shotgun

Transcriptional Regulation

During tracheal development, the tip cells, located at the end of each branch that is going to fuse, extend filopodia to search for targets; later they change their cell shape into a seamless ring, to allow passage of lumen. The cell adhesion molecule E-cadherin accumulates at the site of contact to form a ring that marks the site of lumen entry and is essential for the fusion. DE-cadherin expression in tip cells of a subset of branches is dependent on escargot, a zinc finger gene expressed in all tip cells. Such escargot mutant tip cells failed to adhere to one another and continue to search for alternative targets by extending long filopodia. These cells also develop an abnormal cuticle in their blind-ended structure and their terminus floats freely in the body. escargot positively regulates transcription of the DE-cadherin gene, shotgun. Overexpression of DE-cadherin rescues the defect in one of the fusion points in escargot mutants, demonstrating an essential role of DE-cadherin in target recognition and identifying escargot as a key regulator of cell adhesion and motility in tracheal morphogenesis (Tanaka-Matakatsu, 1996).

Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (Zw-3), and Armadillo (Arm), work sequentially; Wg and Dsh negatively regulate Zw-3, which in turn down-regulates Arm. To biochemically analyze interactions between the Wg pathway and Drosophila E-cadherin (DE-cadherin) which binds to Arm, Dsh, Zw-3, and Arm, were overexpressed in the Drosophila wing disc cell line, clone 8, which responds to Wg signal. Dsh overexpression leads to accumulation of Arm primarily in the cytosol and elevation of DE-cadherin at cell junctions. Overexpression of wild-type and dominant-negative forms of Zw-3 decreases and increases Arm levels, respectively, indicating that modulation in Zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevates DE-cadherin levels, suggesting that Dsh-induced DE-cadherin elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative Zw-3-, and truncated Arm-induced accumulation of DE-cadherin protein is accompanied by a marked increase in the steady-state levels of DE-cadherin mRNA, suggesting that transcription of DE-cadherin is activated by Wg signaling. In addition, overexpression of DE-cadherin elevates Arm levels by stabilizing Arm at cell-cell junctions (Yanagawa, 1997).

The JAK/STAT signaling pathway, renowned for its effects on cell proliferation and survival, is constitutively active in various human cancers, including ovarian. JAK (Hopscotch) and STAT are required to convert the border cells in the Drosophila ovary from stationary, epithelial cells to migratory, invasive cells. The ligand for this pathway, Unpaired (Upd: Outstretched), is expressed by two central cells within the migratory cell cluster. Mutations in upd or jak cause defects in migration and a reduction in the number of cells recruited to the cluster. Ectopic expression of either Upd or JAK is sufficient to induce extra epithelial cells to migrate. Thus, a localized signal activates the JAK/STAT pathway in neighboring epithelial cells, causing them to become invasive (Silver, 2001).

In order to gain further insight into the mechanism by which STAT regulates border cell migration, the expression of a number of proteins that are highly expressed in border cells was examined, some of which are also required for migration. The first gene identified as playing a critical role in border cell migration was slow border cells (slbo). slbo encodes Drosophila C/EBP, a basic region/leucine zipper transcription factor. Slbo protein expression is undetectable in stat mutant border cells, which were identified using a positive clone marking system known as MARCM. This result was confirmed by examining several additional proteins, the expression of which is reduced in slbo mutant border cells. In wild-type stage 8 and 9 egg chambers, FAK expression is upregulated in migratory border cells. Border cells that lack stat exhibit reduced levels of FAK. In wild-type stage 9 egg chambers, DE-cadherin (Shotgun) is enriched throughout the border cell cluster and is expressed to the highest level in the polar cells. Stat mutant border cells exhibit reduced DE-cadherin expression compared to wild-type border cells of the same cluster. The polar cells, though mutant, do not show reduced DE-cadherin staining, which is also true of slbo mutants. Additional downstream targets of slbo, including PZ6356 and zinc finger transcription factor jing, are also reduced in stat mosaic clones. Thus, even the few mutant cells that are recruited to the cluster fail to express many border cell proteins required for migration. The effect is specific because expression of Taiman, a protein that is required for border cell migration but is independent of the slbo pathway, was not altered. Mosaic clusters containing a mixture of wild-type and mutant cells show variable migration defects. On average, the extent of migration is proportional to the number of wild-type cells in the cluster (Silver, 2001).

Egg chambers from females heterozygous for any of the stat alleles have a semi-dominant border cell migration phenotype. Advantage was taken of this slight haploinsufficiency to test for dominant genetic interactions with other genes required for border cell migration. Dominant genetic interactions were observed with slbo, hop, and upd alleles. A mutation in the gene coding for DE-cadherin, shotgun, also exhibited a dominant interaction with stat. These interactions appeared to be specific, since stat does not interact with other known border cell migration genes, such as tai, jing, or PZ6356 (Silver, 2001).

At the time of normal border cell migration, expression of Drosophila E-cadherin (DE-cadherin: Shotgun) increases within the border cells, and Drosophila C/EBP (Slbo) is required for this elevation of DE-cadherin expression. Drosophila ß-catenin, known as Armadillo (Arm), colocalizes with DE-cadherin in both wild-type and mutant egg chambers. To determine whether jing function is also required for proper accumulation of DE-cadherin and Arm, egg chambers containing jing mutant border cells were stained with antibodies against DE-cadherin or Arm, and the staining was compared to wild-type and slbo mutant border cells. In wild-type border cell clusters, staining for DE-cadherin and Arm is strongest in the central cells known as polar cells, which express FASIII and in the junctions between border cells. The staining is somewhat less intense and punctate in appearance at the interfaces between border cells and nurse cells. In slbo mutant clusters, DE-cadherin and Arm staining is only detected in the central polar cells. Border cells mutant for jing exhibit normal expression of both DE-cadherin and Arm. Thus jing function, unlike slbo, is not required for either DE-cadherin or Arm expression. In all cases FASIII staining is normal, indicating normal polar cell fate (Liu, 2001).

DE-cadherin expression is required for border cell migration and is reduced in slbo mutant border cells, but not in jing mutant border cells. Yet expression of Jing is able to rescue the migration defect associated with the slbo hypomorph. DE-cadherin expression in the P[hs-jing];slbo/slbo egg chambers was examined to determine whether the cells were able to migrate despite the absence of DE-cadherin expression or, alternatively, whether high levels of Jing were able to restore DE-cadherin expression. DE-cadherin expression in the border cells is restored in early stage 9 slbo;hs-jing egg chambers, following expression of Jing, but not at later stages (Liu, 2001).

Thus DE-Cadherin expression is not affected in jing mutant clones, though it is reduced in slbo mutants. DE-cadherin expression may require the presence of either jing or slbo. In slbo mutants, expression of a jing-lacZ reporter is also reduced, and DE-cadherin expression is affected. However in jing mutants, slbo expression does not appear to be reduced and DE-cadherin expression is unaffected. However DE-cadherin expression may require that some Slbo protein is present since over-expression of Jing does not rescue the strong female sterile combination of slbo alleles (LY6/e7b) even though it does rescue the weaker allele (slbo¹). This selective rescue has also been observed with hs-breathless, which rescues the mild but not the strong female sterile slbo alleles. To date only hs-slbo has been observed to rescue the border cell migration defects associated with the strongest female sterile alleles of the slbo locus. Thus jing cannot completely substitute for slbo, consistent with the observation that there are multiple downstream targets of slbo with essential roles in border cell migration (Liu, 2001).

Among the diverse cellular processes taking place during oogenesis, the delamination and migration of border cells (BCs), a group of anterior follicle cells, represent a powerful model to study cell invasion in a normal tissue. During stage 9 of oogenesis, BCs detach from the outer epithelium to invade the germline cyst compartment. The BC cluster contains two centrally located polar cells surrounded by approximately six outer border cells and undergoes a nearly 6-hour long posteriorward migration to reach the anterior part of the growing oocyte. Together with centripetal cells, they assemble the micropyle, a specialized structure required for sperm entry. domeless was isolated in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, dome is required in the germarium for the polarization of follicle cells during encapsulation of germline cells. In this process, dome controls the expression of the apical determinant Crumbs. In contrast to the ligand Upd, whose expression is limited to a pair of polar cells at both ends of the egg chamber, dome is expressed in all germline and follicle cells. However, Dome protein is specifically localized at apicolateral membranes and undergoes ligand-dependent internalization in the follicle cells. dome mutations interact genetically with JAK/STAT pathway genes in border cell migration and abolish the nuclear translocation of Stat92E in vivo. dome functions downstream of upd and both the extracellular and intracellular domains of Dome are required for JAK/STAT signaling. Altogether, the data indicate that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis (Ghiglione, 2002).

The dramatic, early follicle cell phenotype contrasts with the essentially normal phenotype of dome mutant cells observed in later stage egg chambers. In this case, follicle cells are viable and divide normally. A similar, dual phenotype has been reported in crumbs mutant chambers. After initial polarization of the follicle cells in the germarium, Crumbs is no longer required and its loss has no visible effects. Importantly, dome controls Crumbs expression in follicle cells, thus providing a novel link between the JAK/STAT signaling pathway and epithelial polarity (Ghiglione, 2002).

In addition to its early function in the germarium, dome is required for the normal expression of several follicle cell markers, including DE-cadherin and Fas3. It is important to note that despite a clear defect in the expression of these markers, dome mosaic egg chambers are morphologically normal. However, because completely mutant egg chambers cannot be obtained because of the early effect of dome in the germarium, one cannot rule out the possibility that large mutant clones would lead to abnormal development of egg chambers (Ghiglione, 2002).

The pattern of epithelial markers in dome mutant cells indicates that the JAK/STAT pathway is active in all follicle cells, a notion that is reinforced by the wide expression of nuclear Stat92E. How is Dome activated during egg chamber development and does this activation follow the same profile at all stages? Given the restricted pattern of upd expression in the egg chamber and its dramatic effect upon overexpression, it is unlikely that Upd is able to signal long distances in the follicular epithelium of late stage egg chambers. Rather, a model by which the JAK/STAT pathway plays a pre-patterning function is favored, acting early during egg chamber development to activate DE-cadherin and Crumbs expression. This view is consistent both with the expression pattern of upd and the distribution of Dome-containing vesicles described in this study. The formation of endogenous vesicles can be promoted by Upd, and a gradient of such vesicles is present around polar cells. Strikingly, these vesicles, which likely indicate active signaling through Dome, are widespread at early stages and become more restricted later on. It is proposed that during early development, the Upd signal produced by anterior and posterior polar cells contributes to the differentiation of all follicle cells. At this stage, Upd would be more diffusible than later, as suggested by the pattern of Dome intracellular vesicles. The study of the mechanisms controlling Dome activation and Upd activity will require additional tools to directly detect Upd, as, for example, Upd-GFP fusion proteins (Ghiglione, 2002).

This study has revealed several new findings about the function of dome and the JAK/STAT pathway during oogenesis. Future work will help to understand how Upd and Dome initially interact at the cell surface and transduce the signal to downstream JAK/STAT pathway members (Ghiglione, 2002).

In an attempt to identify gene targets of ash2, an expression analysis was performed by using cDNA microarrays. Genes involved in cell cycle, cell proliferation, and cell adhesion are among these targets, and some of them are validated by functional and expression studies. Genes involved in cell adhesion and/or development of the neural system (i.e., FasII, mfas, Ama, Lac, and shg) are two of the main classes regulated by ash2. Even though trithorax proteins act by modulating chromatin structure at particular chromosomal locations, evidence of physical aggregation of ash2-regulated genes has not been found. This work represents the first microarray analysis of a trithorax-group gene (Beltran, 2003).

IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin

Remodelling of tissues depends on the coordinated regulation of multiple cellular processes, such as cell-cell communication, differential cell adhesion and programmed cell death. During pupal development, interommatidial cells (IOCs) of the Drosophila eye initially form two or three cell rows between individual ommatidia, but then rearrange into a single row of cells. The surplus cells are eliminated by programmed cell death, and the definitive hexagonal array of cells is formed, which is the basis for the regular pattern of ommatidia visible in the adult eye. This cell-sorting process depends on the presence of a continuous belt of the homophilic cell adhesion protein DE-cadherin at the apical end of the IOCs. Elimination of this adhesion belt by mutations in shotgun, which encodes DE-cadherin, or its disruption by overexpression of DE-cadherin, the intracellular domain of Crumbs, or by a dominant version of the monomeric GTPase Rho1, prevents localisation of the transmembrane protein IrreC-rst to the border between primary pigment cells and IOCs. As a consequence, the IOCs are not properly sorted and supernumerary cells survive. During the sorting process, Notch-mediated signalling in IOCs acts downstream of DE-cadherin to restrict IrreC-rst to this border. The data are discussed in relation to the roles of selective cell adhesion and cell signalling during tissue reorganisation (Grzeschik, 2005).

