InteractiveFly: GeneBrief

Trapped in endoderm-1: Biological Overview | References

BIOLOGICAL OVERVIEW

Despite significant progress in identifying the guidance pathways that control cell migration, how a cell starts to move within an intact organism, acquires motility, and loses contact with its neighbors is poorly understood. This study shows that activation of the G protein-coupled receptor (GPCR) Trapped in endoderm 1 (Tre1) directs the redistribution of the G protein Gβ as well as adherens junction proteins and Rho guanosine triphosphatase from the cell periphery to the lagging tail of germ cells at the onset of Drosophila germ cell migration. Subsequently, Tre1 activity triggers germ cell dispersal and orients them toward the midgut for directed transepithelial migration. A transition toward invasive migration is also a prerequisite for metastasis formation, which often correlates with down-regulation of adhesion proteins. Uniform down-regulation of E-cadherin causes germ cell dispersal but is not sufficient for transepithelial migration in the absence of Tre1. These findings therefore suggest a new mechanism for GPCR function that links cell polarity, modulation of cell adhesion, and invasion (Kunwar, 2008).

Cell migration plays a important role during a variety of processes such as development, immune defense, and metastasis. The coordinated migration of different kinds of cells in space and time gives rise to the three germ layers and the three-dimensional architecture of different organs and organisms. Cells of the immune system migrate through blood vessels and tissues to reach infected sites; and cancer cells migrate away from their tissues of origin to ectopic places during metastasis. Thus far, the basic mechanisms of cell migration have been elucidated mostly from in vitro studies in solitary cells. Cell migration in living, multicellular organisms, however, is likely much more complex. At the onset of directed migration, cells not only have to acquire motility but also have to be able to perceive specific, directional migration cues. During their journey, migrating cells may be required to detect and interpret multiple, possibly conflicting guidance cues, and must coordinate their adhesion to surrounding cells to reorient, pause, and move in a directed fashion while targets change. Finally, at the end, cells have to know when they have reached their target and cease their motility (Kunwar, 2008).

Significant progress has been made in identifying guidance molecules, receptors, and intracellular mediators that act during directed migration. G protein-coupled receptors (GPCRs) have been widely studied for their role in directional migration (Doitsidou, 2002; Ara, 2003; Knaut, 2003; Kunwar, 2003b; Molyneaux, 2003; Kunwar, 2006). Cells use GPCRs to detect and migrate toward higher concentrations of chemoattractants. Immune cells and germ cells, for example, express the chemokine receptor CXCR4 and follow the distribution of the chemokine SDF1 (stromal cell-derived factor 1) (Kunwar, 2008).

Lymphocytes use sphingosine-1-phosphate receptors to egress from lymphoid tissues, where S1P levels are higher. Despite significant progress in identifying the guidance molecules, receptors, and intracellular mediators that act during directed migration, the cellular and molecular mechanisms that initiate cell migration are only poorly understood. At the start of migration, cells need to acquire motility, may lose cell adhesion with neighboring cells, and are required to gain the ability to respond directionally to external cues. The detailed cellular transformations, the temporal sequence of these events, and the relative influence caused by intrinsic and extrinsic cell information are the focus of this study (Kunwar, 2008).

Drosophila germ cells provide a genetically tractable system to visualize and follow individual germ cells as they start directed migration. The onset of directed germ cell migration coincides with the transepithelial migration of germ cells through the primordium of the future midgut. Evidence for a germ cell autonomous function for transepithelial migration came from the identification of a novel GPCR trapped in endoderm 1 (tre1; Kunwar, 2003a). Maternal tre1 RNA is present in germ cells, and tre1 function is required there. General cell motility and the movements of germ cells toward the gonad do not depend on Tre1, which suggests that Tre1 specifically regulates the onset of migration (Kunwar, 2008).

To understand the cellular mechanisms underlying the onset of directed migration, two-photon imaging was used to visualize the cellular transformations that occur in vivo as germ cells migrate through the midgut epithelium. Comparison of wild-type and tre1 mutant germ cells suggests that regulated activation of the Tre1 GPCR controls three phases of early migration: polarization of germ cells, dispersal into individual cells, and transepithelial migration. Germ cell polarization leads to a redistribution of cell-cell adherens proteins, such that Drosophila E-cadherin (DE-cadherin) levels are reduced from the leading edge of the migrating cells and accumulate in the tail region. Tre1 likely signals via the G proteins Gγ1 and Gβ13f as well as Rho-1, since Gβ and Rho-1 protein localization is detected in the tail region, and deletion of their function specifically in germ cells causes the same phenotype as mutation in tre1. These results suggest a novel function for GPCR signaling in initiating cell migration by polarizing the migrating cell. This polarization leads to the redistribution of signaling components and adherens proteins and may trigger cell dispersal and directed migration (Kunwar, 2008).

