglycerol. Since PLC-mediated production of diacylglycerol can be promoted by both receptor tyrosine kinase activation and by activation of receptor-coupled heterotrimeric G proteins, it is possible that the nucleotide-exchange activity of DRhoGEF2 is stimulated by signals transduced by both of these types of receptors. The presence of a PDZ domain in DRhoGEF2, suggests that it may interact with additional signaling proteins. Therefore, it appears that the GEF activity of DRhoGEF2 may be regulated by multiple upstream signals (Barrett, 1997).

It is postulated that Fog acts via the G alpha protein Concertina to activate DRhoGEF2, thereby promoting Rho1 activation and consequent actin rearrangements. Significantly, the Drosophila G alpha subunit, Concertina, exhibits the strongest sequence similarity to the mammalian Galpha12 and Galpha13 proteins, which mediate the activation of Rho by LPA. Thus, it appears likely that a Rho-mediated signaling pathway linked to heterotrimeric G proteins has been evolutionarily conserved (Barrett, 1997 and references).

Why do cells of distinct fates (for example, presumptive mesodermal cells or presumptive posterior midgut cells) require a G-protein coupled signal to initiate gastrulation? The answer may lie in the fact that cell commitment to a certain fate does not in itself provide a mechanism for the cytoskeletal modifications that are necessary for invagination. Secretion of a ligand that functions to excite G-protein coupled receptors would be required to signal cytoskeletal changes that drive gastrulation. Such signaling could also act to synchronize cell activity and ensure graded responses by cells somewhat removed from the ventral midline. Cell signaling mechanisms might be required for the rapid completion of the invagination process. Invagination of the ventral furrow is completed in about 10 minutes, and invagination of the posterior midgut invagination takes about 20 minutes (Costa, 1994).

Because gastrulation starts without the G-protein coupled mechanism, it is suggested that two separate mechanisms initiate apical constriction of cells during invagination. One mechanism that does not require fog operates in the most ventral and posterior cells, respectively, of the mesodermal and posterior midgut primordia. A second mechanism, taking place subsequent to the first, requires a secreted ligand and G-protein coupled receptors, as well as fog. Such a mechanism is postulated, since fog mutant flies do, in fact, initiate invagination. It is not unusual in biological systems that what appears to be a unitary process is actually staged. In these cases, a later process, in this case secretion of a ligand, often appears as a cellular response to an earlier process (initiation of gastrulation).

Regulation of epithelial morphogenesis by the G protein-coupled receptor mist and its ligand fog

Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. This study developed an assay to recapitulate morphogenetic shape changes in individual cultured cells and used RNA interference-based screening to identify Mist (Methuselah-like 1<), a Drosophila G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. These data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes (Manning, 2013).

The Fog pathway is a well-understood example of how transcriptional programming is translated into cell behavior, but the picture of how this process is regulated was incomplete. The data now support a model in which the Drosophila GPCR Mist can act in the Fog morphogenetic pathway, providing a role for GPCRs in morphogenesis. Mist may function as the sole receptor for Fog. Alternatively, there may be other receptors that act with Fog either as co-receptors with Mist or in different developmental processes. One possibility is the GPCR CG31660, which has been identified as a candidate by deletion mapping. Further detailed analysis of the phenotypes of fog, t48, mist, cg31660, and rhoGEF2 mutants in living embryos, and cell biological and biochemical characterization of the relationship of Mist to other putative proteins in the pathway, will help define the array of cell behaviors controlled by each protein. The embryo also provides a venue to establish the epistatic relationships of Fog and Mist by combining loss of function and misexpression of Fog pathway members (Manning, 2013).

The data also allow the completion of the connection between the mesoderm transcriptional program given by the transcription factors Twist and Snail and the cellular machinery involved in triggering epithelial folding. Mist is the first downstream transcriptional target of Snail that is required for ventral furrow invagination. The data, in combination with data from others, provide a model for how the branches of the Twist and Snail regulatory pathway are ultimately integrated, by driving independently patterned, yet overlapping, expression of a ligand-receptor pair. Twist activates production of Fog and T48 for ventral furrow invagination and reinforces Snail expression in the presumptive mesoderm cells. Snail, in turn, promotes mist expression, either directly or indirectly (Manning, 2013).

A model is favored in which Fog is secreted and activates Mist through autocrine signaling, leading to activation of Cta, recruitment of RhoGEF2 to the apical membrane through T48, and localized contractility through the Rho pathway. It is also possible, however, that Mist acts independently of Fog in different cellular processes, such as basal expansion or cell shortening along the apical-basal axis. Additionally, other receptors may mediate some of the effects of Fog. The process can function correctly with either ubiquitously expressed Fog or ubiquitously expressed Mist. The coexpression of Fog and Mist may help make the patterned morphogenetic process of ventral furrow formation more robust, with possible subtle effects in timing or coordination. The broader implications of these concepts will be important and exciting to explore in the future (Manning, 2013).