To summarise, IrreC-rst is colocalised with DE-cadherin in epithelial cells of pupal eye discs, and misdistribution of adherens junction components induces the mislocalisation of IrreC-rst, which then affects sorting of IOCs. However, although DE-cadherin forms a continuous belt in the apical regions of all cells (including all IOCs) in wild-type discs, IrreC-rst colocalises with DE-cadherin only at the border between 1° pigment cells and IOCs. What factor(s) might be responsible for the spatial restriction of IrreC-rst to this border? It has recently been shown that the removal of Notch or Delta function during cell-sorting results in the ubiquitous distribution of IrreC-rst to all plasma membranes and the prevention of programmed cell death. This study analysed whether this might be the result of defective DE-cadherin localisation. Antibody staining reveals no influence of Notch on the continuous apical localisation of DE-cadherin, but shows that IrreC-rst now colocalises with the latter on all plasma membranes of the IOCs. This suggests that Notch acts downstream of DE-cadherin in the control of IrreC-rst localisation. It is therefore tempting to speculate that it is the Notch pathway, which provides local signalling between the lattice cells to direct cell death, that prevents the accumulation of IrreC-rst at the borders between IOCs and thus restricts its localisation to the 1°/IOC cell boundary (Grzeschik, 2005).

Pattern formation in the Drosophila eye disc depends on a well-balanced system of signals that promote either the survival or the death of cells, mediated by the EGF and Notch receptor pathways, respectively. In addition, the morphogenetic events, which take place in a single-layered epithelium, crucially depend on factors that regulate the maintenance of cell polarity and cell shape, and modulate cell adhesion. Sorting of interommatidial cells (IOCs) during pupal development, which results in the conversion of several parallel rows of cells into a single ring, requires the weakening of pre-existing adhesive cell contacts and the establishment of new ones without interrupting the epithelial integrity of the tissue. During tissue morphogenesis, epithelial cells use different strategies to modify their adhesive contacts. One of these consists of regulating the amount and/or distribution of the homophilic cell-adhesion molecule E-cadherin, one of the central components of the adherens junctions. The first in vivo evidence for this kind of regulation came from the analysis of the Drosophila egg chamber. There, the localisation of the oocyte at the posterior pole depends on a higher level of expression of DE-cadherin in the oocyte and the posterior follicle cells, when compared with the nurse cells and other follicle cells. Differential adhesion can also be regulated by alterations in the composition or activity of intracellular binding partners, or by the integration of various other molecules into the adhesive complexes. No change in the distribution of the adherens junction components DE-cadherin and alpha-catenin could be detected in wild-type discs undergoing rearrangements of the IOCs. This behaviour contrasts with epithelial rearrangements during morphogenesis of the Drosophila tracheal system, which are associated with alterations in the amount of DE-cadherin, controlled by Drosophila Rac, another member of the Rho GTPase family. This in turn suggests that other cytoplasmic or transmembrane proteins are involved in the modulation of adhesion in IOCs. The adhesion protein IrreC-rst is involved in the control of the cell sorting process. Its predominant localisation at the border between primary pigment cells and IOCs (at the 1°/IOC border) has been suggested to provide an attractive interface that controls sorting. According to this proposal, IOCs tend to maximise their contacts with primary pigment cells. Failure to restrict IrreC-rst to this border results in the inability to sort the IOCs properly. Although IrreC-rst behaves as a homophilic adhesion molecule when expressed in cell culture, data from expression analysis argue for the presence of a different, as yet unknown, partner in the primary pigment cell (Grzeschik, 2005).

Of particular interest is the relationship between the localisation of DE-cadherin, a component of the zonula adherens (ZA) and IrreC-rst. In wild-type discs IrreC-rst colocalises with DE-cadherin at the 1°/IOC border in the apical ZA of the cell and removal of DE-cadherin completely abolishes IrreC-rst accumulation. Nothing is yet known about how IrreC-rst may integrate into the ZA at this border. In vertebrates, the Ca⁺⁺-independent cell adhesion molecule nectin, a transmembrane protein of the immunoglobulin superfamily, has been implicated in the organisation of cadherin-based adherens junctions, tight junctions and synapses. It is recruited into cadherin-based adherens junctions through interactions with the PDZ domain of l-afadin, an F-actin-binding protein. Intriguingly, the C-terminal sequence of IrreC-rst (T-A-V) matches the consensus binding site for class I PDZ domains (S/T-X-V). Interestingly, the protein encoded by the mutant allele irreC-rst^CT, which lacks the C-terminal 175 amino acids of the wild-type form, is no longer recruited into the ZA. It is, however, unlikely that IrreC-rst acts as a general adhesion molecule in IOCs of pupal eye discs, because the epithelial tissue structure is stable in the absence of irreC-rst function, as deduced from the formation of the continuous apical belt of DE-cadherin in irreC-rst mutants (Grzeschik, 2005).

The continuous belt of DE-cadherin can be disrupted by a number of different genetic conditions, such as overexpression of the membrane-bound intracellular domain of Crumbs, of DE-cadherin itself, or of a dominant-negative version of the monomeric GTPase Rho1. Overexpression of the membrane-bound intracellular domain of Crumbs in embryonic epithelia has been shown to lead to a redistribution of DE-cadherin throughout the plasma membrane and the formation of multilayered tissues. By contrast, IOCs overexpressing Crb_intra exhibit a fragmented DE-cadherin belt, which remains localized in the apical zone of the cells, and apicobasal organisation and tissue integrity are not affected. This suggests that IOCs may contain additional adhesion components which are independent of, or less affected by, Crb. Support for this view comes from the phenotype of discs lacking crb function, in which the apical belt of DE-cadherin expression is fragmented, yet there is no major effect on polarity or adhesion of the epithelium: the cells undergo nearly normal sorting and IrreC-rst is still restricted to the membrane at the 1°/IOC border. Overexpression of Crb_{intraDeltaERLI} (lacking four C-terminal amino acids [ERLI] of its short cytoplasmic domain, which serve to recruit a multiprotein complex that forms apical to the zonula adherens) does not interfere with sorting, suggesting that a protein complex similar to the one that controls apicobasal polarity in embryonic epithelia (which includes Stardust, DPATJ and D-Lin7) contributes to the development of the dominant phenotype (Grzeschik, 2005).

Overexpression of DE-cadherin similarly results in the fragmentation of the adhesion belt and defects in cell sorting. In various tissues, overexpression of full-length DE-cadherin can also reduce Wingless signalling by sequestering Armadillo from the cytoplasmic pool, thus making it unavailable to transduce the Wingless signal. However, the possibility that the defects in sorting are the result of a suppression of Wingless signalling can be excluded. Inactivation of components of the Wingless pathway in eye imaginal discs induces the initiation of ectopic morphogenetic furrows, and this phenotype was not observed upon overexpression of DE-cadherin. Overexpression of DE-cadherin in eye discs therefore seems to interfere with adhesion, rather than Wingless signalling (Grzeschik, 2005).

Rho GTPases play central roles in the organisation of the actin cytoskeleton and in cell adhesion. In mammals, inhibition of Rho activity results in the removal of cadherins from epithelial cell junctions, while increased Rho activity induces an invasive and metastatic phenotype. Members of the Rho GTPase family are recruited into the adherens junctions by direct interactions with junctional components. Thus, in Drosophila, Rho1 localises to the adherens junctions and interacts directly with alpha-catenin and p120^ctn, a homologue of ß-catenin. As in pupal epithelia expressing a dominant-negative form of Rho1, Rho1 mutant embryos exhibit a diffuse distribution of components of the ZA, such as DE-cadherin and alpha- and ß-catenin. Rho1 may either act directly on the accumulation of cadherins at the junctions, or indirectly by recruiting accessory proteins, which then modulate the amounts or activity of junctional and/or cytoskeletal proteins. Rho1 plays a different role in tracheal epithelia insofar as its inactivation does not disrupt DE-cadherin localisation, but rather interferes with the formation of the apical surface and the tracheal lumen (Grzeschik, 2005).

Although it is evident that DE-cadherin plays a crucial role in the accumulation of IrreC-rst at the adherens junctions, other mechanisms are required to explain the asymmetric localisation and restriction of the latter to the 1°/IOC boundary. It has been speculated that an as yet unknown ligand expressed in the primary pigment cell may account for this restricted accumulation. As an alternative, but not mutually exclusive model, it is suggested that signalling between the IOCs, mediated by Notch, which is expressed in IOCs during pupal development, prevents the accumulation of IrreC-rst at their borders. Interplay between adhesion and signalling molecules also directs other processes in which cellular polarisation is involved in tissue remodelling. The growth of the wing imaginal disc along the proximodistal axis, for example, is the result of cell shape changes and cell rearrangements during pupal development, which are controlled by the atypical cadherins Fat and Dachsous, as well as Four-Jointed, which is assumed to be a secreted molecule. This process, in turn, is responsible for the asymmetric localisation of components that control planar polarity, such as Frizzled, Dishevelled or Strabismus, that serves to ensure that bristles and hairs adopt a common orientation. During germ band elongation in the Drosophila embryo, adherens junction remodelling in intercalating ectodermal cells is facilitated by the polarised expression of non-muscle myosin II at the anteroposterior and of Bazooka at the dorsoventral cell boundaries. Future experiments will demonstrate whether cell sorting in pupal eye discs makes use of any of the components known to be involved in these processes (Grzeschik, 2005).

Function of the ETS transcription factor Yan in border cell migration: Regulation of Shotgun activity

Invasive cell migration in both normal development and metastatic cancer is regulated by various signaling pathways, transcription factors and cell-adhesion molecules. The coordination between these activities in the context of cell migration is poorly understood. During Drosophila oogenesis, a small group of cells called border cells (BCs) exit the follicular epithelium to perform a stereotypic, invasive migration. The ETS transcription factor Yan is required for border cell migration and Yan expression is spatiotemporally regulated as border cells migrate from the anterior pole of the egg chamber towards the nurse cell-oocyte boundary. Yan expression is dependent on inputs from the JAK/STAT, Notch and Receptor tyrosine kinase pathways (Egfr and Pvr) in border cells. Mechanistically, Yan functions to modulate the turnover of DE-Cadherin-dependent adhesive complexes to facilitate border cell migration. These results suggest that Yan acts as a pivotal link between signal transduction, cell adhesion and invasive cell migration in Drosophila border cells (Schober, 2005).

The dynamic expression of Yan is crucial for BC migration, as indicated by the migratory defects associated with both gain- and loss-of-function alleles of yan. Analysis of mutations in the JAK/STAT and Notch signaling pathways reveals that they are required for the expression of at least two transcription factors that are crucial for BC migration and which themselves influence DE-Cad activity. Slbo is specifically expressed in BCs and enhances shg transcription. Yan, by contrast, is expressed in anterior terminal cells, but becomes upregulated in BCs at the time they exit from the epithelium to become migratory. Yan might enhance DE-Cad turnover to facilitate the transition from an immobile epithelial state to a migratory one. Enhanced BC migration defects of hypomorphic slbo mutant egg chambers overexpressing Yan further underscore their interaction to regulate DE-Cad expression and BC migration (Schober, 2005).

Is the function of Yan to facilitate the transition of BCs from an epithelial to a migratory state, or to promote their motility? Although E-Cadherin is often downregulated as cells transit from an epithelial to a mesenchymal-like migratory state, this may not be the case in BCs, since DE-Cad is strongly expressed in BCs and shg mutant BCs fail to migrate. However, BCs mutant for yan or Ecdysone hormone co-receptor taiman (tai) accumulate ectopic DE-Cad-containing adhesive complexes. Consistent with these observations, ectopic stimulation of PVR in BCs, which enhances tai mutant BC migration defects, also results in elevated, cortical DE-Cad staining. Even though the observed BC migration defects in these mutants might not be due to altered surface levels of DE-Cad only, it was found that overexpression of DE-Cad alone can cause migration impaired BCs. E-cadherin not only mediates homophilic cell-cell adhesion but also functions together with its binding partners as a key regulator of the cortical actin cytoskeleton. It is therefore interesting to note that follicle cells overexpressing DE-Cad show severely enhanced filamentous actin staining (Schober, 2005).