To visualize germ cell migration in developing embryos, a germ cell-specific expression system, which translates the actin-binding domain of Moesin fused to EGFP under the control of nanos regulatory sequences, was used (Sano, 2005). Germ cells appear motile soon after their formation at the blastoderm stage (stage 5, 2 h and 10 min to 2 h and 50 min after egg laying [AEL]), as they produce small protrusions away from their neighbors. Despite this apparent motility, germ cells only rarely (1-2 germ cells per embryo) separate from their neighbors and migrated directly through the underlying blastoderm cells. Subsequently, during gastrulation (stage 7-8, 3 h to 3 h and 40 min AEL), as germ cells are internalized together with the invaginating posterior midgut primordium, they round up and show less protrusive activity. At stage 9 (3 h and 40 min to 4 h and 20 min AEL), germ cells are found inside the midgut primordium in a tight cluster; they are in close contact with each other and show little contact with the surrounding somatic midgut cells. During this stage, germ cells started to reorganize, changed their shape, and take on a highly polarized morphology. Using electron microscopy, a radial organization of germ cells within the midgut is clearly visible, with the large germ cell nuclei pointed toward the midgut while fine membranous material, apparently corresponding to the tail region, fills the inside of the cluster. This organization orients the leading edge of each germ cell toward the surrounding midgut primordium. Next, the germ cells lose adhesion to each other, and extensions reach from the germ cells toward the midgut epithelium (Kunwar, 2008).

Subsequently, germ cells disperse as they migrated through the midgut primordium to reach the basal side of the midgut cells by stage 10 (4 h and 20 min to 5 h and 20 min AEL). Long cytoplasmic extensions connected germ cells with each other immediately after the onset of transepithelial migration. As germ cells transmigrate through the midgut epithelium, they appear completely individualized, display amoeboid behavior, and are polarized with a broad lagging edge and actin localized at the leading edge. On average, individual germ cells transmigrate the midgut within 40 min from the onset of polarization. Tracking of individual germ cells showed that they disperse radially and transmigrate in all directions through the pocket of the midgut epithelium After transmigration, germ cells reorient on the midgut toward the dorsal side of the embryo, sort into two bilateral groups, and migrate toward the gonadal mesoderm, which forms on either side of the embryo (Kunwar, 2008).

Tre1 encodes an orphan GPCR that is required maternally in germ cells for their migration through the midgut epithelium (Kunwar, 2003a). In embryos from tre1 mutant females, (hereafter referred to as 'mutant embryos'), germ cells fail to cross the midgut epithelium. This phenotype could result from a defect in the acquisition of motility by germ cells or in their ability to polarize, disperse, or transmigrate. To distinguish between these possibilities, tre1 mutant germ cells were observed live by in vivo imaging. At stage 5, tre1 germ cells show small protrusions and sporadically cross the blastoderm with a similar frequency to the wild type (Kunwar, 2003a). In striking difference to the wild type, however, the tre1 germ cell cluster does not reorganize at stage 9 and fails to transmigrate to the midgut. Mutant germ cells do not polarize, and remain in a tight, disorganized group in which germ cells fail to interact with the surrounding midgut cells (Kunwar, 2008).

To begin to understand how Tre1, an orphan GPCR, initiates germ cell migration, it was asked whether Tre1 function is mediated by trimeric G protein activation in germ cells. It was found that only a single Gγ (Gγ1) and a single Gβ (Gβ13f) subunit are provided maternally (Fuse, 2003). Loss of maternal Gβ13f or Gγ1 function causes defects in gastrulation, which precluded an immediate analysis of germ cell migration (Kunwar, 2008).

However, it was possible to rescue the gastrulation defect through early zygotic, soma-specific expression of the respective G protein. This genetic manipulation allowed testing for a germ cell-specific role of these G proteins, since early Drosophila germ cells are transcriptionally silent, and germ cells thus depend completely on the maternally provided G proteins. In embryos rescued for the gastrulation defect, Gβ13f mutant germ cells are unable to disperse and migrate through the midgut epithelium, and thus resembled the tre1 phenotype. Gγ1 mutants showed a similar although slightly weaker phenotype likely caused by residual function of the Gγ1^N159 allele used, which lacks the C-terminal isoprenylation site required for membrane anchoring. These results suggest that germ cell transepithelial migration requires Tre1-mediated canonical G protein signaling (Kunwar, 2008).

For Gα proteins, focus was placed in particular on the role of the single D. melanogaster Gα12/13A homologue, encoded by concertina (cta), because this subfamily of G proteins has been shown to regulate cell migration and metastatic invasion and to directly interact with E-cadherin and Rho1 (Huber, 2005; Kelly, 2006a; Kelly, 2006b). Cta protein is present in the germ cells and maternal loss of cta causes a gastrulation defect similar to Gβ13f and Gγ1 (Parks, 1991). Again, it was possible to rescue the gastrulation phenotype by early, somatic Cta expression, as described for Gγ1 and Gβ13f. In contrast to findings with Gβ and Gγ mutants, however, cta mutant germ cells migrated normally to the gonad. To confirm this result, mutant cta germ cells derived from cta mutant mothers were transplanted into wild-type embryos. It was found that cta germ cells migrated to the gonad with similar efficiency as transplanted wild-type control germ cells. Thus, Gα12/13 does not act as the sole mediator of Tre1 GPCR activation. Analysis of the available mutants in other Gα proteins did not reveal a single Gα protein that mediates the Tre1 signal, which perhaps indicates that redundant or overlapping functions of Gα proteins act downstream of Tre1 (Kunwar, 2008).

The observation that both Gβ13f and Gγ1 are required for germ cell dispersal and transepithelial migration suggests that Tre1 function in germ cells is mediated by a G protein-dependent pathway, akin to the requirement for GPCR signaling seen during the directed migration of Dictyostelium discoideum amoeba and in neutrophils toward a chemokine gradient. To determine how Tre1 signaling may affect downstream components, the localization of Gβ13f protein was analyzed, as well as the localization of Rho1, which has been shown to affect germ cell transepithelial migration in wild-type and tre1 mutant germ cells (Kunwar, 2003a). It was found that Gβ13f and Rho1 proteins were localized uniformly along the cell membrane of wild-type germ cells at the blastoderm stage (Kunwar, 2008).