Signaling by Folded gastrulation is modulated by mitochondrial fusion and fission

Mitochondria are increasingly being identified as integrators and regulators of cell signaling pathways. Folded gastrulation (Fog) is a secreted signaling molecule best known for its role in regulating cell shape change at the ventral furrow (VF) during gastrulation in Drosophila. Fog is thought to signal via a G-protein coupled receptor, to effect downstream cytoskeletal changes necessary for cell shape change. However, the mechanisms regulating Fog signaling that lead to change in cell morphology are poorly understood. This study describes identification of proteins involved in mitochondrial fusion and fission as regulators of Fog signaling. Pro-fission factors were found to function as enhancers of signaling, while pro-fusion factors were found to have the opposite effect. Consistent with this, activation of Fog signaling was seen to result in mitochondrial fragmentation and inhibiting this process could attenuate Fog signaling. The findings here show that mitochondria, through regulation of fusion-fission, function as downstream effectors and modulators of Fog signaling and Fog dependent cell shape change (Ratnaparkhi, 2013).

Fog is a secreted signaling molecule known to regulate apical constriction of cells at the ventral furrow during gastrulation in Drosophila. This study describes a genetic screen conducted to identify regulators of Fog signaling using the adult wing as an assay system. Fog is expressed in the wing disc; heterozygous mutant combinations of fog, cta and dRhoGEF2, result in deformed wings. Given a functional role for Fog in wing development, it seemed relevant to screen for interactors using the wing as an assay system. Through the identification of proteins involved in mitochondrial fusion and fission as regulators of Fog signaling, this study shows, for the first time, a role for the mitochondria as a downstream effectors of Fog signaling (Ratnaparkhi, 2013).

Overexpression of Fog leads to mitochondrial fission. A similar phenotype was observed upon expression of a constitutively active form of concertina suggesting that this effect is a consequence of the signaling pathway and not Fog protein per se. Conversely, knockdown of Fog results in different mitochondrial morphologies all of which are associated with excessive or enhanced fusion. In the present study, expression of fog dsRNA lead to formation of highly filamentous, 'donuts' shaped or fused amorphous forms of mitochondria. Whether these different morphologies arise due to differences in the extent of fog knockdown is not clear at this point (Ratnaparkhi, 2013).

Fog mediated mitochondrial fission is dependent on Drp1 (Dynamin related protein 1). Inhibiting fission through down regulation of drp1 suppresses Fog signaling. Consistent with this, knock down of drp1 in cells that respond to Fog in the blastoderm, leads to invagination defects at the ventral furrow. Interestingly drp1 has been identifed as a differentially expressed factor at the ventral furrow. Injection of dsRNA against drp1 was found result in ventral furrow defects ranging from mild to severe. In the experiments described here, the ventral furrow phenotype due to transgenic knockdown of drp1 were not as severe as those observed in fog mutants; one reason for this could be insufficient expression of drp1 dsRNA. Relatively stronger effects were seen in embryos in which expression of the RNAi was carried out at 28°. Some of these phenotypes were also observed in drp1^KG03815 mutants. However, the posterior midgut primordium (PMG) phenotype associated with fog loss-of-function was not seen in these mutants. One reason for this could be the presence of maternal drp1 mRNA that compensates for the loss of any zygotic drp1 expression. Nonetheless, the observation that inhibiting mitochondrial fission results in fog loss-of –function like phenotypes correlates well with observed interaction between fog and drp1 and suggests that mitochondrial fission is necessary for coordinated cell shape change at the ventral furrow (Ratnaparkhi, 2013).

The differential effect of fog gain-of-function and loss-of-function on mitochondrial morphology also indicates that the system might be used by the cell to 'sense' and distinguish one scenario from the other, pointing towards a very sensitive role for mitochondria in regulating Fog signaling. In this context, it would be interesting to test if the production of reactive oxygen species (ROS) or any other 'mitochondrial output' is altered in response to Fog signaling, since this could be an additional parameter the cell could use, to respond appropriately to Fog. It would also be interesting to see if the involvement of mitochondria in regulation of cell shape change is more widespread that previously known. This will need to be tested more rigorously. It would also be interesting to determine if the effect of fog on mitochondrial morphology is context dependent (Ratnaparkhi, 2013).

Generation of cell asymmetry and its regulation by mitochondrial fusion and fission pathways has been previous observed in migrating lymphocytes. In these cells, activation of G-protein signaling in response to chemokines was found to result in the redistribution and accumulation of mitochondria in the uropod or non migrating front to provide the ATP essential for actomyosin contraction during migration. The process of redistribution was shown to dependent on mitochondrial fission such that inhibiting fission lead to a loss of cell polarization and inhibition of migration. More recently, in another study, mitochondrial fission mediated by drp1 was found to be necessary and sufficient for delamination of cells of the amnioserosa (AS) during dorsal closure- a process that occurs via acto-myosin contraction. While these studies together with the one described in this study suggest a wider role for mitochondria in regulating acto-myosin based contraction and cell shape change, fragmentation of mitochondria in response to Fog may be required for relocalization of mitochondria to the site of action to provide the necessary energy for apical constriction (Ratnaparkhi, 2013).