The experiments revealed that DE-Cad was elevated in yan mutant BCs and suppressed upon expression of UAS-yan^ACT, suggesting that Yan controls, at least in part, DE-Cad expression in BCs. These observations find further support in the partial rescue of slbo-Gal4::UAS-yan^ACT-induced BC migration defects upon co-expression of UAS-DE-Cad. How does Yan affect DE-Cad expression in BCs? Although the function of Yan as a transcriptional repressor in various tissues suggests that it may act as a transcriptional regulator of shg, no change was detected in shg transcription in yan mutant follicle cells. However, increased membrane dye FM1-43 incorporation in Drosophila SL2 cells overexpressing Yan^ACT, and a decrease in incorporation after yan^RNAi, suggests a change in endocytic activity. E-Cadherin has been found in endocytic compartments and endocytosis has been speculated to modulate E-Cadherin activity regulation during morphogenetic movements. Interestingly, blocking endocytosis by the expression of dominant-negative Rab5 leads to severe BC migration defects and increased DE-Cad staining. Consistent with these observations, expression of shg under a heterologous promoter has been shown to rescue shg mutant BC migration defects, suggesting that the dynamic expression of DE-Cad in BCs might depend on both transcriptional and post-transcriptional mechanisms. Based on these results, a model is favored whereby Yan might, at least in part, function to regulate DE-Cad turnover, possibly through the transcriptional regulation of as-yet-unidentified components of the endocytic machinery (Schober, 2005).

Drosophila exocyst components Sec5, Sec6, and Sec15 regulate E-Cadherin trafficking from recycling endosomes to the plasma membrane

Loss of function of the Drosophila exocyst components in epithelial cells results in E-Cadherin (Shotgun) accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits Shotgun delivery to the membrane. Rab11 and Armadillo interact with Sec15 and Sec10, respectively. These results support a model whereby the exocyst regulates E-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located (Langevin, 2005).

In budding yeast, the exocyst has been proposed to tether post-Golgi vesicles to the membrane of the growing bud prior to fusion. This model is supported by several observations. (1) Exocyst components localize both on post-Golgi vesicles and on the bud membrane (Boyd, 2004). Analogously in Drosophila, Sec5 and Sec15 localize along the lateral membrane and on the REs. (2) Mutations in genes encoding components of the exocyst complex lead to the accumulation of post-Golgi vesicles (Novick, 1980). Analogously, Sec5, Sec6, and Sec15 loss of function leads to an enlargement of the recycling endosome (RE) compartment; this enlargement interpreted as an accumulation of RE vesicles. (3) The localization of Sec8p and Exo70p at the growing bud, i.e., the site of polarized exocytosis, depends on the function of the other exocyst components. Analogously, Sec5 is localized along the lateral membrane, where E-Cadherin delivery is affected, and its localization along the cortex depends on Sec6. It is therefore proposed that in Drosophila epithelial cells, Sec5, Sec6, and Sec15 act by tethering vesicles originating from the recycling endosomal compartment to the lateral membrane of epithelial cells, as a prerequisite for their exocytosis (Langevin, 2005).

In epithelial cells, Arm and E-Cadherin colocalize to the AJs of the ZA as well as along the lateral membrane. In the absence of Sec5, Sec6, and Sec15 function, E-Cadherin trafficking is affected and E-Cadherin accumulates in the RE. Similarly, in the absence of arm, E-Cadherin fails to localize at the membrane and localizes in the RE. The identification of an interaction between Arm and Sec10 is therefore consistent with a model whereby this interaction provides a landmark at the site where Arm is enriched in order to deliver E-Cadherin from the recycling endosomes. Nevertheless, Arm may play an additional role in stabilizing E-Cadherin at the AJs. A direct demonstration of the function of Arm in regulating the delivery of E-Cadherin will therefore require the identification of arm mutant alleles that do not perturb its function as a regulator of E-Cadherin stabilization and only affects its interaction with Sec10 (Langevin, 2005).

In the absence of Sec5, Sec6, or Sec15 function, E-Cadherin delivery to the lateral membrane is inhibited and E-Cadherin accumulates in the REs. Furthermore, E-Cadherin was found to transcytose in a Sec5-dependent manner from the lateral membrane of epithelial cells to the apical AJs. Therefore, this study reveals at least a role of the exocyst in the recycling of E-Cadherin from the lateral membrane to the apical AJs. Furthermore, the strong reduction of E-Cadherin present on the lateral membrane is interpreted as a failure to recycle E-Cadherin from the lateral membrane back to the lateral membrane, which cannot be compensated for by the delivery of newly synthesized E-Cadherin to the lateral membrane. The loss of E-Cadherin on the lateral membrane may also lead to a reduction of E-Cadherin delivery at the AJs. This may have also contributed to the loss of epithelial cell polarity observed in some of the sec5 mutant epithelial cells (Langevin, 2005).

In polarized MDCK cells, the apical REs are well known as a site of sorting during endocytic and transcytotic transport. The REs have also been shown to serve as an intermediate during the transport of newly synthesized proteins from the Golgi to the plasma membrane in nonpolarized MDCK cells. Similarly, upon overexpression of GFP-E-Cad in HeLa cells, E-Cad transits from the Golgi to the Rab11 endosomes. Nevertheless, the existence of such a pathway remains to be established in polarized MDCK cells. In fact, the overexpression of a dominant-negative form of Rab11 leads to sequestration of E-Cadherin in the REs, but whether sequestered E-Cadherin represented newly synthesized or recycled E-Cadherin was not determined. The existence of such a Golgi-to-RE pathway also remains to be established in Drosophila epithelial cells. If so, a role of the exocyst in regulating the delivery of newly synthesized E-Cadherin from the Golgi to the lateral membrane via the REs remains plausible (Langevin, 2005).

Whether the exocyst regulates E-Cadherin localization in mammalian cells has not been directly analyzed. However, E-Cadherin is proposed to act as a regulator of the localization of the exocyst complex in polarizing mammalian cells since E-Cad- and Nectin-2α-dependent cell-cell contacts were proposed to recruit the exocyst complex in order to promote the growth of the lateral epithelial cell domain. The current study suggests that upon the recruitment of the exocyst complex by E-Cadherin, the exocyst promotes the delivery of more E-Cadherin to the lateral membrane during the establishment of apico-basal polarity. In fact, several reports can be reconciled with a function of the exocyst in regulating the transport of E-Cadherin in mammalian cells. Thus, polarized exocytosis of E-Cad to the lateral membrane is dependent upon its interaction with Arm. And, as stated above, REs have shown to serve as an intermediate during the transport of E-Cad from the Golgi to the lateral membrane where E-Cadherin, β-Catenin, and α-Catenin form the AJs. Furthermore, the overexpression of a dominant-negative form of Rab11 impairs the delivery of E-Cadherin to the lateral membrane. Consistent with the exocyst regulating trafficking from the REs, exocyst components also localize on the REs, and Sec15 is an effector of Rab11. Finally, E-Cadherin and catenins are associated with exocyst components (Langevin, 2005 and references therein).

In conclusion, this work provides evidence for a conserved role of the exocyst in regulating the delivery of E-Cadherin from REs to sites on the plasma membrane and in thereby contributing to the maintenance of epithelial cell polarity (Langevin, 2005).

Drosophila Cip4 and WASp define a branch of the Cdc42-Par6-aPKC pathway regulating E-Cadherin endocytosis

Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. This study shows that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. The Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. Altogether These results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue (Leibfried, 2008).

Cdc42 has been implicated in the regulation of polarity establishment in the early Drosophila embryo. The function was shown to be dependent upon the interaction of Cdc42 with the Baz-Par6-aPKC complex that promotes the exclusion of Lgl through Lgl phosphorylation by aPKC. However, the role of Cdc42 in epithelial tissue is unlikely to depend only on its regulation of aPKC because aPKC was shown to be dispensable for apico-basal polarity establishment in the Drosophila embryo. The role of Cdc42 in mammalian epithelial cells has so far been examined by the expression of constitutively active and dominant-negative forms of Cdc42, and such an examination has led to conflicting results in establishing the exact role of Cdc42 in apico-basal polarity maintenance. Nonetheless, they point toward an important role of Cdc42 in the regulation of polarized trafficking. The possible role of Cdc42 in polarized trafficking in epithelial cells was further strengthened by the identification of Cdc42 and the Par complex as regulators of endocytosis in both mammalian cells and C. elegans. Nevertheless, the precise role of Cdc42 and the Par complex in the regulation of endocytosis has remained poorly understood except in migrating cells in which the Par complex was shown to inhibit integrin endocytosis via Numb (Leibfried, 2008).

Cdc42 and its effector Drosophila Cip4 have been found to regulate E-Cad endocytosis and that their loss of function is associated with the formation of long tubular endocytic structures similar to what is observed upon blocking Dynamin function. It is therefore proposed that in Drosophila epithelial cells, Cdc42 controls the early steps of E-Cad endocytosis via Cip4. Because Cdc42, aPKC, and Par6 loss of function are associated with similar defects in E-Cad and Cip4 localization, a simple model is favored, in which the loss of aPKC or Par6 activity disrupts Cdc42 localization or activity and in turn prevents Cip4 function (Leibfried, 2008).

The identified role of PCH family of protein stems in part from the biochemical analysis of Toca-1 as a regulator of actin polymerization. Toca-1 is necessary to activate actin polymerization and actin comet formation downstream of PIP2 and Cdc42 in a WASp-dependent manner (Ho, 2004). On the basis of elegant biochemical assays, Toca-1 was further shown to be necessary to alleviate the WIP inhibitory activity on WASp, in order to allow efficient Arp2/3 activation by WASp (Ho, 2004). Toca-1 was proposed to play an essential role in the fine spatial and temporal regulation of actin polymerization in both cell migration and vesicle movement. Cip4 has been implicated in microtubule organizing center (MTOC) polarization in immune natural killer cells (Banerjee, 2007), a process in which Cdc42 and the Par complex are also involved. Importantly, because Cip4 was shown to bind microtubules, the interaction between Cdc42 and Cip4 might indicate that Cip4 might also be an effector of Cdc42-Par complex in the regulation of MTOC polarization (Leibfried, 2008).

In mammalian cells, regulation of endocytic-vesicle formation has been proposed to be dependent upon both branched actin-filament formation and Dynamin. The role of WASp and Arp2/3 in the regulation of E-Cad endocytosis may therefore indicate that Cip4, which is also known to form dimers, can promote vesicle scission by recruiting Dynamin and promoting actin polymerization via WASp. Therefore, it is proposed that Cip4 and WASp act as a link between Cdc42-Par6-aPKC and the early endocytic machinery to regulate E-Cadherin endocytosis in epithelial cells (Leibfried, 2008).

Csk-deficient boundary cells are eliminated from normal Drosophila epithelia by exclusion, migration, and apoptosis; Loss of Csk functions leads enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion

The construction and maintenance of normal epithelia relies on local signals that guide cells into their proper niches and remove unwanted cells. Failure to execute this process properly may result in aberrant development or diseases, including cancer and associated metastasis. This study shows that local environment influences the behavior of dCsk-deficient cells. Broad loss of dCsk leads to enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion. Loss of dCsk in discrete patches leads to a different outcome: epithelial exclusion, invasive migration, and apoptotic death. These latter phenotypes required sharp differences in dCsk activity between neighbors; dE-cadherin, P120-catenin, Rho1, JNK, and MMP2 mediate this signal. Together, these data demonstrate how the cellular microenvironment plays a central role in determining the outcome of altered dCsk activity, and reveal a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells (Vidal, 2006).

The mechanisms that regulate organ size and shape are not well understood, but recent studies have pointed to the importance of local interactions between neighboring cells. For example, in the process known as 'cell competition', cells with relatively higher proliferative rates actively eliminate their neighbors by programmed cell death. Conversely, apoptotic cells send proliferative signals to their neighbors to compensate for their loss. In this way, normal tissue size is achieved. The misregulation of such mechanisms may contribute to the development of cancer, since most solid tumors arise from intact epithelia and are resistant to size-control signals. Tumors are particularly dangerous when linked to metastasis, a process in which cells leave the primary tumor and invade distant tissues. These processes are best understood within the context of an intact epithelium, in which the full range of cell interactions is retained. Work in Drosophila has provided an important in situ view of the action of oncogenes within epithelia (Vidal, 2006).