At stage 9, as wild-type germ cells polarize, Gβ13f and Rho1 proteins are down-regulated along the germ cell membranes facing the midgut, and become highly enriched in the tail region. In early germ cells, Gβ13f and Rho1 proteins are uniformly distributed in tre1 mutants similar to the wild type; in contrast to the wild type, however, this uniform distribution persists during stage 9. These results suggest that Tre1 receptor activation leads to germ cell polarization in part by causing the redistribution of downstream signaling molecules away from the leading edge and accumulation in the tail (Kunwar, 2008).

tre1 mutant germ cells fail to disperse at the onset of the migration, which suggests that tre1 regulates adhesion molecules in germ cells. DE-cadherin is a good candidate, since it is deposited maternally in the early embryo. The role of DE-cadherin was first tested in the adhesion of wild-type germ cells. For this analysis, a newly identified partial loss-of-function allele of Drosophila E-cadherin encoded by the shotgun (shg) gene, which allows normal oogenesis, was used. In embryos derived from shg^A9-49 mutant ovaries, germ cells did not organize into a radial cluster. Instead, germ cells separated from one another prematurely, at early stage 8 (3 h and 10 min to 3 h and 40 min AEL) compared with stage 10 in the wild type (4 h and 20 min to 5 h and 20 min AEL). This dispersal phenotype was observed in embryos from homozygous germ line clones, in which embryonic patterning defects were rescued by a wild-type shg⁺ copy from the father (M^-Z⁺). This suggests that DE-cadherin is required autonomously in germ cells, since they are transcriptionally quiescent and thus likely depend exclusively on maternally contributed DE-cadherin. These results indicate that DE-cadherin is required for germ cell-germ cell adhesion in the wild-type embryo (Kunwar, 2008).

To understand how DE-cadherin is regulated in the dispersal step, the distribution of DE-cadherin was analyzed in wild-type germ cells. It was found that DE-cadherin as well as α and β catenins were initially uniformly present along the germ cell membrane but become enriched in the tail region during germ cell polarization (Kunwar, 2008).

In stark contrast, DE-cadherin remained uniformly distributed along the cell surface in tre1 mutant embryos. To quantitate the levels, the fluorescent intensity of DE-cadherin staining on the cell membrane of wild-type and tre1 mutant germ cells was compared. It was found that DE-cadherin is distributed uniformly and that levels are similar in wild-type and mutant germ cells at stage 5, before migration, whereas the levels are reduced along the leading edge membrane of wild-type germ cells compared with tre1 mutant germ cells at stage 9. These results suggest that tre1 activation leads to a reduction of DE-cadherin along the leading edge and restricts it in the tail region (Kunwar, 2008).

In shg mutants, early dispersal of germ cells does not lead to premature migration through the midgut, as would be expected if release of germ cell-germ cell adhesion via E-cadherin was the only trigger for transepithelial migration. Instead, shg mutant germ cells moved through the midgut slightly later during stage 10 than wild-type germ cells. This delay phenotype is less penetrant compared with the precocious dispersal phenotype and could be caused by an impaired ability of the shg^A9-49 mutant germ cell to migrate at this and subsequent stages (Kunwar, 2008).

To test directly if Tre1 acts via DE-cadherin in transepithelial migration, embryos were generated that lacked tre1 and maternal shg^A9-49 function. The germ cells in these embryos dispersed early, thus displaying a phenotype similar to shg^A9-49 mutants; 80% of tre1, shg^A9-49 double mutant embryos showed precocious dispersal as opposed to 0% in the tre1 mutant embryos (Kunwar, 2008).

However, even these dispersed germ cells were not able to transmigrate through the midgut in tre1, shg^A9-49 double mutant embryos, thereby resembling tre1 mutant germ cells. This suggests that loss of germ cell-germ cell contact may not be sufficient to trigger transepithelial migration. To test this idea further, germ cell-germ cell contact was disrupted independent of E-cadherin function by reducing the germ cell number. Alleles of the maternal effect gene tudor (tud) were used to reduce the number of germ cells in the embryo to a single germ cell. Such single, tud mutant germ cells migrated through the midgut and invariably reached the gonad. These germ cells had normal morphology and appeared polarized. Next, mutant embryos lacking both tre1 and maternal tud were analyzed. In the absence of tre1, single germ cells were left inside the midgut and did not migrate to the gonad. Thus, whereas germ cell individualization requires Tre1-mediated down-regulation of DE-cadherin, Tre1 activity has additional roles in transepithelial migration (Kunwar, 2008).

This study has used live imaging to explore the mechanisms by which germ cells acquire motility and traverse the midgut epithelium. It was found that before transepithelial migration, germ cells polarize toward the midgut and down-regulate E-cadherin from the leading edge and accumulate E-cadherin in the tail region. This polarization requires Tre1 GPCR activity. It is proposed that GPCR-mediated polarization triggers germ cell dispersal and orients germ cells toward the midgut for directed transepithelial migration (Kunwar, 2008).