How might Fog regulate mitochondrial fragmentation? One possibility is that activation of Fog signaling modulates expression or localization of drp1 in a manner that promotes mitochondrial fission. Drp1 is largely a cytoplasmic protein, which gets recruited to the mitochondria during fission. Many studies on mammalian drp1 have shown the recruitment to be regulated by post-translational modifications such as phosphorylation and sumoylation. However, based on the interaction between Fog and actin regulators it is likely that actin may be involved in mediating fission. Recent studies have shown that actin can regulate recruitment of Drp1 to mitochondria in a context dependent and thus control the process of fission. In another study excessive stable F-actin was shown to accumulate Drp1 and prevent it from localizing to mitochondria in a process dependent on the non-muscle Myosin II resulting in long elongated mitochondria. The suppression of Fog mediated mitochondrial fission by expression of WASP, an actin nucleator, suggests that a similar mechanism could be operating in this context as well (Ratnaparkhi, 2013).

This study was initiated by identification of rhomboid-7 as a suppressor of fog. At this point, it is unclear how Rho-7, a pro-fusion factor might interact with Fog signaling. It is likely that the interaction is independent of its role in mitochondrial fusion. This will need further investigation (Ratnaparkhi, 2013).

Quantitative control of GPCR organization and signaling by endocytosis in epithelial morphogenesis

Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII; Zipper) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist, that activate G proteins, the small GTPase Rho1, and the kinase Rok. Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells. The GPCR Smog activity is concentrated on two different apical plasma membrane compartments, i.e., the surface and plasma membrane invaginations. Using fluorescence correlation spectroscopy, the surface of the plasma membrane was probed, and it was shown that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is regulated by the kinase Gprk2 and beta-arrestin-2 that clears active Smog from the plasma membrane. When Fog concentration is high or endocytosis is low, Smog rearranges in homo-clusters and accumulates in plasma membrane invaginations that are hubs for Rho1 activation. Lastly, this study found higher Smog homo-cluster concentration and numerous apical plasma membrane invaginations in the mesoderm compared to the ectoderm, indicative of reduced endocytosis. Dynamic partitioning of active Smog at the surface of the plasma membrane or plasma membrane invaginations has a direct impact on Rho1 signaling. Plasma membrane invaginations accumulate high Rho1-guanosine triphosphate (GTP) suggesting they form signaling centers. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that regulate differential Rho1 and MyoII activation in the Drosophila embryo (Jha, 2018).

Tissue morphogenesis requires control over changes in cell shape and cell-cell contacts, which depend on the spatiotemporal regulation of actomyosin contractility. In Drosophila embryos, mesoderm invagination is driven by apical constriction, a geometric cell shape change facilitated by medial-apical Myosin II activation. In the ectoderm, tissue extension arises from cell-cell intercalation, whereby cells undergo neighbor exchange through the polarized remodeling of cell junctions. Junction remodeling is driven by medial-apical MyoII contractile pulses and MyoII planar polarized accumulation (Jha, 2018).

Actomyosin contractility is regulated by conserved signaling pathways. MyoII regulatory light chain is activated by Rho-kinase (Rok) downstream of the small GTPase Rho1, which in turn is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). This conserved pathway was shown to be under the direct control of signaling at the cell surface, such as Celsr in vertebrate neural tube formation and G protein-coupled receptors (GPCRs) in early Drosophila embryos. The GPCRs Mist (Manning, 2013) and Smog (Kerridge, 2016) transduce signals from the secreted ligand Fog in the Drosophila presumptive mesoderm (Mist and Smog) and ectoderm (Smog). Medial-apical MyoII activation progresses downstream of hetero-trimeric G proteins Gα12/13, Gβ13F, and Gγ1 in both mesoderm and ectoderm. In the mesoderm, high medial-apical MyoII activation is under a stable regime that ensures persistent apical constriction, while in the ectoderm, intermediate medial-apical MyoII activation is under a pulsatile regime that enables cell-cell intercalation. Therefore, to understand how quantitative activation of MyoII is generated and its temporal dynamics encoded, it is necessary to decipher the regulation of GPCR signaling (Jha, 2018).

Differential MyoII activation in the mesoderm and ectoderm is partly imparted by the ligand Fog, co-expression of Mist and Smog in the mesoderm, as well as by the mesoderm-specific transmembrane protein T48, which enhances apical recruitment of RhoGEF2 and, thereby, is proposed to potentiate Rho1 and MyoII activation. High Fog expression in mesoderm activates high MyoII, while in the ectoderm low Fog expression leads to low activation of MyoII. However, in general, ligand availability is one of several mechanisms impacting GPCR activation and signaling. Various cell culture studies have focused on the other modalities that regulate GPCR signaling. The major regulators of GPCR signaling are G protein-coupled receptor kinases (GRKs) that phosphorylate GPCRs and trigger signal termination, by allowing β-arrestin binding and recruitment of other adaptor proteins. In turn, β-arrestins direct activated receptors to clathrin-coated pits and remove them from the plasma membrane by endocytosis. While removal of activated GPCRs from the plasma membrane via endocytosis terminates GPCR signaling, it also reduces the number of receptors present on the surface for ligand stimulation. This effectively sets a quantitative control over GPCR signaling via endocytosis. Drosophila has only one non-visual GRK (Gprk2) and one non-visual β-arrestin-2 (kurtz). Gprk2 mutant mothers show aberrant contractility in the mesoderm lateral cells, and it was suggested that Gprk2 attenuates Fog-dependent MyoII activation in these cells. Eggs lacking Kurtz display cuticle phenotypes and suggest gastrulation defects. These data indicate that Kurtz plays a role with Gprk2 to terminate Fog signaling and could control Rho1 and MyoII via GPCR endocytosis. Its function in the mesoderm and ectoderm has not been addressed (Jha, 2018).