Src family kinases (SFKs) are active in a broad range of cancer types, including tumors of the breast, colon, and hematopoietic systems. SFK activity typically increases as tumorigenesis progresses and is associated with metastatic behavior. The major inhibitor of SFK activity is C-terminal Src kinase (Csk) and its paralog Chk; these may act as tumor suppressors in, e.g., breast cancer, presumably through their ability to inhibit Src activity and perhaps other pathways. Drosophila Csk acts primarily or exclusively through Src pathway regulation, and the reduction of dCsk activity by itself led to increased organ size, organismal lethality, and increased cell proliferation due to a failure to exit the cell cycle. However, neither Csk loss nor Src activation has been clearly linked to early events in tumorigenesis, bringing into question the role of Csk/Src in proliferation in vivo. Instead, Src is currently thought to be a major player in the metastatic events that occur later in oncogenesis. How Csk or Src promotes the metastatic behavior of cells in situ remains largely unknown (Vidal, 2006).

This study analyzed the phenotypes of dCsk in the context of developing epithelia. The outcome of a cell's loss of dCsk is linked to its cellular microenvironment. When dCsk activity is reduced broadly in the developing eye or wing, the result is overproliferation, inhibition of apoptosis, and decreased cell adhesion. Tissue integrity is retained, but dCsk cells become inappropriately mobile and fail to maintain their appropriate contacts. The outcome of these effects is an overgrown and mispatterned adult tissue. By contrast, loss of dCsk in discrete patches results in epithelial exclusion, invasive migration through the basal extracellular matrix, and eventual apoptotic death; these events occur exclusively at the boundary between dCsk and wild-type cells. Further emphasizing the unique nature of cells at this boundary, a specific requirement was found for a signal that includes Drosophila orthologs of E-cadherin, P120-catenin, RhoA, JNK, and the metalloprotease MMP2. Hence, this study explores the mechanisms by which the cellular microenvironment can direct different behaviors of cells, both in the regulation of apoptosis and epithelial integrity. It also uncovers a mechanism for the removal of abnormal cells from a normal epithelium (Vidal, 2006).

This study shows that reducing dCskactivity results in a blockade of apoptosis and downregulation of cellular adhesion. The work is consistent with the view that Csk is a tumor suppressor that acts at multiple steps. Mutations in the locus encoding the Csk paralog Chk have been described in breast tumors, and, in this study, it has been observed that human Chk can functionally replace dCsk. Therefore the experimental advantages of developing Drosophila imaginal epithelia were used to explore specific aspects of dCsk function that are relevant to the behavior of tumor cells (Vidal, 2006).

Visualization studies suggest that a reduction in dCsk activity leads to a failure of cells to stably retain associations with their neighbors, resulting in prolonged cell movement as cells slide across each other in a manner not observed in wild-type tissue. This may reflect a failure to establish stable junctions, excess cell motility, or both. Recent work has demonstrated a critical and dynamic role for the cadherin-based apical junctions in patterning the Drosophila retina. Misexpressing dE-cadherin prevents patterning defects in GMR>dCsk-IR retinas, suggesting that dCsk cells have reduced dE-cadherin function. Links between Csk, Src, cadherins, and junctional integrity have been reported in mammalian cell culture, and an association has been observed between Drosophila Src42A and dE-cadherin during embryonic development (Takahashi, 2005). The data are consistent with this view: misexpression of a kinase-dead form of Src42A leads to a disruption in the localization of the dE-cadherin-associated protein Armadillo; also, reduced Armadillo levels observed in dCsk retinas is rescued by dE-cadherin misexpression. Together, these data suggest that altering dCsk/Src activity affects cell movements by decreasing dE-cadherin adhesion (Vidal, 2006).

The mechanism by which dCsk alters dE-cadherin function is not clear, but it is relevant to note that Src activation can shift cadherin-based cell adhesion from a 'strong' to a 'weak' adhesive state in mammalian cultured cells. Phosphorylation of cadherins and catenins may mediate 'inside-out' signaling that can alter the adhesive strength of the homophilic bond between cells (reviewed in Gumbiner, 2005). Evidence for such a mechanism has been provided for integrin-mediated focal adhesions (reviewed in Hynes, 2002), and Src activity can alter focal adhesions (Yeatman, 2004). However, normal basal membrane architecture was observed in dCsk cells, as assessed both by anti-integrin staining and by transmission electron microscopy, indicating that at least the gross structure is not affected (Vidal, 2006).

The ability of dCsk to influence cell proliferation, apoptosis, and cell adhesion is consistent with its ability to direct tissue overgrowth: reducing dCsk activity throughout a tissue (or the entire organism) leads to significantly enlarged tissues. This ability demonstrates that dCsk can participate in the mechanisms that set tissue size. A small number of other proteins have been implicated in this process, including Salvador, Hippo, and Lats/Warts, which show phenotypes that are strikingly similar to dCsk. Furthermore, dCsk can directly phosphorylate Lats/Warts (Stewart, 2003) in vitro (Vidal, 2006).

However, reduction of dCsk activity shows some important differences. Mutations in salvador, hippo, or lats/warts lead to an increase in Diap1 levels, which, in turn, blocks apoptotic cell death. By contrast, reductions in dCsk does not significantly alter Diap1 protein levels. Furthermore, although both Hippo and dCsk are required to exit the cell cycle, the cell cycle profile from hippo mutant cells is normal, while dCsk cells contain a significant shift toward G2/M (Read, 2004; Stewart, 2003). Perhaps the most striking difference is the effects of these factors on discrete mutant patches. While broad loss of dCsk activity leads to expanded tissues, surprisingly discrete patches of dCsk tissue are eliminated by neighboring cells. Unlike salvador, hippo, or lats/warts, clonal patches of dCsk cells fail to survive to adulthood. The effects of dCsk reduction are more similar to those reported for the tumor suppressor gene scribble. The scribble locus encodes a component of the septate junction that regulates cell polarity and proliferation; mutant cells display neoplastic overgrowth in a homotypic environment, but are removed by JNK-dependent apoptosis in discrete clonal patches abutting wild-type tissue (Vidal, 2006).

This work provides evidence that neighboring wild-type tissue provides a locally nonautonomous signal that leads to the removal of dCsk mutant cells. For example, FRT-derived clones of dCsk cells were out-competed by neighbors with normal levels of dCsk: this was most easily seen by the clonally related 'twin spot' of wild-type tissue that was consistently larger than the few surviving dCsk clones. In contrast, FRT-mediated dCsk clones that encompassed the entire eye survived and overproliferated. In the developing wing, cells at the periphery of sd>dCsk-IR or ptc>dCsk-IR expression domains were preferentially removed by apoptosis. This death is dependent not on absolute dCsk activity, but on the juxtaposition of cells that are starkly different in their levels of dCsk. Small differences, for example across the ptc>dCsk-IR or omb>dCsk-IR graded expression domains, did not trigger cell death (Vidal, 2006).

This translocation and death of dCsk-IR cells at the patched/wild-type boundary requires at least two steps. At boundaries with wild-type tissue, dCsk cells initially lose their apical profile, shift downward, and eventually become basally excluded from the epithelium. Such excluded cells then migrate away from the boundaries in both directions and eventually die by apoptosis. These events are strikingly reminiscent of those described for tumor cells undergoing metastasis. Altered activity of both Csk and Src has been implicated in a broad variety of tumors. Typically, however, increased Src activity is associated with later events in tumorigenesis, particularly metastasis. Although the connections between high Src activity and metastases are not understood, they likely include Src's ability to break cell-cell junctions and increase cell motility. Another hallmark of metastatic behavior is the ability to degrade basal extracellular matrix: this study also demonstrate a functional requirement for MMP2 activity during the translocation of mutant cells out of the wing epithelium (Vidal, 2006).

While evidence supports the view that the activity of Csk -- and presumably Src and perhaps other effectors -- can regulate metastatic behavior, it alone is not sufficient. First, reducing dCsk activity by itself is not sufficient to allow migrating cells to survive; the data suggest that most or all eventually die. This is consistent with previous work highlighting the importance of a 'two-hit' model to allow for stable tumor overgrowth and metastasis. A second mutation that prevents apoptotic cell death would be minimally required. Second, all cells within a discrete dCsk patch are not equivalent: cells at the boundary of the clone that border cells of strongly differing dCsk levels are exclusively prone to release from the epithelium. This work predicts that cells at the borders of some human tumors are especially prone toward metastatic behavior. Metastasis is often the most serious aspect of a tumor, and approaches that address the metastatic behavior of cells may need to take into account the properties of cells at the periphery. Understanding whether and how these cells are unique may help to more effectively target therapeutic intervention (Vidal, 2006).

In addition to enabling a detailed examination of dCsk cells and their behavior within an epithelium, this model system permitted identification of signaling components that are necessary to execute the aberrant cell mobility and cell death. The results indicate important roles for dE-cadherin, dP120ctn, Rho1, dJnk, and MMP2 (Vidal, 2006).

JNK-dependent apoptosis is required for a broad palette of related mechanisms such as cell competition in developing tissues and the removal of scribble mutant cells. JNK signaling is also associated with the movement of cells within epithelia, including dorsal closure in Drosophila and in mammals. Interestingly, JNK activity is required for the synthesis of MMP2 by v-Src-transformed mammalian cells (Vidal, 2006).

JNK activity can be triggered by several upstream signaling factors, including the small GTPases of the Rho family, and genetic data provide a link between dCsk, dJnk, and Rho1. Rho family proteins are key regulators of cell shape and motility. They also promote the cytoskeletal rearrangements required for epithelial-to-mesenchymal transitions (EMTs), and it is noted that dCsk boundary cells show a number of features that are reminiscent of EMTs. In Drosophila, Rho1 was found to induce an 'invasive' phenotype in wing disc cells, but, in this study, it was demonstrated that, similar to dCsk boundary cells, ptc>Rho1 misexpressing cells also undergo apoptotic death. Most importantly, halving the genetic dose of Rho1 strongly suppresses discrete loss of dCsk, but does not appreciably affect broad loss. Thus, Rho1 activity is linked to dCsk, and activation of Rho1 is sufficient to phenocopy both the apoptotic and migratory phenotypes of dCsk cells located near wild-type tissue (Vidal, 2006).

Previous work in mammalian cell culture has provided direct links between Src and P120-catenin, between cadherins and P120-catenin, and between RhoA and P120-catenin; the latter two interactions have been reported in Drosophila tissue culture systems as well. This study further supports links between these factors in dCsk boundary cells. Interestingly, although normal levels of both dP120ctn and Rho1 were required for the efficient removal of dCsk boundary cells, they were not required for the phenotypes resulting from broad loss of dCsk. The requirement for p120ctn specifically in boundary cells may explain why, although it is the only ortholog present in Drosophila, dP120ctn (Drosophila p120-catenin) is not required for organism viability (Vidal, 2006).

Both Src and P120-catenins are known to directly interact with cadherins, and, in fact, a role was demonstrated for dE-cadherin/Shotgun in the removal of dCsk cells. A model is postulated in which the loss of dCsk results in the remodeling of the zonula adherens, presumably by the phosphorylation of catenins and dE-cadherin itself by Src. Src activation is known to switch cadherin from a strong adhesive state to a weak one, providing one potential explanation for why dCsk retinal cells displayed reduced cell adhesion in situ. One critical question regarding cadherins is whether they have signaling roles that are independent of their adhesive properties. Perhaps relevant to this point, it was surprising to find that reducing dE-cadherin function leads to a suppression of the effects of dCsk-IR at the boundary. A simple dCsk-IR-mediated reduction in dE-cadherin adhesion would be enhanced by further reducing dE-cadherin activity, suggesting that dE-cadherin may provide an active signal that promotes boundary cells' release from the epithelium. If such a signal does exist, neighboring wild-type cells must trigger it, either through their own endogenous dE-cadherin or through a separate, local signal. Why are multiple (3-4) rows affected? The results are consistent with the creation of a successive new boundary as the previous row of cells descends, although other longer-range signals cannot be ruled out (Vidal, 2006).