A requirement for GPCR signaling during the directed migration toward a chemokine gradient has been described in detail in D. discoideum amoeba and in mammalian neutrophils. The events underlying signal transduction leading to the polarization of migrating cells have been worked out extensively in these cells. The first localized response to receptor activation is the enrichment of the activated G protein βγ subunits, which results in the activation of phosphoinositide 3 (PI3) kinase. As a consequence of chemokine sensing, the PI3 kinase product phosphatidylinositide 3,4,5-tris phosphate (PIP3) becomes localized to the leading edge, and the phosphatase PTEN (phosphatase and tensin homolog) moves to the lagging edge in a Rho dependent manner (for review see Affolter, 2005). These signaling events organize the cytoskeleton leading to cellular polarization and directional movement. The current studies suggest a new mechanism by which GPCR signaling initiates directed cell migration. Activation of Tre1 causes a redistribution of G protein β, the GTPase Rho1, DE-cadherin, and other adherens junction components to a small region in the tail of the germ cells. The decrease in DE-cadherin from the leading edge of germ cells causes a loss of adhesion across the broad leading edge of the germ cells and causes germ cell polarization toward the midgut. This localization event may thereby convert an adherent group of cells into directionally migrating individuals. Tre1 belongs to a family of GPCRs that includes Moody in D. melanogaster and GPR84 in mouse and human (Bainton, 2005; Bouchard, 2007). Based on the results with Tre1, this family may act to regulate cellular polarity and adhesion, a view in line with the proposed function of Moody in epithelial morphology at the blood-brain barrier, and with GPR84, which was recently described to be up-regulated in microglia upon infection (Kunwar, 2008).

How could Tre1 activation cause DE-cadherin redistribution? Regulation of E-cadherin is widely attributed to play an important role in metastasis and in the epithelial-to-mesenchymal transition that occurs during gastrulation and neural crest migration. In these systems, it has been proposed that E-cadherin is regulated by transcriptional repression or by Gα12/13-mediated uptake and turnover (Huber, 2005; Kelly, 2006a; Kelly, 2006b). The data suggest the presence of a different mode of regulation, since neither transcriptional regulation nor Gα12/13 activity seem to be required for the regulation of DE-cadherin in germ cells. An attractive mechanism for DE-cadherin down-regulation could be the control of its endocytosis by Tre1. During zebrafish gastrulation, Rab GTPases have been shown to control E-cadherin turnover and the adhesion of mesendodermal cells (Ulrich, 2005). A role for Rab proteins in germ cell migration has yet to be demonstrated. This study found the same localization pattern for Gβ13f, Rho1, and DE-cadherin in the wild type, and this pattern is disrupted in tre1 mutant germ cells. This suggests a role for G protein and Rho1 activation in the polarization of DE-cadherin in germ cells (Kunwar, 2008).

Tre1 also affects transepithelial migration independently of global DE-cadherin regulation. Uniform down-regulation of DE-cadherin or loss of germ cell-germ cell contact in single cells are neither sufficient to trigger precocious transepithelial migration in the wild type nor able to suppress the tre1 transepithelial migration phenotype. One possibility is that the localized activation of Tre1 and polarized down-regulation of DE-cadherin at the leading edge would orient germ cells radially toward the midgut. This radial orientation would allow germ cells to respond to additional guidance cues required for directed transepithelial migration. Although these additional guidance cues may not depend on DE-cadherin, they require G protein signaling and Tre1 (Kunwar, 2008).

A function for E-cadherin in controlling adhesion and migration has been studied extensively in the progression of tumor metastasis and the development of epithelial-mesenchymal transitions (EMTs). Cells undergoing metastasis and EMTs express lower levels of E-cadherin, and the loss of E-cadherin promotes invasion of tumor cells. The loss of E-cadherin in these cases promotes the disruption of E-cadherin-mediated cell adhesion between epithelial cells, allowing these cells to spread and migrate, and is often triggered through induction of the transcriptional repressors Twist and Snail in response to inductive signals. However, in the case of germ cell dispersal, the effect of Tre1 on DE-cadherin is not transcriptional because DE-cadherin is provided maternally in the germ cells. These data suggest that Tre1 GPCR signaling might regulate the turnover or cellular distribution of DE-cadherin-mediated adhesion complexes in a polarized fashion. It is possible that in addition to transcriptional mechanisms, such a polarized regulation also functions during EMT and metastasis (Kunwar, 2008).

Drosophila primordial germ cell migration requires epithelial remodeling of the endoderm

Trans-epithelial migration describes the ability of migrating cells to cross epithelial tissues and occurs during development, infection, inflammation, immune surveillance, wound healing and cancer metastasis. This study investigated Drosophila primordial germ cells (PGCs), which migrate through the endodermal epithelium. Through live imaging and genetic experimentation it was demonstrated that PGCs take advantage of endodermal tissue remodeling to gain access to the gonadal mesoderm and are unable to migrate through intact epithelial tissues. These results are in contrast to the behavior of leukocytes, which actively loosen epithelial junctions to migrate, and raise the possibility that in other contexts in which migrating cells appear to breach tissue barriers, they are actually exploiting existing tissue permeability. Therefore, the use of active invasive programs is not the sole mechanism to infiltrate tissues (Seifert, 2012).

Cells undergoing trans-epithelial migration (TEM) must overcome the specialized cell-cell junctions of epithelial cells that form occluding barriers and obstruct cell migration. During leukocyte extravasation, a well-studied model of TEM, interactions between leukocytes and endothelial cells through ligand-receptor binding trigger a loosening of endothelial cell-cell junctions, allowing leukocytes to squeeze through these sites of reduced endothelial contact. Although the active remodeling of cell-cell contacts by migrating cells is an established mechanism of TEM, migrating cells in other systems might employ additional mechanisms (Seifert, 2012).