Conventionally, GPCR signaling from the plasma membrane is thought to occur via ligand binding and subsequent signal transduction via G proteins that relay the information to the interior of the cell. Apart from GPCR endocytosis, the localization of GPCR within the cell membrane will influence GPCR signaling. Lateral movement of GPCRs within the plasma membrane is often restricted to specific nano-domains, suggesting that selective compartmentalization is necessary for efficient signaling as it can increase GPCR localization and clustering. GPCR clustering in the form of homo- and hetero-oligomers has been reported to control both signal amplification as well as receptor recycling. Whether the main role of GPCR clustering is for chaperoning active receptors for transport or to control GPCR signaling specificity remains unclear, especially during development. To understand GPCR signaling during tissue morphogenesis, it is important to elucidate both the clustering of GPCRs at the plasma membrane and the role of endocytosis (Jha, 2018).

This study investigated the quantitative regulation of the GPCR Smog signaling by endocytosis in both the ectoderm and the mesoderm. Fog was shown to promote homo-clusters of Smog, while endocytosis rapidly removes Smog homo-clusters from the surface of the plasma membrane in the ectoderm. Dynamic partitioning of active Smog homo-clusters in two plasma membrane compartments, the surface or the plasma invaginations, was shown to directly impact Rho1 and MyoII activation. In the mesoderm, numerous apical plasma membrane invaginations and high Smog homo-clusters correlate with high Rho1 and MyoII activation compared to the ectoderm (Jha, 2018).

Epithelial cells exhibit different types of cell deformations owing to quantitative control over cell contractility that arises from contraction of the actomyosin cytoskeleton. GPCR signaling relays information conveyed by tissue-specific factors in the mesoderm and ectoderm to control this quantitative regulation during tissue morphogenesis. Rho1-dependent activation of MyoII during both apical constriction in the mesoderm and cell-cell intercalation in the ectoderm is controlled by GPCR signaling. Activation of the GPCR Smog underlies Rho1 activation in both mesoderm and ectoderm. It is believed that differential regulation of the GPCR Smog and other GPCRs underlies these tissue-specific differences in MyoII activation. This partly relies on the fact that Fog, the activating ligand, is present at higher levels in the mesoderm than in the ectoderm. This work sheds new light on this process by probing the plasma membrane organization and distribution of Smog in conditions that affect both endocytosis and production of the ligand Fog (Jha, 2018).

Probing the ectodermal cells with FCS, Smog homo-clusters on the surface of apical plasma membrane is reported and this process depends on Fog. When Fog is absent, such as in a fog-dsRNA, the brightness per Smog::GFP unit is lower, suggesting that Fog induces the formation of Smog homo-clusters. Dynamic exchange of homo-clustered Smog occurs sbetween the surface and plasma membrane invaginations. This dynamic distribution of Smog between the two plasma membrane compartments is strongly dependent upon both the rate of Smog endocytosis and Fog concentration. Increasing Fog concentration or reducing Smog endocytosis enhances the presence of Smog homo-clusters in apical plasma membrane invaginations, which results in an apparent decrease in Smog homo-clusters at the cell surface. When Fog concentration is high under conditions where Smog endocytosis is reduced, for example, when β-arrestin-2 is knocked down, Smog homo-clusters accumulate at the surface as well as in the plasma membrane invaginations. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that control the Smog cluster formation and influence the distribution of active Smog in different plasma membrane compartments. Importantly, this controls the quantitative activation of Rho1 and MyoII. Under low-endocytosis regimes (Gprk2 or β-arrestin-2 knockdowns in the ectoderm), high levels of active Rho1 accumulate in the apical plasma membrane invaginations. It is proposed that the apical plasma membrane invaginations are signaling hubs, where signaling components could concentrate, give rise to high G protein signaling (e.g., Gα12/13), and sustain high MyoII activation. The size and stability of these signaling invaginations is tuned by endocytosis, and they may provide a means to control the strength and persistence of signaling. Pulsatile active Rho1 in the ectodermal cells requires intermediate Rho1 activation. In the ectoderm, low Fog expression and rapid Smog endocytosis by Gprk2 and β-arrestin-2 lead to intermediate activation of Rho1. In turn, intermediate Rho1 activation at the apical plasma membrane creates the conditions required for self-organized actomyosin dynamics associated with pulsation (Jha, 2018).