It is noted that reducing dCsk activity by itself is not sufficient to direct stable tumor overgrowth, supporting the importance of a 'two-hit' model in Drosophila. Loss of the junction protein Scribble showed similar phenotypes to dCsk, including apoptosis, but was found to confer survival and metastatic-like behavior to cells in the presence of an activated Ras isoform. Interestingly, coexpression of dE-cadherin prevents this metastatic behavior (Vidal, 2006).

Finally, how can dP120ctn and Rho1 promote release of dCsk near wild-type boundaries but not act similarly with other dCsk cells? One source of information is the cadherins themselves: the boundary creates an interface of cadherins that have been exposed to different levels of Csk and, presumably, Src activity. This unusual interface may generate the needed dE-cadherin signal. Importantly, recent work has noted a change in the subcellular localization of P120-catenin and E-cadherin specifically at the border of human tumor tissues. At the time that ptc>dCsk-IR boundary cells lose their apical profiles, this study found that dP120ctn is relocalized to the cytoplasm. These results again emphasize the possibility that cells at tumor boundaries pose a special risk of undergoing epithelial-to-mesenchymal-like transitions and metastatic behavior. Metastasis is often the most serious complication of progressing tumors. Targeting therapies to this aspect of cancer may benefit from considering boundary cells and their potentially distinctive properties (Vidal, 2006).

Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary via E-cadherin trafficking

All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. This study shows that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (ß-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs, through which E-cadherin transits, are tightly associated with the fusome. It is proposed that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology (Bogard, 2007).

The first clue that Rab11 plays important roles in early oogenesis in Drosophila came from immunostaining experiments that revealed strong expression of endogenous Rab11 and a fully functional Rab11::GFP in GSCs, cystoblasts and young (2-4- and 8-cell) germline cysts. Strikingly, the proteins were concentrated as discrete dots on the fusome, which electron microscopy and photobleaching studies have shown is highly vesicular and rapidly exchanged with other membrane stores. Triple-stain experiments showed that some of these dots also contained E-cadherin, which has been shown to transit though Rab11-positive recycling endosomes (REs) en route to the plasma membrane in some cells. High-magnification images showed that the Rab11 (and, more rarely, E-cadherin) dots were often nestled into cavities within the fusome. Such Rab11-harboring cavities are visible in the fusomes of all examined GSCs, cystoblasts and young germline cysts, not only in the ovary but also in the testes. In view of the well-described enrichment of Rab11 in REs, it is proposed that these Rab11- and E-cadherin-harboring cavities are REs and are therefore referred to as FREs (fusome-associated REs) (Bogard, 2007).

These studies indicate that Rab11 maintains GSC identity through polarized trafficking of E-cadherin and, possibly, other cargoes that reinforce essential GSC-niche contacts. These studies further indicate that Rab11 is required for fusome localization and asymmetric GSC division and suggest a feedback linkage between these events and E-cadherin trafficking. Although Rab11 has been implicated in the trafficking of E-cadherin in other cells, there are no other cases in which such trafficking has been correlated with a biological response. It will be of interest to determine whether Rab11 is required for the maintenance of stem cells in other systems and whether such maintenance involves E-cadherin trafficking or the trafficking of other adhesion molecules. It will also be of interest to determine the role of Rab11 in other E-cadherin-dependent cell behaviors, particularly as Rab11, at least in Drosophila, is expressed in only a small subset of E-cadherin-expressing cells (Bogard, 2007).

Tre1 GPCR initiates germ cell transepithelial migration by regulating Drosophila melanogaster E-cadherin

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. 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. 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. 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. 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. 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. Cta protein is present in the germ cells and maternal loss of cta causes a gastrulation defect similar to Gβ13f and Gγ1. 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. 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. 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).

Regulation of post-embryonic neuroblasts by Drosophila Grainyhead: shotgun is a dowstream target of Grainyhead

The Drosophila post-embryonic neuroblasts (pNBs) are neural stem cells that persist in the larval nervous system where they proliferate to produce neurons for the adult CNS. These pNBs provide a good model to investigate mechanisms regulating the maintenance and proliferation of stem cells. The transcription factor Grainyhead (Grh), which is required for morphogenesis of epidermal and tracheal cells, is also expressed in all pNBs. This study shows that grh is essential for pNBs to adopt the stem cell program appropriate to their position within the CNS. In grh mutants the abdominal pNBs produced more progeny while the thoracic pNBs, in contrast, divided less and produced fewer progeny than wild type. Three candidates were investigated to determine whether they could mediate these effects; the neuroblast identify gene Castor, the signalling molecule Notch and the adhesion protein E-Cadherin. Neither Castor nor Notch fulfills the criteria for intermediaries, and in particular Notch activity is dispensable for the normal proliferation and survival of the pNBs. In contrast E-Cadherin, which has been shown to regulate pNB proliferation, is present at greatly reduced levels in the grh mutant pNBs. Furthermore, ectopic expression of Grh is sufficient to promote ectopic E-Cadherin and two conserved Grh-binding sites were identified in the E-Cadherin/shotgun flanking sequences, arguing that this gene is a downstream target. Thus one way Grh could regulate pNBs is through expression of E-cadherin, a protein that is thought to mediate interactions with the glial niche (Almeida, 2005).

Previous studies have shown that E-Cadherin is necessary for normal pNB proliferation. These studies show that expression of a dominant negative E-Cadherin in the neural and glial cells reduces the number of progeny produced by pNBs to <25% of wild type. Expression in the ensheathing glia alone led to more minor reduction, arguing that the protein is needed in both glia and pNBs. As reported previously, strong expression of E-Cadherin was detected in the pNBs and their adjacent progeny. In grh³⁷⁰ however, the levels of E-Cadherin in the thoracic region of the CNS were dramatically reduced. Several pNBs lack significant E-Cadherin expression all together, others retained some expression but at much lower levels compared to wild type. Similar effects were seen in clones mutant for another loss of function grh allele, grh^B32. In lineages homozygous for grh^B32 there was a variable reduction in E-Cadherin compared to neighbouring wild-type lineages (Almeida, 2005).

The effects on Cadherin contrast with those on Notch, where expression in the thoracic pNBs remains robust in grh³⁷⁰, arguing against an indirect effect resulting from changes in size. To further test this, it was asked whether Grh is sufficient to promote E-Cadherin expression when expressed elsewhere in the CNS. A pros::Gal4 driver line was used that directs high levels of expression in neurons and lower expression in the pNB lineages. When this was used to drive expression of the CNS isoform of grh, high levels of ectopic E-Cadherin were detected, particularly in many of the embryo-derived neurons that are normally devoid of E-Cadherin expression at these stages. Neither Castor nor Notch expression was altered under these conditions. Therefore Grh appears to be an activator of E-Cadherin expression. However, ectopic Grh was not sufficient to direct additional proliferation under the conditions tested (Almeida, 2005).

The genomic sequence flanking the E-Cadherin gene (shotgun, shg) was examined for consensus Grh binding sites using two different strategies. Grh binds as a dimer. In recent studies of Grh family proteins a consensus target-site was derived (WCHGGTT). Eight matches to this consensus are present in the genomic region spanning from 1 kb upstream of the shg transcript (another gene, CG10540, starts 944 bp upstream of shg) to 5 kb downstream. A second search using a weighted matrix revealed 13 matches within 5 kb of shg. A comparison of the two sets of putative sites identified four common matches: AAACAGGTTA (−300); AAACAGGTAA (+275); ATACTGGTTT (2650 bp downstream, Shg2); CAACAGGTAG (3131 bp downstream, Shg1). The latter two are 100% conserved between D. melanogaster and the five other Drosophila species for which sequence is available. To confirm that these two sites are recognised by Grh, a stringent assay was used where their ability to compete with a well-characterised, high affinity site, Gbe2 from the Dopa decarboxylase gene, was tested. Both sites were able to compete, reducing the amount of probe bound by 51% (Shg1) and 75% (Shg2) when present at 40× molar excess. The presence of these conserved sites indicates therefore that shg/E-Cadherin is likely to be a direct target of Grh. However, E-Cadherin cannot be the only target, since it was not possible to rescue the grh³⁷⁰ mutant phenotype by supplying E-Cadherin via an exogenous driver (GrhNB::Gal4/UAS::E-Cadherin) (Almeida, 2005).

Although the data show that Cadherin is regulated by Grh, they do not resolve unequivocally whether it is a direct target. Examination of genomic sequence revealed two binding-sites close to the shotgun/E-Cadherin gene that are conserved in other Drosophila species and that are bound by Grh in vitro. Future studies will show whether these sites are essential for shg expression. However, these results are exciting because they provide a link between grh function in the pNBs and in other tissues. Changes were observed in E-Cadherin levels in response to grh in other parts of the animal. There is also an interaction between shg and several genes that act together with grh in epidermal morphogenesis (although no direct genetic interaction was seen between shg and grh itself). Given that the precise levels of E-Cadherin proteins can be critical in shaping the sorting and interactions between cells it will be important to determine whether Grh is required for this regulation. It will also be important to establish whether Cadherins are targets of Grh in other animals, for example in mice where mutations in Grhl3 result in defects in neural tube closure and epidermal integrity (Almeida, 2005).

The defects in the pNB lineages of grh mutants are position dependant. Thus, the thoracic pNBs produce fewer progeny whereas abdominal pNBs proliferate for a more prolonged period. In general, such A/P position dependent patterning is co-ordinated by the homeotic genes and indeed abdA has been shown to regulate the timing of cell death and hence the period of proliferation in the abdominal pNBs, as well as regulating the number of pNBs that persist in abdominal segments. However the phenotype of abdA mutants is significantly different from that of grh; for example the abdominal clusters are much larger and there are no defects in thoracic clusters, so it is unlikely that grh is upstream of abdA. Furthermore, in parallel studies Cenci (2005) has shown that the initiation of AbdA expression still occurs in grh mutants. Therefore it is more likely that grh acts in parallel to the homeotic genes to co-ordinate the pNB program (Almeida, 2005).

Regulation of Shotgun expression: Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development in Drosophila

Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study shows that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless, a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing (Jaiswal, 2006).

Cells of the dorsoventral (DV) boundary in the wing imaginal disc synthesize Wg. The DV boundary marks the distal end of the growing appendage, while the future hinge region, displaying Wg expression in two concentric rings, marks the proximal wing. The lacZ reporter of the quadrant enhancer of vestigial (vg), Q-vg-lacZ marks the entire distal wing [i.e. the presumptive wing blade (pouch) (Jaiswal, 2006).

In optical sections of the imaginal disc epithelium, AJs are visualized in the XY or XZ planes based on immunolocalization of DE-Cad and ß-catenin/Arm, besides binding with fluorochrome conjugated Phalloidin to F-actin. Both ß-catenin/Arm and DE-Cad display characteristic upregulation across the DV boundary along the PD axis of the wing imaginal disc. Optical sections along the XY plane reveal higher levels of DE-Cad localization and narrower apical circumferences in the AJs of cells flanking the DV boundary when compared with those of the more proximally located cells. Optical sections along the XZ plane further confirmed upregulation of DE-Cad. Thus, along the PD axis of the wing disc, cell shapes and DE-Cad levels are graded (Jaiswal, 2006).

Whether the PD gradient of cell shape and DE-Cad levels are linked to Wg signaling was tested. Somatic clones displaying constitutive Wg signaling (induced by overexpression of Dsh or of a degradation resistant variant of ß-catenin/Arm, Arm^S10) induce cell-autonomous upregulation in the levels of DE-Cad and apical cell constrictions. Somatic clones expressing secreted Wg, however, are expected to induce non-cell-autonomous effects. Indeed, these clones induced non-cell autonomous and graded upregulation in the levels of DE-Cad in the AJs and changes in apical cell shapes. In the presumptive hinge region, Wg overexpression produces a more striking pattern of non-cell autonomous changes in cell shapes: cells neighboring the Wg-expressing cells appear to organize as whorls around the former and display epithelial misfolding (Jaiswal, 2006).

Furthermore, expression of GPI-anchored DFz2 receptor GPI-DFz2, which compromises Wg signaling, obliterates the characteristic PD gradient in the levels of DE-Cad and F-actin in the AJs. Finally, loss of Wg expression in the DV boundary of wing imaginal disc of N^ts mutants grown at a restricted temperature also abolishes the PD gradient of DE-Cad and apical cell shapes. To further test if apical cell constrictions are linked to elevated levels of DE-Cad in AJs, DE-Cad was expressed in somatic clones. These clones were apically constricted, consistent with the role of DE-Cad/E-Cad in remodeling cell shape and tissue architecture. These results thus link Wg signaling to the PD gradient in the levels of DE-Cad and apical cell shapes in the wing imaginal discs (Jaiswal, 2006).