In many species, primordial germ cells (PGCs), which are responsible for producing gametes, migrate through several tissues within the embryo as they travel to the site where the gonad will form. In Drosophila, PGCs start their active migration by penetrating an epithelium comprising endodermal cells. After crossing the endoderm, PGCs reorganize on the basal surface of the future midgut and migrate into the mesoderm where they contact and adhere to the somatic gonadal precursor cells. Migration through the endoderm requires activation of the G protein-coupled receptor (GPCR) Trapped in endoderm 1 (Tre1) within PGCs . However, the signal that activates the Tre1 receptor is currently unknown. Transplantation studies indicate that the timing of PGC migration is dictated by the developmental stage of the endoderm, not by the developmental stage of the PGCs, consistent with a model in which the endoderm produces the Tre1 signal leading to PGC migration. PGCs do not initiate migration in embryos mutant for serpent (srp), a GATA transcription factor required for endoderm specification, further supporting this model (Seifert, 2012).

Concomitant with PGC migration, the endoderm undergoes epithelial remodeling as part of an epithelial to mesenchymal transition (EMT). Discontinuities in circumferential adherens junctions and intercellular spaces between endodermal cells observed by electron microscopy have been postulated to act as exit sites for PGCs, although their functional role has never been tested. In srp mutants EMT does not occur, nor does the epithelium display intercellular gaps. Therefore, it is unknown whether the physical transformation of the endoderm from epithelium to mesenchyme is required for PGC migration or if the endoderm is responsible for generating a Tre1 signal leading to PGC migration (Seifert, 2012).

To determine the role of the endoderm during PGC migration this study assessed the ability of PGCs to migrate in genetic backgrounds that perturb epithelial remodeling, endodermal specification or epithelial maintenance. The results indicate that PGCs are incapable of migrating through intact epithelial tissues and are dependent on developmentally regulated epithelial remodeling, which causes discontinuities in the endoderm that allow PGCs to migrate. It was found that two independent programs are required for PGC migration through the endoderm: first, activation of Tre1 within PGCs as part of an autonomous migratory program; and second, disruption of the endodermal epithelium, which generates spaces within the tissue for PGC migration (Seifert, 2012).

Collectively stabilizing and orienting posterior migratory forces disperses cell clusters in vivo

Individual cells detach from cohesive ensembles during development and can inappropriately separate in disease. Although much is known about how cells separate from epithelia, it remains unclear how cells disperse from clusters lacking apical-basal polarity, a hallmark of advanced epithelial cancers. Using live imaging of the developmental migration program of Drosophila primordial germ cells (PGCs), this study shows that cluster dispersal is accomplished by stabilizing and orienting migratory forces. PGCs utilize a G protein coupled receptor (GPCR), Tre1, to guide front-back migratory polarity radially from the cluster toward the endoderm. Posteriorly positioned myosin-dependent contractile forces pull on cell-cell contacts until cells release. Tre1 mutant cells migrate randomly with transient enrichment of the force machinery but fail to separate, indicating a temporal contractile force threshold for detachment. E-cadherin is retained on the cell surface during cell separation and augmenting cell-cell adhesion does not impede detachment. Notably, coordinated migration improves cluster dispersal efficiency by stabilizing cell-cell interfaces and facilitating symmetric pulling. This study demonstrates that guidance of inherent migratory forces is sufficient to disperse cell clusters under physiological settings and present a paradigm for how such events could occur across development and disease (Lin, 2020).

The developmental migration of Drosophila PGCs is an excellent model to study how actomyosin contractility is deployed to separate cells from non-epithelial clusters. PGCs are a group of 30-40 cells born at the posterior of the embryo. During gastrulation, PGCs are swept into the interior of the embryo, where they reside as a tight cluster in a rosette configuration enveloped by the endoderm. PGCs subsequently separate and individually transmigrate through the endoderm as it undergoes a developmentally programmed epithelial-to-mesenchymal transition (EMT). How cluster separation is achieved mechanistically remains elusive. Known autonomous proteins required for PGC cluster dispersal are the orphan G-protein-coupled receptor (GPCR), Tre1 and its associated Gβγ subunit, consisting of Gβ13F and Gγ1, as well as the small Rho GTPase, RhoA, suggesting the involvement of contractile forces. However, actomyosin dynamics during PGC cluster dispersal remain uncharacterized and how Tre1 influences the spatiotemporal dynamics of these networks is unknown (Lin, 2020).

This work harnesses two-photon live imaging to provide an in vivo description of how actomyosin contractility is deployed to disperse cell clusters lacking apical-basal polarity under physiological conditions. In contrast to current models of epithelial delamination, cluster dispersal does not involve a sustained downregulation of cell-cell adhesion or augmented force production and is surprisingly robust to increased levels of adhesion. Rather, inherent migratory forces are co-opted to liberate cells. This is accomplished through the sensing of a directed migration cue via the GPCR, Tre1. Tre1 signaling stabilizes and orients migratory polarity radially from the cell cluster, thereby positioning posterior myosin II dependent contractile forces towards cell-cell interfaces in the cluster interior. This collective radial polarity stabilizes cell-cell interfaces and enables symmetric tugging, increasing the efficiency of cluster dispersal (see PGC clusters disperse by directing individual migratory polarity outward to collectively remove cell-cell adhesions). Symmetric tugging, however, is not absolutely necessary for cell separation, as individual WT PGCs can still detach from tre1 PGC clusters, albeit less efficiently. Subsequent detachment requires sustained pulling on cell-cell adhesions provided by a stable migratory polarity. Thus, randomly migrating cells, equally capable of contractile force production, are unable to separate because they do not pull on cell-cell adhesions in a given orientation for a sufficient period of time. A caveat to this model is that this study has not directly shown that migrating PGCs exert posterior pulling forces, as this is technically challenging at the depth where PGC cluster dispersal occurs. However, posterior pulling forces have been clearly demonstrated in various cell types utilizing a rearward driven 3D migration mode which closely resembles PGC migration in Drosophila (Lin, 2020).