This study also points to the possibility of tissue level regulation of endocytosis and plasma membrane compartmentalization of GPCRs. Large apical plasma membrane invaginations are observed in the mesoderm compared to the ectoderm. In the mesoderm, Smog accumulates in larger, more numerous, apical plasma membrane invaginations, and it displays larger Smog homo-clusters compared to in the ectoderm. In the mesoderm, Rho1 and MyoII activation is higher. Another GPCR, Mist produced in the mesoderm, works synergistically with Smog to boost Rho1 and MyoII activation (Manning, 2013). This is also due to the expression of another GPCR Mist in the mesoderm and to Fog being present at higher levels in the mesoderm. Ectodermal cells have similar properties of high Smog homo-clusters when Fog is overexpressed and GPCR endocytosis is slowed down. An intriguing possibility is that Smog and potentially Mist endocytosis is downregulated in the mesoderm compared to the ectoderm. Interestingly, the E3 ubiquitin ligase Neuralized (Neur), which is uniformly expressed in the embryo, is inhibited in the ectoderm by the small proteins of the Bearded (Brd) family. Brd genes are repressed by the mesoderm transcription factor Snail, so that Neur is only active in the mesoderm. In a Brd mutant, where Neur becomes active in the ectoderm, MyoII activation is increased and Neur degradation or repression in the mesoderm following Brd overexpression both reduce MyoII activation. Previous studies have shown that the E3 ubiquitin ligase targets β-arrestin-2 for ubiquitination and degradation, and, thereby, it affects endocytosis and signaling by GPCRs. It is possible that GPCR endocytosis could be reduced in the mesoderm due to increased Neur activity in this tissue. This may depend on the downregulation of several target proteins, such as β-arrestin-2 (Jha, 2018).

Selective compartmentalization of GPCR on the plasma membrane as in the case of large apical plasma membrane invaginations can increase the concentration and the probability of GPCR clustering and oligomerization. The current data suggest that the dynamic modulation of GPCR signaling can be achieved by a change in their cluster/oligomer formation. Receptor oligomerization may enlarge the signaling capacities by the recruitment of more downstream signaling components during GPCR signaling. G proteins are reported to be expressed at low concentration, and selective compartmentalization of GPCRs on the plasma membrane further increase the probability of GPCR clustering and oligomerization for efficient signaling. Investigation of G protein activation by different GPCRs in vivo will be needed to test if a similar mechanism is in place during epithelial morphogenesis (Jha, 2018).

Structure-function analysis of beta-arrestin Kurtz reveals a critical role of receptor interactions in downregulation of GPCR signaling in vivo

Arrestins control signaling via the G protein coupled receptors (GPCRs), serving as both signal terminators and transducers. Previous studies identified several structural elements in arrestins that contribute to their functions as GPCR regulators. However, the importance of these elements in vivo is unclear, and the developmental roles of arrestins are not well understood. An in vivo structure-function analysis of Kurtz (Krz), the single ortholog of mammalian beta-arrestins in the Drosophila genome, was carried out. A combination of Krz mutations affecting the GPCR-phosphosensing and receptor core-binding ('finger loop') functions (Krz-KKVL/A) resulted in a complete loss of Krz activity during development. Endosome recruitment and bioluminescence resonance energy transfer (BRET) assays revealed that the KKVL/A mutations abolished the GPCR-binding ability of Krz. The isolated 'finger loop' mutation (Krz-VL/A), while having a negligible effect on GPCR internalization, severely affected Krz function, suggesting that tight receptor interactions are necessary for proper termination of signaling in vivo. Genetic analysis as well as live imaging demonstrated that mutations in Krz led to hyperactivity of the GPCR Mist (also known as Mthl1), which is activated by its ligand Folded gastrulation (Fog) and is responsible for cellular contractility and epithelial morphogenesis. Krz mutations affected two developmental events that are under the control of Fog-Mist signaling: gastrulation and morphogenesis of the wing. Overall, these data reveal the functional importance in vivo of direct beta-arrestin/GPCR binding, which is mediated by the recognition of the phosphorylated receptor tail and receptor core interaction. These Krz-GPCR interactions are critical for setting the correct level of Fog-Mist signaling during epithelial morphogenesis (Chai, 2019).

This study reveals the developmentally important structural elements in the Drosophila β-arrestin Krz. Several lines of evidence, obtained in vivo and in cultured Drosophila cells, support a view that the residues most critical for Krz function during development include the phosphate-sensing region (disrupted by the KK/A mutation) and the finger loop (disrupted by the VL/A mutation). These motifs are engaged in direct interactions with the phosphorylated GPCR tail and the receptor core region, respectively, and both contribute to the tight binding of β-arrestins to the receptor. Since such interactions mediate efficient uncoupling of the receptor from G proteins, it appears that the primary function of Krz in development is to inhibit GPCR signaling via receptor desensitization (Chai, 2019).

The combined mutation Krz-KKVL/A resulted in a complete inactivation of Krz as a developmental regulator, however the disruption of the two key phosphate-sensing residues together with an additional phosphate-binding residue (KKR/A), leaving the finger loop intact, was not sufficient to eliminate Krz zygotic function. Structural studies suggest that the G proteins and the finger loop of arrestins make contacts with the same region in GPCRs. Therefore, the binding of arrestins' finger loop to the receptor directly interferes with the GPCR/G protein interactions, and mutations of this motif are expected to disrupt the ability of arrestins to uncouple GPCRs from G proteins. In support of this view, this study found that embryos from Krz-VL/A-rescued females showed gastrulation defects and cuticular phenotypes that were similar to the ones observed for krz¹, the strongest loss of function allele of krz. These findings suggest that the finger loop region of Krz is critical for its in vivo functions. It is noted that embryos obtained from Krz-KK/A-rescued females aborted development before cellularization, which is a phenotype that is even stronger than that observed in maternal krz¹ mutants. The KK/A mutation may thus have a dominant-negative effect, possibly due to a global hyperactivity and dysregulation of multiple GPCRs (Chai, 2019).