Somatic clones with altered cell-cell adhesion sort out from their neighbors and display smooth clone borders. Indeed, somatic clones displaying gain of Wg signaling owing to Dsh or Arm^S10 misexpression sort out from their neighbors and display smooth clone borders, akin to those misexpressing DE-Cad. Wg signaling may alter cell-cell adhesion by enhancing recruitment of ß-catenin/Arm to the AJs and/or by its transcriptional input. In many cell types, for example, expression of cadherins rather than the levels of catenins appears to be the rate-limiting step of Catenin-Cadherin complex formation at AJs and cell-cell adhesion. Wild type ß-catenin/Arm (Arm^S2), when overexpressed, does not transduce Wg signaling. Somatic clones overexpressing Arm^S2 display 'wiggly' clone borders, unlike those expressing Dsh or Arm^S10. Thus, expression of ß-catenin/Arm alone, without a concomitant enhancement of Wg signaling, fails to alter cell-cell adhesion. Cell-cell adhesion in wing imaginal disc epithelium is therefore likely to be regulated by transcriptional input from Wg signaling (Jaiswal, 2006).

To test if canonical Wg signaling regulates DE-Cad expression, the response of its lacZ reporter, DE-Cad-lacZ, was examined. Cells receiving high threshold of Wg signaling in the wing imaginal discs, as in those flanking the DV boundary, displayed higher levels of DE-Cad-lacZ reporter activity when compared with those further away from the source of Wg expression. Furthermore, somatic clones expressing Arm^S10 or Dsh display cell-autonomous activation of the DE-Cad-lacZ. Finally, clones expressing the secreted Wg induce non-cell-autonomous activation of DE-Cad-lacZ: i.e., in cells within and surrounding the clones. Together, these results suggest that regulation of DE-Cad by the long-range activity of the Wg morphogen sets up the PD gradient of cell-cell adhesion and cell shape in the distal wing (Jaiswal, 2006).

Somatic clones lacking Ft (ft^-/ft^-), marked by loss of GFP, display overgrowth and altered cell-cell adhesion with characteristic circular and smooth clone borders, unlike the 'wiggly' borders of their wild type (ft⁺/ft⁺) twins that are marked by brighter GFP. Furthermore, cells lacking Ft displayed upregulation of DE-Cad in their AJs and DE-Cad-lacZ. By contrast, when Ft was overexpressed, levels of both DE-Cad or DE-Cad-lacZ were downregulated. Besides, following overexpression of Ft in the posterior wing compartment, cells flanking the DV boundary displayed wider apical circumferences when compared with those of the anterior wing compartment. These results suggest that Ft regulates DE-Cad expression, cell-cell adhesion and apical cell shapes in the distal wing (Jaiswal, 2006).

The results suggest that by regulating DE-Cad expression, Wg signaling integrates cell-cell adhesion with tissue growth and pattern. Regulation of DE-Cad expression could be a prevalent mechanism for coordination of the emerging pattern in an organ primordium with the spatial control of its cell-cell adhesion. For example, DE-Cad levels are also upregulated in cells flanking the stripe of cells along the AP boundary that express the morphogen Decapentaplegic (Dpp); misregulation of Dpp signaling also affects DE-Cad expression. The Ft tumor suppressor, by contrast, negatively regulates DE-Cad expression in the distal wing. This may also explain the inverse correlation between the levels of DE-Cad in AJs and the activity of Ft. Thus, besides its heterophilic binding with Ds, Ft controls cell-cell adhesions at AJs by regulating DE-Cad expression (Jaiswal, 2006).

Apart from cell-cell adhesion, DE/E-Cad regulation may impact a variety of other cellular processes and developmental mechanisms. E-Cad has been shown to mark the sites of actin assembly on cell surface. Cadherin complexes regulate cytoskeletal networks and cell polarity, while disruption of AJ associated components affects asymmetric cell division. Fat1, a mammalian homolog of Drosophila Ft, modulates actin dynamics. Interestingly, Ft also regulates orientated cell division (OCD) in imaginal epithelium, which is mirrored by orientation of the spindles of the dividing cells; OCD may also regulate organ shape along the PD axis. Misregulation of DE-Cad may thus affect the cytoskeleton and produce OCD phenotype in ft mutant discs (Jaiswal, 2006).

In both loss- and gain-of-function assays, this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained to be the direct targets of Wg, all available evidence so far suggests that these targets positively respond to Wg signaling. These results also show that Ft and Wg signaling intersect and control distal wing growth and pattern, presumably through their opposing regulation of a common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling, Dpp signaling also regulates Q-vg-lacZ; however, its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by its intersection with Wg signaling (Jaiswal, 2006).

The results show that Ft negatively regulates Wg signaling. Loss or gain of Ft induces a telltale sign of perturbations in Wg signaling, namely, changes in the cellular pool of ß-catenin/Arm, consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP) Wnt signaling pathways (Jaiswal, 2006).

One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).

Drosophila wing growth is under dynamic spatial and temporal regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact cell proliferation in their characteristic ways and activate distinct sets of PD markers. Although at a very high threshold, Wg signaling inhibits cell proliferation, at a modest threshold it has been shown to stimulate growth. It is noted that loss of Ft fails to activate Wg targets that demand a high threshold of Wg signaling, e.g., Ac, which is required for wing margin specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ, which responds to a high threshold of Wg signaling, is also not upregulated by loss of Ft. Dll responds to a higher threshold of Wg signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).

Over-proliferation in ft mutant imaginal discs is induced by perturbation of as yet unidentified disc-intrinsic mechanisms that determine the discs' characteristic final sizes. The imaginal discs of ft mutants continue to grow and the extent of their over-proliferation appears to be constrained only by the developmental time available during the extended periods of their larval life. By contrast, growth in wild-type imaginal discs is determinate, which ceases after they attain their predetermined sizes even under conditions of unlimited developmental time; for example, on transplantation into wild-type adult host abdomen that can sustain development. ft mutant imaginal discs thus acquire unlimited proliferative potential, akin to immortalization, a crucial step during tumorigenesis. It is significant that the Ft tumor suppressor downregulates Wg/Wnt signaling, a pathway implicated in cancers. Several orthologs of Ft have been identified in vertebrates with diverse functions. It will thus be interesting to explore if these orthologs of Ft in higher vertebrates also interact with Wnt signaling and thereby behave as tumor suppressors (Jaiswal, 2006).

Zinc transport activity of Fear of Intimacy is essential for proper gonad morphogenesis and DE-cadherin expression

Embryonic gonad formation involves intimate contact between germ cells and specialized somatic cells along with the complex morphogenetic movements necessary to create proper gonad architecture. Gonad formation in Drosophila requires the homophilic cell-adhesion molecule Drosophila E-cadherin (DE-cadherin), and also Fear of Intimacy (FOI), which is required for stable accumulation of DE-cadherin protein in the gonad. In vivo structure-function analysis is presented of FOI that strongly indicates that zinc transport activity of FOI is essential for gonad development. Mutant forms of FOI that are defective for zinc transport also fail to rescue morphogenesis and DE-cadherin expression in the gonad. Expression of DE-cadherin in the gonad is regulated post-transcriptionally and foi affects this post-transcriptional control. Expression of DE-cadherin from a ubiquitous (tubulin) promoter still results in gonad-specific accumulation of DE-cadherin, which is strongly reduced in foi mutants. This work indicates that zinc is a crucial regulator of developmental processes and can affect DE-cadherin expression on multiple levels (Mathews, 2006).

It has been unclear whether ZIP family members regulate developmental processes by acting as zinc transporters or through some other unidentified function. These data now indicate that FOI regulates gonad formation through its zinc transporter activity. The ability of the mutant forms of FOI to rescue gonad morphogenesis and DE-cadherin expression corresponds directly with their ability to function as zinc transporters. Mutations that strongly affect the zinc transport activity of FOI (e.g., H554A) also strongly reduce the ability of FOI to rescue gonad morphogenesis and DE-cadherin expression. Mutations that only partially affect the zinc transport activity of FOI (e.g., Y646A) retain some ability to rescue gonad morphogenesis and DE-cadherin expression. If FOI affects gonad formation through a function separate from zinc transport, identification of conserved residues that affect these two activities independently would have been expected. This was not the case. Indeed, even single amino acid changes in very different regions of FOI (e.g., D308A and H554A) affect both zinc transport and gonad morphogenesis. It is concluded that the zinc transporter function of FOI is essential for gonad morphogenesis and regulation of DE-cadherin. This reveals a crucial role for zinc regulation in development and suggests that other ZIP family members with developmental roles (e.g., zebrafish LIV1) may also act via zinc transport (Mathews, 2006).

In vivo analysis is also informative for revealing domains that are essential for FOI function. Even though the N-terminal extracellular domain of FOI shows little sequence conservation with other family members, and some ZIP family members lack an extended N-terminal domain, this domain is nevertheless important for FOI function. In addition to their TM character, the specific sequence of the TM domains is crucial for FOI function. Mutations that are not predicted to affect the TM structure of FOI, such as mutating a single acidic residue in TM2 (D308A) or replacing TM6-8 of FOI with similar TM domains from the related protein CATSUP (CAT TM6-8), still disrupt the in vivo rescue activity of FOI. Finally, several characteristics were analyzed of the highly conserved HELP domain in FOI (which may or may not have TM structure). The predicted amphipathic alpha-helical nature of this domain appears to be crucial, since altering the pattern of acidic residues (D551A/D558A and E584A/E588A/D591A) or inserting a helix contorting proline residue (T557P) disrupts FOI function. In addition, conserved histidines in this domain are essential (H554A and H583A/H587A), and mutating even a single histidine has a dramatic effect in vivo. Since FOI is a zinc transporter, it is likely that the specific sequences of the TM domains form the proper membrane pore for zinc, while histidines in the N-terminal and HELP domains act to coordinate zinc before and during transport (Mathews, 2006).

shg and foi are both required for proper gonad and tracheal morphogenesis, and foi regulates DE-cadherin expression in the gonad. DE-cadherin protein levels are not reduced in foi mutants simply because the gonad has failed to coalesce; other mutations blocking gonad coalescence do not affect DE-cadherin. Thus, it is likely that foi affects DE-cadherin more directly and this is an important aspect of how foi functions in gonad and tracheal development. In support of this, it was found that expression of DE-cadherin was sufficient to partially rescue foi mutant gonads (Mathews, 2006).

As both DE-cadherin protein and shg RNA levels are reduced in foi mutant gonads, whether foi affects DE-cadherin transcription was investigated. Analysis of a shg enhancer-trap suggests that some aspects of DE-cadherin regulation by foi may be at the transcriptional level. Recently, it has been shown that a related ZIP protein, zebrafish LIV1, can regulate the activity of the Zn-finger transcription factor SNAIL, which may also influence E-cadherin expression (Yamashita, 2004; Mathews, 2006).

However, although a majority of studies focus on transcriptional regulation of E-cadherin, it is likely that this essential cell-adhesion molecule is often regulated at many levels, including through post-transcriptional and post-translational mechanisms. This study presents clear evidence that DE-cadherin is regulated at the post-transcriptional level in the embryonic gonad. Expression of DE-cadherin from a general tubulin promoter (tub-DE-cad) is sufficient to restore gonad-specific DE-cadherin protein accumulation in shg mutants. Recent work suggests that DE-cadherin localization within the ovary is also regulated partly through a post-transcriptional mechanism. Thus, post-transcriptional regulation may be sufficient to generate tissue-specific patterns of DE-cadherin expression in many contexts. tub-DE-cad is much less able to restore DE-cadherin protein to the gonad in foi mutants. This indicates that FOI is required for positive, post-transcriptional regulation of DE-cadherin. One component of this regulation is likely to act on shg RNA stability, since foi affects the gonad-specific accumulation of shg RNA from tub-DE-cad, but does not affect the activity of the tubulin promoter. Thus, the steady-state pattern of shg RNA accumulation does not merely reflect shg promoter activity but may have a significant post-transcriptional component. In principle, zinc could regulate the activity of RNA-binding proteins that affect RNA stability in the same way it regulates DNA-binding transcription factors. In addition, DE-cadherin may be further regulated at the protein level in the gonad, such as through regulation of translation or protein stability (Mathews, 2006).