Mechanistically, the migration-based cluster dispersal mechanism described in this study harbors many commonalities with hepatocyte growth factor (HGF) mediated epithelial scattering. Myogenic precursors induced to delaminate by ectopic application of HGF maintain expression of N-cadherin, the cardinal adhesion molecule originally linking them to the dermomyotome. Similarly, HGF induced scattering of Madin-Darby canine kidney (MDCK) cells does not involve direct alterations in E-cadherin. Instead, HGF promotes motility and strengthens cell-ECM attachment through integrins, which in turn generate a local increase in tension on cell-cell adhesions until they are physically disrupted. PGCs, on the other hand, continue to utilize E-cadherin to adhere to another cellular substrate, the surrounding endoderm, to pull away from each other. For both PGCs and HGF stimulated MDCK cells, the absence of free space to migrate is sufficient to block dispersal (Lin, 2020).

Cell ensembles frequently exhibit collective migration during development and disease. How is group cohesion maintained if contractile migratory forces are sufficient to disrupt cell-cell adhesion? In collectively migrating squamous cell carcinoma (SSC) cells, primary colorectal cancer explants, and Xenopus neural crest, actomyosin contractility is enriched along the group perimeter and is actively suppressed from cell to cell interfaces in SSC cells to prevent cell detachment. This suppression relies on Discoidin domain receptor 1 (DDR1), which acts in a noncanonical manner at cell-cell interfaces by recruiting Par3 and Par6 to control the localization of RhoE to antagonize RhoA activity. Depletion of DDR1 and other members of the complex result in elevated levels of active Myosin II at cell-cell margins and individual cell migration away from the group, leading to group dispersal. This is strikingly similar to the mechanism uncovered in this study during PGC cluster dispersal and that of HGF mediated cell scattering. Recent work has also revealed an alternative strategy to reduce RhoA signaling at cell-cell junctions of collectively migrating SSC cells through Snail dependent expression of claudin-11, which activates Src to recruit p190RhoGAP to cell-cell interfaces (Li, 2019). In collectively migrating Drosophila border cells, E-cadherin tension is also reduced in the interior of the migrating cell cluster. Thus, while the suppression of contractile forces at cell-cell contacts appears to be a general principle to ensure cohesion in collectively migrating cell populations, PGCs actively direct actomyosin contractility towards cell-cell interfaces to separate. It will be interesting to assess whether pathological cell aggregates can be coaxed to disperse through a similar mechanism (Lin, 2020).

This work demonstrates that cluster dispersal can be driven by the concerted reorientation of migratory actomyosin forces towards cell-cell interfaces. For PGCs, this is accomplished by sensing a directed migration cue through a GPCR, Tre1. However, in principle, any migratory cue, such as ECM or substrate stiffness, could be sufficient to mediate this reorientation. Subsequent cell-cell separation does not require any alterations of the inherent actomyosin forces driving migration. Rather, these forces must be applied on cell-cell junctions for a sufficient period of time, highlighting a potential safeguard against erroneous cell detachment. An open question is the identity of the Tre1 ligand. Given that PGCs are directed to migrate toward the endoderm within a tightly enclosed pocket, the ligand is likely to be surface bound, which would concur with the known roles of Tre1 in orienting neuroblast division56 and immune cell extravasation. Overall, it is anticipated that directed motility-based separation will have general relevance for individual cell detachment events from clustered cell groups lacking apical-basal polarity in development and disease (Lin, 2020).

Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells

In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. This study reports the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. It is proposed that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target (Kunwar, 2003a).

A gain-of-function screen was conducted using the GAL4/UAS system to upregulate genes specifically in the germ cells and then defects in germ cell migration were assayed. To drive expression, the nanos-GAL4-VP16 (nos-GAL4) transgene was used to maternally localize the GAL4-VP16 transcriptional activator specifically to germ plasm and primordial germ cells. Of 2,300 lines screened, one, EP1631, gave the most striking phenotype, causing large numbers of germ cells to scatter throughout the embryo. At stage 11, when germ cells in the wild-type have largely associated with the mesoderm, germ cells expressing EP1631 were very disorganized, and although many cells were near the SGPs, some cells migrated far past their mesodermal targets and into the ectoderm. At later stages, many germ cells were found at ectopic locations, often resulting in gonads with as few as one germ cell, instead of the 12-15 found per gonad normally. This phenotype was only observed when EP1631 was expressed in germ cells. Overexpression of EP1631 in a number of other migratory tissues, such as gut, mesoderm, central nervous system (CNS), trachea, or crystal cells, did not affect germ cell migration, nor were significant somatic defects observed in these embryos (Kunwar, 2003a).