A major GPCR system involved in early Drosophila embryogenesis is the Fog signaling pathway. After several steps of signal transduction, activation of Fog signaling culminates in phosphorylation of Sqh, which controls acto-myosin contractility and mediates apical constrictions. Setting a proper level of cellular contractility is required for gastrulation movements. Consistent with a recent report, this study found that loss of krz resulted in excessive accumulation of Sqh-GFP in the mid-apical region of ventral cells, indicating overactivation of Fog signaling. Live imaging showed that mid-apical accumulation of Sqh-GFP persists much longer in krz maternal mutants than in wild type embryos, resulting in a stalled and aberrant gastrulation. It is noted that the area of cells undergoing apical constriction was wider in krz¹ maternal mutants, which may be explained in part by ectopic activity of the Toll signaling pathway, that may lead to an expansion of Fog and Mist expression domains downstream of Twist activation. However, medial-apical Sqh-GFP localization persisted in krz¹ mutants even in cells along the ventral midline, suggesting that Krz is required to limit the activity of the Fog pathway within the normal domain of its activation. In support of this view, maternal knockdown of krz by RNAi did not result in an expansion of Twist expression, yet led to abnormal mid-apical accumulation of Sqh (Chai, 2019).

Fog signals through its receptors Mist and Smog, and Krz may control Smog signaling at the level of endocytosis. Analyses of Krz mutants that were expected to disrupt clathrin and AP2 interactions (Krz-ΔLIQLD and Krz-F/A, respectively) showed that neither of these mutations affected Krz functions. Both Krz variants rescued krz¹ homozygotes to adulthood, did not have maternal effect, and internalized into endosomes with CrzR upon ligand activation, though Krz-Δ;LIQLD and the double Krz-Δ;LIQLD ​+ ​F/A mutant did show a mild reduction in the percentage of cells containing endosomes. It is possible that Krz relies on other structural elements to mediate GPCR endocytosis that were not included in this analysis, such as a short motif present in Krz that resembles the second clathrin-binding region in the long splice variant of β-arrestin-1. However, it is also possible that the endocytic function of Krz is secondary to the role of direct receptor engagement. Consistent with this view, it was found that mutation of the finger loop (Krz-VL/A) resulted in a strong reduction in Krz function, without affecting endocytosis. Deletion of the finger loop region in rat β-arrestin-1 also did not affect its ability to internalize GPCRs but impaired its ability to uncouple the receptor from G proteins. These results suggest that Krz-mediated attenuation of GPCR and G protein signaling via direct receptor interactions is critical during early development in Drosophila, though receptor internalization may also be involved as an additional regulatory step (Chai, 2019).

In addition to controlling gastrulation, the Fog-Mist signaling is involved in the development of the wing, and the data show that Krz-mediated regulation of this pathway also occurs in this tissue. The effects of the phospho-sensing and finger loop mutations were even more pronounced in the wing, as neither Krz-KK/A nor Krz-VL/A were capable of effectively suppressing the gain of function Fog phenotype. Thus, Krz controls Fog-Mist signaling in the embryo and in the wing, and perhaps in other tissues where this pathway guides epithelial morphogenesis. It was previously reported that loss of krz had no significant effect on wing development. It is possible that the discrepancy with the current results was due to the use of the specific GAL4 drivers used in that study (638-GAL4 and nub-GAL4), that may be expressed in a more restricted pattern or at a lower level than the MS1096-GAL4 driver that this study used. Consistent with the observation of Krz requirement in wing development, homozygous mutant krz¹ flies that are rescued with the elavC155-GAL4 driver, which is primarily expressed in the nervous system, have abnormal wings - a result that this study confirmed (Chai, 2019).

Phosphorylation of GPCRs by G protein-coupled receptor kinases (GRKs in mammals, Gprk2 in Drosophila) is a required step for the recognition of the activated receptor by arrestins. Previously, mutations in Gprk2 were shown to result in gastrulation defects that are remarkably similar to the phenotypes observed in krz maternal mutants, suggesting that phosphorylation of GPCRs by Gprk2 is required for the binding of Krz to the receptors. In support of this view, the Krz-KK/A mutant, which disrupts the phosphate-sensing residues, lost its ability to suppress the Fog overexpression phenotype in the wing and exhibited a strong maternal effect. This study also found that overexpression of Gprk2 itself could suppress overactive Fog signaling in the wing. Gprk2 and Krz thus operate in concert to inhibit GPCR signaling (Chai, 2019).