Recent in vivo work on several ZIP proteins suggests that these zinc transporters play essential roles in development and disease that may broadly involve regulation of cell-cell adhesion. In zebrafish, regulation of SNAIL by LIV1 is essential for the anterior migration of zebrafish organizer cells and may regulate E-cadherin expression in this tissue (Yamashita, 2004). According to this model, LIV1 activates SNAIL activity, which leads to downregulation of E-cadherin and the decreased cell adhesion necessary for cell migration (Yamashita, 2004). Interestingly, SNAIL is also thought to be an important regulator of E-cadherin during the progression and metastasis of certain cancers, such as breast cancer. As a tumor gains metastatic potential, SNAIL expression is upregulated and E-cadherin is downregulated. Since human LIV-1 is strongly expressed in breast cancer cell lines, and has been implicated in breast cancer metastasis, it may function to activate the activity of SNAIL as a transcriptional repressor of E-cadherin, again allowing for cell migration and metastasis. A similar, but opposite, relationship may exist in the Drosophila tracheal system, where the SNAIL family member Escargot (ESG) is a positive regulator of E-cadherin during the fusion of neighboring tracheal branches. Since FOI is also required for this process, FOI may act by promoting ESG activity. In this case, FOI and ESG would activate DE-cadherin expression, which is necessary for cell-cell attachment during tracheal branch fusion. In the gonad, FOI is also positively required for DE-cadherin expression. Although ESG is present in the gonad, no changes have been observed in DE-cadherin expression during gonad coalescence in esg mutants, indicating that some other target for regulation by FOI and zinc must exist in this tissue. An important theme in the action of ZIP proteins may be to influence the activity of zinc-regulated transcription factors, with cell-cell adhesion molecules being important targets of such regulation. However, it was found that additional, post-transcriptional mechanisms are crucial in the gonad for regulation of DE-cadherin protein expression by FOI. Thus, it will be very important to analyze the contribution of post-transcriptional regulation of E-cadherin to other developmental and disease processes. Indeed, there is even evidence that the same crucial factor, SNAIL, can influence post-transcriptional regulation (Mathews, 2006).

An important issue relevant to the role of zinc and zinc transporters in development and disease is whether they play an instructive or permissive role. Is zinc merely required at a minimum threshold level in various tissues or does regulation of intracellular zinc concentration play a signaling role at specific times and places? Existing evidence suggests that zinc may play an instructive role. Both Drosophila foi and zebrafish LIV1 have highly tissue-specific patterns of expression and affect the development of selected tissues, while others remain unaffected. Mammalian ZIP and Cation Diffusion Facilitator family members also have tissue-specific expression patterns. Thus, zinc transporters have the necessary spatial and temporal resolution to play an instructive role. In addition, zinc transporters have clear roles as modulators of intracellular signals. They have the capacity to modulate signaling pathways, for example the ras pathway, and can influence transcription factor activity and gene expression. Because the zinc transport activity of FOI is crucial for its developmental role, it is likely to act by modulating zinc concentration. Thus, zinc has the potential to be an important and dynamically regulated signaling molecule during development and adult homeostasis (Mathews, 2006).

dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion

The Drosophila dysfusion basic-helix-loop-helix-PAS transcription factor gene is expressed in specialized fusion cells that reside at the tips of migrating tracheal branches. dysfusion mutants were isolated, and genetic analysis of live embryos revealed that mutant tracheal branches migrate to close proximity but fail to recognize and adhere to each other. Misexpression of dysfusion throughout the trachea further indicated that dysfusion has the ability to both inhibit cell migration and promote ectopic tracheal fusion. Nineteen genes whose expression either increases or decreases in fusion cells during development were analyzed in dysfusion mutant embryos. dysfusion upregulates the levels of four genes, including the shotgun cell adhesion protein gene and the zona pellucida family transmembrane protein gene, CG13196. Misexpression experiments with CG13196 result in ectopic tracheal fusion events, suggesting that it also encodes a cell adhesion protein. Another target gene of dysfusion is members only, which inhibits protein nuclear export and influences tracheal fusion. dysfusion also indirectly downregulates protein levels of Trachealess, an important regulator of tracheal development. These results indicate that fusion cells undergo dynamic changes in gene expression as they switch from migratory to fusion modes and that dysfusion regulates a discrete, but important, set of these genes (Jiang, 2006; full text of article).

The isolation of dys mutants allowed detailed phenotypic analysis using time-lapse confocal microscopy of live embryos. Migration and the presence of filopodia during DB branching appeared normal in dys mutant embryos. The two DB fusion cells moved close together, and their filopodia were observed to touch. However, unlike wild-type cells, the dys mutant fusion cells failed to stably adhere. Thus, no fusion occurred, and ultimately the branches retracted from one another. Additional insight into the role of dys emerged from experiments in which dys was misexpressed throughout the trachea. Ectopic fusion events were observed, which is consistent with the dys mutant phenotype and indicates that dys promotes fusion cell recognition/adhesion. In addition, dys misexpression results in a strong reduction in both tracheal branching and formation of MAb 2A12 luminal material at fusion sites. The reduction in branching suggests that another possible function of dys is to inhibit migration in preparation for fusion. The reduction in MAb 2A12 luminal material at sites of normal and ectopic fusion suggests that the fusion process is incomplete. This can be explained for the ectopic fusion results by proposing that Dys activates genes that can mediate fusion but not lumen formation. Since the ectopic cells are not fusion cells, the additional lumen-forming functions would not be present. In contrast, since lumen formation was also defective at normal fusion sites, it is possible that Dys overexpression inhibits lumen formation. In summary, the genetic and misexpression experiments suggest that dys is activated late in the branching process to inhibit migration and promote branch recognition and adhesion. It may also play additional roles after branches join (Jiang, 2006).

Since dys encodes a transcription factor, it is expected that it functions by regulating gene expression. Previous work had identified several genes prominently expressed in fusion cells, as well as additional trachea-expressed genes whose fusion cell expression was low or absent. This paper further shows that a number of prominent trachea-expressed genes are also downregulated in fusion cells, indicating that this is a common occurrence. Expression of 19 genes was assayed in dys mutant embryos to identify Dys target genes. RNA levels of four genes (CG13196, CG15252, mbo, and shg) were reduced. In contrast, Trh protein levels, which normally decline in fusion cells, increased in dys mutants. These results were confirmed by dys misexpression experiments, in which CG13196 and CG15252 were increased and Trh protein levels declined. Despite dys expression in all tracheal fusion cells, there exist branch-specific differences in Dys-regulated gene expression. CG13196 is expressed in all fusion cells, and dys is required for its expression. In contrast, shg is upregulated in DB and DT fusion cells, but only DB upregulation requires Dys, an effect also seen for Esg. CG15252 is expressed only in DT fusion cells, and this restriction may be due to the positive or negative action of branch-specific transcription factors, such as Spalt major (DT specific, positively acting) or Kni and Knrl (non-DT branches, negatively acting) (Jiang, 2006). All of the dys misexpression defects (and thus probably the mutant defects) require Dys DNA binding, since deletion of the dys basic region, and presumably its ability to bind DNA, abolishes the tracheal defects. Although trh RNA levels decline in fusion cells along with protein levels, the RNA reduction is not dys dependent. Thus, dys likely regulates transcription of a gene that regulates Trh protein levels. Similarly, the requirement of Dys DNA binding to regulate Trh protein levels does not support a model in which Trh levels are reduced as a consequence of Dys competing for their common dimerization partner, Tgo, since this is unlikely to require DNA binding (Jiang, 2006).

The recognition/adhesive properties promoted by dys may be mediated by two Dys target genes, shg and CG13196. Shg is a well-studied adhesion protein, and CG13196 encodes a ZP transmembrane protein, although its function and subcellular localization are unknown. Misexpression of CG13196 results in ectopic fusion events consistent with it playing a role in cell adhesion. Thus, one key role of dys may be to promote tracheal fusion by controlling expression of two or more cell adhesion protein genes. They could work together in the same cellular process or in different aspects of tracheal fusion, lumen formation, or function. The identification of members only (mbo) as a transcriptional target of Dys is intriguing, since mbo mutants have a tracheal fusion defect and it attenuates protein nuclear export. Although the fusion cell protein cargo regulated by mbo is unknown, it presumably includes proteins that are localized to nuclei in fusion cells (Jiang, 2006).

The two major transcription factors studied to date that control fusion cell transcription and development are esg and dys. esg precedes dys during fusion cell development and controls expression of dys in DBs and GBs but not DTs (the case for LTs is unknown, since fusion cells die in esg mutants. dys itself does not affect esg expression. The tracheal fusion phenotypes of both genes are similar. The DT is the one branch that still fuses in both esg and dys mutants, although both show constrictions at the sites of DT fusion. Previous work on esg revealed that, genetically, it is required for both activation of fusion cell gene expression and repression of terminal cell gene expression in fusion cells. In this study, it was found that dys constitutes a transcriptional pathway that carries out a subset of esg functions, focused on upregulating expression of genes involved in cell adhesion and protein localization, although future work may uncover additional target genes. Since a large number of genes are either activated or downregulated in tracheal fusion cells, it will be important to continue genetic and molecular studies to determine which genes are targets of Esg and Dys and whether their control is direct or indirect (Jiang, 2006).

One model of Drosophila dys function is that dys acts as a developmental timer near the end of tracheal branching to inhibit migration and promote cell adhesion and fusion. The adhesion component works, in part, through activation of shg and (possibly) CG13196. The inhibition of migration has only been postulated from misexpression experiments and needs to be confirmed by alternative approaches. It is also important to note that the switch from migration to fusion can also include changes in gene expression that are independent of dys. For example, it is shown here that RNA levels of btl, a gene required for tracheal migration, are downregulated in fusion cells in a dys-independent mode. dys is expressed in a variety of Drosophila embryonic cell types, including leading edge, brain, gut, and anal pad, and the mammalian ortholog is prominently expressed in the brain. However, the function of dys in these cell types is unknown, although a potential connection between tracheal fusion cells and both migrating neuronal axon growth cones and leading-edge cells is worth investigating. The role of dys in controlling fusion cell behavior suggests that it is worthwhile to look in tissues that undergo branching morphogenesis, such as the vertebrate lung and vascular system, for regulatory proteins expressed in tip/fusion cells that control the migration, recognition, and fusion properties of their branches (Jiang, 2006).

Protein Interactions

DE-Cadherin is functionally similar to classic vertebrate cadherins. For example, it is associated with alpha-Catenin and beta-Catenin (Armadillo), and protected from trypsin digestion only in the presence of Ca2+, as is the case for many classic cadherins. Transfection of S2 cells with the DE-Cadherin cDNA enhances their Ca(2+)-dependent cell aggregation. Antibodies to this molecule inhibit aggregation, not only in the transfectants but also of early embryonic cells (Oda, 1994).

A series of Armadillo mutants were generated and examined and expressed in Drosophila embryos. Although DE-cadherin and alpha-catenin bind to Armadillo independent of one another, binding of both is required for the function of adherens junctions, that is, mutations that block alpha-Catenin and Cadherin-binding block junction formation. E-cadherin appears to bind in the Armadillo repeat region; this region is required for localization to the adherens junction. alpha-Catenin binding is eliminated by deletion of the region between the N-terminus and the repeats. There are two separate regions of Armadillo critical for Wingless signaling. Mutant Arm proteins deleted in the central repeats or lacking the N-terminal alpha-catenin-binding site all localize prominently at higher levels to cell nuclei in cells responding to Wingless signal. Some of the proteins deleted for parts of the central repeat region require Wingless signal to accumulate in the nucleus while others do not. Endogenous Armadillo normally accumulates in the nucleus and it may act there in transducing Wingless signal. Armadillo's roles in adherens junctions and Wingless signaling are independent. Mutant proteins lacking the domain between the C-terminal region and the Arm repeats retain function in adherens junctions but lack function in Wingless signal transduction. Phosphorylation changes in the Arm protein are detected only in mutant proteins with deletions in the Arm repeats region. It is concluded that the region essential for alpha-catenin binding is not essential for Wingless signaling. The Arm repeats region are essential both for adherens junction function and for Wingless effector function (Orsulic, 1996).

