folded gastrulation: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - folded gastrulation

Synonyms -

Cytological map position - 20A4--20B3

Function - secreted ligand

Keyword(s) - gastrulation, dorsal-ventral patterning

Symbol - fog

FlyBase ID: FBgn0000719

Genetic map position - 1-[66]

Classification - novel protein

Cellular location - secreted

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Urbansky, S., González Avalos, P., Wosch, M. and Lemke, S. (2016). Folded gastrulation and T48 drive the evolution of coordinated mesoderm internalization in flies. Elife 5. PubMed ID: 27685537
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. How such morphogenetic differences are genetically encoded and whether they have provided specific developmental advantages is unclear. This study identified two genes, folded gastrulation and t48, which in the evolution of fly 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 the fruit fly Drosophila melanogaster 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.

Lim, B., Levine, M. and Yamakazi, Y. (2017). Transcriptional pre-patterning of Drosophila gastrulation. Curr Biol 27(2): 286-290. PubMed ID: 28089518
Gastrulation of the Drosophila embryo is one of the most intensively studied morphogenetic processes in animal development. Particular efforts have focused on the formation of the ventral furrow, whereby approximately 1,000 presumptive mesoderm cells exhibit coordinated apical constrictions that mediate invagination. Apical constriction depends on a Rho GTPase signaling pathway (T48/Fog) that is deployed by the developmental regulatory genes twist and snail. It is thought that coordinate mesoderm constriction depends on high levels of myosin along the ventral midline, although the basis for this localization is uncertain. This study employed newly developed quantitative imaging methods to visualize the transcriptional dynamics of two key components of the Rho signaling pathway in living embryos, T48 and Fog. Both genes display dorsoventral (DV) gradients of expression due to differential timing of transcription activation. Transcription begins as a narrow stripe of two or three cells along the ventral midline, followed by progressive expansions into more lateral regions. Quantitative image analyses suggest that these temporal gradients produce differential spatial accumulations of t48 and fog mRNAs along the DV axis, similar to the distribution of myosin activity. It is therefore proposed that the transcriptional dynamics of t48 and fog expression foreshadow the coordinated invagination of the mesoderm at the onset of gastrulation.
Jha, A., van Zanten, T. S., Philippe, J. M., Mayor, S. and Lecuit, T. (2018). Quantitative control of GPCR organization and signaling by endocytosis in epithelial morphogenesis. Curr Biol 28(10): 1570-1584.e1576. PubMed ID: 29731302
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) 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.
Cell shape change controlled by folded gastrulation

Model of fog function in controlling cell shape change: The patterning gene twist (twi) specifies mesodermal fate of the ventral cells. These cells in turn activate transcription of folded gastrulation, resulting in the production and secretion of Fog protein from the apical side of the cell. Reception of Fog signal results in localized activation of Rho kinase which in turn activates the contractility of myosin with actin. This local source of actomyosin contractility drives myosin to the apical side of the cell. The actin-myosin cytoskeleton is tethered to the cell surface through adherens junctions. The continued contraction of apical actin-myosin exerts further force on the adherens junctions, pulling them close together, and resulting in the apical constriction of the cells and consequent gastrulation (Dawes-Hoang, 2005).

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 drp1KG03815 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).


cDNA length - 3.7 kb

Bases in 5' UTR - 849

Bases in 5' UTR - 849

Bases in 3' UTR - 612


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).

folded gastrulation: | Evolutionary Homologs Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 December 2013 

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