While Krz has been implicated in the regulation of several signaling pathways, this study highlights the developmental importance of Krz in controlling its prototypical targets, GPCRs. In all contexts examined in this study, Krz plays an inhibitory role, which is likely mediated by direct engagement of the activated GPCRs via the two-part recognition of the phosphorylated receptor tail and its core. It will be of interest to examine how Krz coordinates its involvement in the various pathways active in the early embryo, such as the GPCR, ERK, and Toll signaling pathways, and whether these pathways are differentially affected by the mutations examined in this study. Of note, it was found that the pre-activating mutation Krz-R/E did not affect Krz interaction with the Toll pathway regulator Cactus, but did increase the association of Krz with ERK. With regard to ERK signaling, it was previously shown that Krz inhibits ERK activation downstream of receptor tyrosine kinases in Drosophila development. One question for future studies is to determine whether Krz is involved in the positive signaling events downstream of GPCRs, given the recent interest in developing G protein- and β-arrestin-biased ligands that modulate ERK activation and can increase the specificity and efficacy of GPCR-directed therapies. Rescue of krz¹ homozygous animals by both human β-arrestins shows significant functional conservation and suggests that Drosophila Krz can serve as a platform for modeling mammalian β-arrestin functions (Chai, 2019).

This work has used the Drosophila β-arrestin Kurtz (Krz) to identify conserved structural elements in the β-arrestin molecule that are functionally important in vivo. Some of the previously identified elements appear to be dispensable. Nonetheless, it was revealed that two regions in Krz are critical for its activity: a phosphate sensing motif which interacts with the phosphorylated GPCR tail, and a 'finger loop' region that directly contacts the GPCR core. The finger loop mutation (VL/A) is notable because it severely affected Krz function without having a significant effect on its ability to internalize a GPCR in endosomes. This study showed that GPCR-binding Krz mutations disrupt its ability to limit the activity of the Fog-Mist signaling pathway that plays a key role during epithelial morphogenesis events, such as embryo gastrulation and folding of the wing epithelium. These studies thus uncovered the structural motifs in β-arrestins that are critical for their developmental functions as GPCR regulators in vivo (Chai, 2019).

Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination

The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. This study shows that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. These data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation (Le, 2021).

MTs have a crucial role in stabilizing apical myosin during epithelial morphogenesis both in early Drosophila embryos and in the Drosophila SG. In the SG, MTs interact with apicomedial myosin via Short stop, the Drosophila spectraplakin, emphasizing a direct interplay between the MT and the apical myosin networks. The data of this study reveal another key role of MTs in regulating protein trafficking to control the apical myosin networks during tissue invagination. During SG invagination, a network of longitudinal MT bundles is observed near the invagination pit. These data show apical enrichment of Rab11 in the same area is MT dependent and that this enrichment is important for forming the apicomedial myosin networks, suggesting a link between localized intracellular trafficking along MTs to apical myosin regulation (Le, 2021).

The dorsal/posterior region of the SG, where Rab11 is apically enriched in a MT-dependent manner, correlates with localized Fog signaling activity that promotes clustered apical constriction. Disruption of MTs or Rab11 knockdown reduces Fog signals in the apical domain of SG cells and causes dispersed Rok accumulation and defective apicomedial myosin formation. It is consistent with a previous study that the absence of Fog signal results in dispersed apical Rok and defects in apicomedial myosin formation. It is therefore proposed that MT- and Rab11-dependent apical trafficking regulates Fog signaling activity to control apical constriction during epithelial tube formation through transporting the Fog ligand. As recycling of membrane receptors to the cell surface plays an important role in regulating overall signaling activity, it is possible that Rab11 is involved in recycling the as yet unidentified SG receptor(s) of Fog to regulate Fog activity in the SG. Indeed, several GPCRs are recycled via Rab11. During epithelial invagination in early Drosophila embryogenesis, the concentration of the Fog ligand and receptor endocytosis by 'β-arrestin-2 have been shown as coupled processes to set the amplitude of apical Rho1 and myosin activation. It is possible that the movement of Fog receptor(s) that have internalized as a stable complex with 'β-arrestin is recycled back to the cell surface by Rab11. The Fog signaling pathway represents one of the best understood signaling cascades controlling epithelial morphogenesis. Although best studied in Drosophila, the pathway components have also been identified in other insects, suggesting a more widely conserved role for Fog signaling in development. Further work needs to be done to fully understand the regulatory mechanisms underlying the trafficking of Fog and its receptor(s) during epithelial morphogenesis (Le, 2021).

Analysis of apicomedial myosin shows that reduced Rab11 function not only causes a decrease of the myosin intensity but also causes myosin to be dispersed rather than forming proper myosin web structures in the apicomedial domain of SG cells. These data support the idea that Rab11 function is required for both concentration and spatial organization of apicomedial myosin. This can be explained by the combined effect of multiple cargos that are transported by Rab11, including Fog, Crb, and E-Cad. Time-lapse imaging of myosin will help determine how the dynamic behavior of apicomedial myosin is compromised when Rab11 function is disrupted (Le, 2021).