EP1631 inserted upstream of the gene CG4322, which encodes a putative seven transmembrane GPCR. In situ hybridization analysis revealed that CG4322 GPCR mRNA is expressed in a variety of migratory cells in the embryo, such as the hemocytes, PMG, caudal visceral mesoderm, and glia. No CG4322 mRNA was detected in germ cells. To determine whether CG4322 plays a role in normal germ cell migration, deletion lines were generated by imprecise P-element excision. It was found that tissues that endogenously express CG4322 transcripts, such as midgut, visceral mesoderm, hemocytes, and glia, showed no gross abnormalities in these mutants. Most importantly, no significant effect was found on germ cell migration. In order to rule out a maternal contribution of CG4322 to germ cell migration, embryos were generated that lacked both maternal and zygotic contribution of the CG4322 GPCR by using the OvoD/Flp technique. These embryos also showed normal germ cell migration. It is therefore concluded that CG4322, while having a dramatic effect when misexpressed in germ cells, does not play a role normally in germ cells (Kunwar, 2003a).

This study has identified a novel Drosophila GPCR, Tre1, that is required for transepithelial migration of germ cells through the PMG epithelium. tre1 RNA is expressed in germ cells, and tre1 acts cell autonomously in germ cells. Transmigration of germ cells through the PMG epithelium is the first active stage of germ cell migration, and specific mutations had previously not been identified for this step. Tre1 GPCR function specifically affects this stage, since 'pioneer' tre1 germ cells that bypass the requirement for transepithelial migration through the PMG are motile and can follow other, later-acting migratory cues. These results suggest that GPCRs play an important role in transepithelial migration of germ cells and lead to the speculation that Tre1 might function in a manner equivalent to the chemokine receptors required for transepithelial migration of leukocytes (Kunwar, 2003a).

The Tre1 GPCR belongs to a new subclass of Rhodopsin family GPCRs. Within this subclass, three fly homologs were identified. Indeed the striking phenotype of one of these homologs, CG4322, when misexpressed in germ cells, led to the discovery of Tre1's role in transepithelial migration. The fact that all three homologs are expressed in migratory cell populations, such as germ cells, hemocytes, glia, and midgut cells, raises the possibility that they may have conserved functions in directional cell migration. While only tre1 mutants cause the transgut migration defect, CG4322 but not tre1 overexpression in germ cells produces a germ cell migration phenotype. These receptors may thus activate different downstream signaling cascades. Alternatively, differences in the extent of their expression levels or their ability to activate the same downstream pathway independent of ligand may cause the differences in migratory response to be observed. Given the expression patterns of the three homologs, it is possible that they function in a partially redundant manner and that they respond to the same ligand. NCBI database analysis identified three uncharacterized Anopheles proteins, which clearly aligned with the respective Drosophila receptors, and there are also vertebrate members of this new family from human, mouse, zebrafish, and Fugu. These are largely uncharacterized GPCRs, and their exact expression pattern, function, or mode of activation is unknown. It is interesting to note, however, that the human family member, EX33, and the mouse homolog, GPR84, are expressed in migratory tissues, including leukocytes, which undergo transepithelial migration. Based on these observations, it is tempting to speculate that this new group of GPCRs might be required for a variety of migratory functions, including transepithelial migration. It will be interesting to see whether these GPCRs also play an important role in germ cell development in other organisms (Kunwar, 2003a).

The studies also identified a likely downstream target of Tre1 GPCR activity. It was found that the ability of germ cells to transmigrate the PMG is affected by mutations in tre1 and by inhibiting Rho1 function. Rho GTPase family members have been shown to mediate GPCR responses through both G protein-dependent and G protein-independent mechanisms. Generally, Rho GTPase mediates signals from G proteins to regulate the actin cytoskeleton to promote adhesion and movement. In Drosophila, Rho1 has been intensively studied for its effect on cell shape changes during gastrulation. Here Rho1 acts downstream of concertina (cta), the Drosophila homolog of G protein β12/13 and a Rho guanine exchange factor, RhoGEF2. Rho1, Cta, and another RhoGEF (Pebble) are present in early germ cells and are thus likely targets to mediate transepithelial migration affected by Tre1. However, because of the maternal-effect gastrulation defect observed in cta mutants and the role of Pebble in blastoderm cytokinesis, it has not yet been possible to investigate their roles in germ cell migration. Interestingly, a few mammalian GPCRs in the Rhodopsin class mediate a response by directly associating with monomeric GTPases, such as Rho1 and ARF, which are involved in the regulation of endocytosis and phagocytosis. This interaction is dependent upon an NPxxY motif in the seventh transmembrane domain of the receptor. All GPCRs of the Tre1 subfamily share the NPxxY domain, suggesting that Rho1 might mediate Tre1 signals through this motif (Kunwar, 2003a).

This study has identified a GPCR, Tre1, required for transepithelial migration. Receptor activity is provided maternally to the germ cells, but the phenotype can also be partially rescued by zygotic expression of the receptor or completely restored by zygotic overexpression of the receptor using the UAS/nos-GAL4 transcription system. While it has been firmly established that the onset of zygotic expression in germ cells is delayed with respect to zygotic expression in the soma, the results suggest that zygotic gene expression is activated in germ cells prior to the onset of germ cell migration. This result, as well as the phenotypes observed after overexpression of Rho1 or the tre1-related gene CG4322, further demonstrates the usefulness of the nos-GAL4 system for the analysis of even very early aspects of germ cell migration and development. The analysis of early germ cells has been hampered by the pleiotropic effects that many of the known signaling molecules exert on oogenesis and early embryogenesis, making it often difficult to assess germ cell migration in an embryo with defective somatic patterning. In the course of these studies using the nos-GAL4 system, many constitutively activated and dominant-negative forms of GTPases were expressed. While other GTPases, such as activated Rac and Rho1, affected the actin cytoskeleton of germ cells and led to migration defects, only dominant-negative Rho1 GTPase gave a specific transepithelial migration defect. Receptors and transducers for most signaling pathways that control many aspects of development were tested, such as FGF, EGF, Notch, Wingless, Hedgehog, Pten, and PI3 kinase in germ cells. Except for the GPCR Tre1 and CG4322, none of them resulted in any type of germ cell migration defect. These data suggest that GPCR signaling is a major determinant in the guidance of Drosophila germ cells. Given the role recently shown for the GPCR CXCR4 in zebrafish and mouse germ cell migration, GPCR signaling may indeed be an evolutionarily conserved aspect of germ cell development (Kunwar, 2003a).