During branching morphogenesis in Drosophila trachea, MTs and dynein motors have a critical role in the proper localization of junctional proteins such as E-Cad. This is consistent with the observations with MT-dependent uniform distribution of E-Cad at adherens junctions in the invaginating SG, suggesting a conserved role of MT-dependent intracellular trafficking in junctional remodeling and stabilization during epithelial tube formation. The data further suggest that the MT networks and Rab11 have key roles in apical distribution of Crb and E-Cad in the SG and that proper levels of apical and junctional proteins are important for apical constriction during SG invagination. Based on these data, it is proposed that MT- and Rab11-dependent apical trafficking of Crb and E-Cad is critical for apical constriction during SG invagination. Alternatively, MTs have an additional role in assembling/anchoring these apical components through the regulation of unidentified molecules. Recent studies in Drosophila mesoderm invagination showed that MTs help establish actomyosin networks linked to cell junction to facilitate efficient force transmission to promote apical constriction. In Ko (2019), however, MT-interfering drugs and RNAi of CAMSAP end-binding protein were used to prevent MT functions and the effect cannot be directly compared with the current data where spastin was used to sever existing MTs. Direct monitoring of MT-dependent transport of Crb and E-Cad during SG invagination will help clarify the mechanism (Le, 2021).

On knockdown of crb or E-Cad, less prominent apicomedial myosin web structures are observed in invaginating SGs, suggesting a requirement of Crb and E-Cad in proper organization of apical actomyosin networks during SG tube formation. Crb acts as a negative regulator of actomyosin dynamics during Drosophila dorsal closure and during SG invagination. It is possible that proper Crb levels are required for modulating myosin activity both in the apicomedial domain and at junctions during SG invagination. Both of which contribute to apical constriction and cell rearrangement, respectively. Anisotropic localization of Crb and myosin was observed at the SG placode boundary, where myosin accumulates at edges where Crb is lowest. Planar polarization of Rok at this boundary is modulated through phosphorylation by Pak1 downstream of Crb. A further test will help understand whether and how Crb might affect junctional myosin dynamics and SG invagination. As contractile actomyosin structures exert forces on adherens junction to drive apical constriction, it is speculated that apical constriction defects on E-Cad RNAi might be due to reduction of cell adhesion and/or of improper force transmission. It will be interesting to determine if the coordination of apical and junctional proteins and apical cytoskeletal networks through intracellular trafficking is conserved during tubular organ formation in general (Le, 2021).

Dhc is also apically enriched in the dorsal/posterior region of the invaginating SG. The data show that knockdown of Dhc64C not only affects Rok accumulation and apicomedial myosin formation but also disrupts MT organization in the SG. These data are consistent with previous findings that cytoplasmic dynein is associated with cellular structures and exerts tension on MTs. For example, dynein tethered at the cell cortex can apply a pulling force on the MT network by walking toward the minus end of a MT. Dynein also scaffolds the apical cell cortex to MTs to generate the forces that shape the tissue into a dome-like structure. In interphase cells, the force generated by dynein also regulates MT turnover and organization (Le, 2021).

In klar mutants, on the other hand, MT organization is not affected in the SG, suggesting that reduction of dynein-dependent trafficking by loss of klar does not cause changes in the MT networks. Notably, although the intensity of apicomedial myosin does not change on Dhc64C knockdown or in the klar mutant background, formation of apicomedial myosin web structures is affected. These data suggest a possible scenario that dynein function is not required for myosin concentration in the apical domain but is only needed for the spatial organization of apicomedial myosin. However, it cannot be ruled out that the zygotic knockdown of Dhc64C by RNAi is not strong enough to affect the intensity of apicomedial myosin. Dhc64C has strong maternal expression and is essential for oogenesis and early embryo development. Embryos with reduced maternal and zygotic pools of Dhc64C showed a range of morphological defects in the entire embryo, some of which were severely distorted. Precise roles for dynein and dynein-dependent trafficking in regulating apicomedial myosin formation remain to be elucidated (Le, 2021).

GENE STRUCTURE

Bases in 5' UTR - 849

Bases in 5' UTR - 849

Bases in 3' UTR - 612

PROTEIN STRUCTURE

Amino Acids - 730

Structural Domains

fog encodes a novel protein with a putative signal sequence (functioning to facilitate secretion) but no potential transmembrane domains. There are nine potential N-linked glycosylation sites and the C-terminal three-quarters of the protein is highly enriched in serine and threonine. There are three potential protease cleavage sites in the Fog sequence. Cleavage at the two amino-terminal sites would produce a central peptide of 109 amino acids; this is in the size range of known peptide ligands of G-protein-coupled receptors (Costa, 1994).

Evolutionary Homologs

Folded gastrulation and T48 drive the evolution of coordinated mesoderm internalization in flies

Gastrulation constitutes a fundamental yet diverse morphogenetic process of metazoan development. Modes of gastrulation range from stochastic translocation of individual cells to coordinated infolding of an epithelial sheet. This study identified two genes, folded gastrulation and t48, which in the evolution of Drosophila gastrulation acted as a likely switch from an ingression of individual cells to the invagination of the blastoderm epithelium. Both genes are expressed and required for mesoderm invagination in Drosophila but do not appear during mesoderm ingression of the midge Chironomus riparius. Early expression of either or both of these genes in C.riparius is sufficient to invoke mesoderm invagination similar to D.melanogaster. The possible genetic simplicity and a measurable increase in developmental robustness might explain repeated evolution of similar transitions in animal gastrulation (Urbansky, 2016).

date revised: 20 December 2013 

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