This study has shown that in addition to providing directional cues for germ cell guidance along somatic tissue, GPCRs play an important role in the transepithelial migration of germ cells. Drosophila germ cells are not unique with regard to transepithelial migration. Primordial germ cells in chick embryos migrate into the vasculature, where they are passively transported by the bloodstream until they transmigrate the endothelium and invade the gonad. Mouse germ cells also undergo transepithelial migration as they move out of the hindgut toward the mesentery. Very little is known about the molecules required for these early migratory events in vertebrates. This study of transepithelial migration in Drosophila may provide the first molecular insight into this process (Kunwar, 2003a).

Search PubMed for articles about Drosophila Tre1

Affolter, M. and Weijer, C. J. (2005). Signaling to cytoskeletal dynamics during chemotaxis. Dev. Cell 9: 19-34. PubMed ID: 15992538

Ara, T., et al. (2003). Impaired colonization of the gonads by primordial germ cells in mice lacking a chemokine, stromal cell-derived factor-1 (SDF-1). Proc. Natl. Acad. Sci. 100: 5319-5323. PubMed ID: 12684531

Bainton, R. J., et al. (2005). moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila. Cell 123: 145-156. PubMed ID: 16213219

Bouchard, C., et al. (2007). G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions. Glia 55: 790-800. PubMed ID: 17390309

Doitsidou, M., et al. (2002). Guidance of primordial germ cell migration by the chemokine SDF-1. Cell 111: 647-659. PubMed ID: 12464177

Fuse, N., et al. (2003). Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions. Curr. Biol. 13: 947-954. PubMed ID: 12781133

Huber, M. A., Kraut, N. and Beug, H. (2005). Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr. Opin. Cell Biol. 17: 548-558. PubMed ID: 16098727

Kelly, P., et al. (2006a). The G12 family of heterotrimeric G proteins promotes breast cancer invasion and metastasis. Proc. Natl. Acad. Sci. 103: 8173-8178. PubMed ID: 16705036

Kelly, P., et al. (2006b). A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion. J. Biol. Chem. 281: 26483-26490. PubMed ID: 16787920

Knaut, H., Werz, C., Geisler, R. and Nusslein-Volhard, C. (2003). A zebrafish homologue of the chemokine receptor Cxcr4 is a germ-cell guidance receptor. Nature 421: 279-282. PubMed ID: 12508118

Kunwar, P. S., et al. (2003a). Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells. PLoS Biol. 1: E80. PubMed ID: 14691551

Kunwar, P.S. and Lehmann, R. (2003b). Developmental biology: Germ-cell attraction. Nature 421: 226-227. PubMed ID: 12529629

Kunwar, P.S., Siekhaus, D. E. and Lehmann, R. (2006). In vivo migration: a germ cell perspective. Annu. Rev. Cell Dev. Biol. 22: 237-265. PubMed ID: 16774460

Kunwar, P. S., Sano, H., Renault, A. D., Barbosa, V., Fuse, N. and Lehmann R. (2008). Tre1 GPCR initiates germ cell transepithelial migration by regulating Drosophila melanogaster E-cadherin. J. Cell Biol. 183(1): 157-68. PubMed ID: 18824569

Li, C. F., Chen, J. Y., Ho, Y. H., Hsu, W. H., Wu, L. C., Lan, H. Y., Hsu, D. S., Tai, S. K., Chang, Y. C. and Yang, M. H. (2019). Snail-induced claudin-11 prompts collective migration for tumour progression. Nat Cell Biol 21(2): 251-262. PubMed ID: 30664792

Lin, B., Luo, J. and Lehmann, R. (2020). Collectively stabilizing and orienting posterior migratory forces disperses cell clusters in vivo. Nat Commun 11(1): 4477. PubMed ID: 32901019

Molyneaux, K. A., et al. (2003). The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival. Development 130: 4279-4286. PubMed ID: 12900445

Parks, S. and Wieschaus, E. (1991). The Drosophila gastrulation gene concertina encodes a G alpha-like protein. Cell 64: 447-458. PubMed ID: 1899050

Sano, H., Renault, A. D. and Lehmann, R. (2005). Control of lateral migration and germ cell elimination by the Drosophila melanogaster lipid phosphate phosphatases Wunen and Wunen 2. J. Cell Biol. 171: 675-683. PubMed ID: 16301333

Seifert, J. R. and Lehmann, R. (2012). Drosophila primordial germ cell migration requires epithelial remodeling of the endoderm. Development 139: 2101-2106. PubMed ID: 22619387

Ulrich, F., et al. (2005). Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin. Dev. Cell 9: 555-564. PubMed ID: 16198297

date revised: 25 December 2020

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