Cell-cell adherens junctions (AJs), comprised of the cadherin-catenin adhesion system, contribute to cell shape changes and cell movements in epithelial morphogenesis. However, little is known about the dynamic features of AJs in cells of the developing embryo. In this study, Dalpha-catenin fused with a green fluorescent protein (Dalpha-catenin-GFP) was constructed, and found to be targeted to apically located AJ-based contacts but not other lateral contacts in epithelial cells of living Drosophila embryos. Using time-lapse fluorescence microscopy, the dynamic performance of AJs containing Dalpha-catenin-GFP in epithelial morphogenetic movements was examined. In the ventral ectoderm of stage 11 embryos, concentration and deconcentration of Dalpha-catenin-GFP occurs concomitantly with changes in length of AJ contacts. In the lateral ectoderm of embryos at the same stage, dynamic behavior of AJs is concerted with division and delamination of sensory organ precursor (SOP) cells. Moreover, changes in patterns of AJ networks during tracheal extension can be followed. Finally, Dalpha-catenin-GFP was used to precisely observe the defects in tracheal fusion in shotgun mutants. Thus, the Dalpha-catenin-GFP fusion protein is a helpful tool to simultaneously observe morphogenetic movements and AJ dynamics at high spatio-temporal resolution (Oda, 1998b).

The lateral ectoderm of living stage 11 embryos of arm-GAL4:UAS-DalphaC-GFP was studied. At early stage 11, many cell divisions occur in the lateral ectoderm. Dividing epithelial cells show dynamic behavior of Dalpha-catenin-GFP. Before cytokinesis, Dalpha-catenin-GFP is distributed in a line at the equator of spherical cells. During cytokinesis, Dalpha-catenin GFP-positive lines of contact between the dividing cell and surrounding cells can be seen. At the end of cytokinesis, new AJ contacts where Dalpha-catenin-GFP has begun to be concentrated appear to be established between the daughter cells. Pairs of divided cells that had delaminated from the surface ectodermal layer were followed. Considering their positions and behavior, these cells seemed to be sensory organ precursor (SOP) cells. After cytokinesis, two daughter cells of a primary SOP cell recover features characteristic of epithelial cells. The cells are indistinguishable in the Dalpha-catenin-GFP distribution pattern from the other ectodermal cells. About 30 min later, the paired SOP cells simultaneously begin to reduce their apical surface area. They constrict their apices smoothly once the constriction begins. It takes 5-10 min for completion of the apical constriction phase. Before entering the constriction phase, Dalpha-catenin-GFP is relatively evenly distributed at apical cell-cell contacts. During the constriction phase, however, Dalpha-catenin-GFP is unevenly distributed at the apical portions of delaminating SOP cells. After SOP cells disappear from the embryo surface, high concentrations of Dalpha-catenin-GFP are left between epithelial cells which have surrounded SOP cells. Eventually, the remaining epithelial cells appear to recover characteristic mesh patterns of Dalpha-catenin-GFP on the apical surface (Oda, 1998b).

These observations seem to reveal a typical series of cellular events in the development of the early embryonic peripheral nervous system (PNS). Division of an SOP cell precedes delamination from the surface ectodermal layer. Although SOP delamination seems to be closely correlated with mitosis, the two events are temporally separate. The delamination is coincident with the apical constriction presumably caused by dynamic functions of AJs. This kind of cellular behavior is commonly observed in a variety of morphogenetic events. For example, the behavior of delaminating SOP cells is reminiscent of that of invaginating presumptive mesodermal cells at the beginning of gastrulation. The invagination process of the mesoderm takes less than 10 min. The time course of mesoderm invagination is comparable to that of SOP delamination. During mesoderm invagination, apically located AJs are broken coinciding with apical constriction. It is possible that a similar AJ disruption event occurs in SOP cells during the apical constriction phase (Oda, 1998b).

Tracheal morphogenesis shows dynamic aspects of DE-cadherin-based cell-cell adhesion. GFP fluorescence of btl-GAL4;UAS-DaC-GFP#3 embryos were observed to directly investigate AJ dynamics during tracheal extension. Concentration of GFP fluorescence can be detected at portions corresponding to AJs in tracheal primordia from stage 11, although signals are also seen uniformly in the cytoplasm but not in nuclei. Tracheal development is not affected by expression of Dalpha-catenin-GFP. Tracheal tissues are located on the inside of the embryo and therefore it has been difficult to examine changes in cell arrangements in living embryos. However, targeted expression of Dalpha-catenin-GFP using btl-GAL4 overcomes this difficulty. Time-lapse observation reveals successive changes in patterns of AJ networks containing Dalpha-catenin-GFP during tracheal extension, although it was still difficult to precisely follow changes in the three-dimensional patterns of AJ networks. Each tracheal primordium elongates along the dorso-ventral axis and the distance between the neighboring tracheal primordia is seen to shorten while the germband is retracting. Tracheal branches extend, coinciding with elongation of Dalpha-catenin GFP-labelled lines. Notably, relatively intense signals for Dalpha-catenin-GFP are often seen around the tips of extending branches. Contact of dorsal trunk (DT) cells between neighboring segments occurs shortly before completion of germband retraction. Soon after the occurrence of this contact, lines of weak Dalpha-catenin-GFP signals can be detected that run through areas of intersegmental DT contact. A previous study has shown that DE-cadherin and Dalpha-catenin are concentrated in tip cells, contributing to the generation of new AJ-based contacts between the cells that give rise to a pore that connects DT lumina. Although it could not be directly determine whether the lines of Dalpha-catenin-GFP accumulation are in tip cells, it is possible that Dalpha-catenin-GFP signals are primary signs of establishing contacts between tip cells of DT (Oda, 1998b).

Dalpha-catenin-GFP was used to analyse tracheal phenotypes of zygotic shg mutants. Mutant embryos could be unambiguously identified by GFP fluorescence in tracheal primordia without any staining. Moreover, targeted expression of Dalpha-catenin-GFP allows clear visualization of morphological defects in the trachea of shg mutants. Epithelial integrity and extension of the tracheal primordia are relatively normal at earlier stages of tracheal development (stages 11-13) probably because of the contribution of maternally supplied functional DE-cadherin molecules. Consistently, considerable amounts of Da-catenin-GFP are detected at apical cell-cell contacts even in shg zygotic mutants although the levels of its accumulation at cell contact sites are reduced compared to those in normal embryos. In normal DT fusion, DE-cadherin-based cell-cell contacts are newly established between tip cells, giving rise to ring-like patterns of cadherin-based junctions, which are visualized by Dalpha-catenin-GFP. In contrast, the process of DT fusion is specifically blocked in shg mutants. At fusion points, tube structures are reduced in diameter or are not constructed at all. Dalpha-catenin-GFP does not accumulate between tip cells of DT in shg mutants probably because a lack of zygotic DE-cadherin causes failure in establishment of new AJ-based contacts between the cells. These results indicate that the accumulation of Dalpha-catenin-GFP at apical contact sites between tip cells is dependent on zygotic expression of normal DE-cadherin. Tracheal fusion is a process in which new apical surface domains facing the lumen are established (Oda, 1998b).

These observations suggested that DE-cadherin is required not only for establishment of AJ-based contacts but also for generation and definition of apical cell domains. In summary, Dalpha-catenin-GFP is a new tool that enables simultaneous visualization of morphogenetic movements and behavior of AJs or the cadherin-based adhesion system in living wild-type and mutant Drosophila embryos. These observations revealed the dynamic performance of AJ-based cell contacts. The methods used in this study will facilitate analysis of the dynamics of cell-cell adhesion at high spatio-temporal resolution in living animals (Oda, 1998b).

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, that then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. These hypotheses were tested in Drosophila by removing endogenous Armadillo. A series of mutant Armadillo proteins with altered intracellular localizations were generated, and these were expressed in wild-type and armadillo mutant backgrounds. Membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. Mutant forms of Armadillo were generated carrying either heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners (Cox, 1999).

Data in vivo suggest that among Arm's known partners, cadherins have the highest affinity, with APC and dTCF (Pangolin) having lower and lowest affinities, respectively. Thus, in embryos with reduced levels of Arm, the remaining Arm is exclusively associated with cadherins, as assayed by immunolocalization and by function. About 70% of cellular Arm is cadherin-associated. When cadherin binding sites are saturated, excess Arm binds to APC/Axin, leading to its destruction and thus preventing accumulation of free Arm. While APC levels, at least in mammalian cells, are low, relative to the total pool of beta catenin, Arm bound to APC is rapidly targeted for destruction, thus opening the way for the binding of additional Arm. Normally the destruction machinery can not only dispose of all non-junctional Arm, but its resources will not even be fully employed, since Arm synthesis can be increased several-fold without biological consequences. However, when the destruction machinery is inactivated either by Wg signal or mutation, Arm is synthesized but not destroyed, and thus levels of Arm rise. APC can bind Arm but in all probability, the APC is rapidly saturated, allowing accumulation of sufficient Arm to allow dTCF to effectively compete for binding. DE-cadherin, dAPC, dTCF and any other possible unknown partners together account for virtually all the Arm in a normal embryo; little if any free Arm is present. This model helps explain the differences in localization of the Armadillo attached to a nuclear localization sequence (Arm-NLS) and Armadillo attached to a nuclear export signal (Arm-NES) in flies and frogs. In Xenopus, added NLS or NES signals dramatically altered Arm's intracellular distribution as expected, while in Drosophila the distribution of wild type Armadillo, Arm-NLS and Arm-NES are indistinguishable. It is proposed that this reflects differences in the level of expression. In flies, mutant Arm accumulates at near wild-type levels, so its binding partners can accommodate the additional protein. Arm bound to cadherin at the plasma membrane is unavailable for nuclear import; likewise Arm in a complex with dTCF is not available for export. Thus Arm-NLS and Arm-NES localization is primarily determined by their binding partners, resulting in a near normal localization. In contrast, Arm-NLS and Arm-NES expression levels in Xenopus likely exceed those of either endogenous beta-catenin or its binding partners. Free Arm is thus accessible to the nuclear import and export machinery, allowing alteration of its localization. Given this, is nuclear localization of Arm a regulated step in Wg signaling in normal cells? The fact that a subset of cells accumulate cytoplasmic but not nuclear Arm suggests that nuclear import may be regulated. In the simplest situation, addition of an NLS ought to promote Arm nuclear accumulation and trigger signaling, while addition of an NES should antagonize signaling. However, heterologous targeting signals have only subtle effects on signaling. Arm-NES signals in the same fashion as does Arm-WT, while only a subset of the Arm-NLS lines are activated for signaling. In the case of Arm-NLS: in cells in which the destruction machinery is on, no free Arm is available for nuclear import or export. In cells with intermediate levels of Wg signaling, the destruction machinery may be slowed, allowing accumulation of cytoplasmic Arm in complex with APC, but not to sufficient levels to saturate APC and allow nuclear import. Only when signaling is fully activated would sufficient free Arm accumulate for nuclear import. Addition of an NLS would thus only alter the balance in cells near the signaling threshold. Further, if nuclear Arm is bound to dTCF, it may be inaccessible to the nuclear export machinery. The mechanisms by which Arm/betacat enters nuclei remain unclear; dTCF-dependent and independent pathways may exist. The recent observation that beta Catenin may mediate its own nuclear transport, independent of importins, further complicates the issue. Additional levels of regulation may occur, beyond the simple regulation of Arm/beta Catenin stability (Cox, 1999 and references).

Specialized cell junctions in epithelia serve as cell-cell adhesion sites and thus contribute to the maintenance of tissue integrity. The Drosophila gene crumbs encodes a transmembrane protein that is required for the biogenesis of the zonula adherens, a belt-like structure encircling the apex of epithelial cells. As previously shown, expression of just the short membrane-bound cytoplasmic domain is sufficient to rescue major defects associated with the loss of crumbs function. The cytoplasmic domain of Crumbs is highly conserved in two putative crumbs homologs in C. elegans. To assess the significance of conserved residues, various point mutations and deletions were introduced into this region. Two functional domains were revealed: an amino-terminal region and the carboxy-terminal amino acids EERLI. Both are necessary for rescue of the crumbs phenotype. The EERLI motif interacts with Discs Lost (now redefined as Drosophila Patj), a cytoplasmic protein containing PDZ domains. Overexpression of the Crumbs cytoplasmic domain induces a transition from the single-layered epithelium to a multilayered tissue. This transition is associated with redistribution of the Drosophila homolog of the cell adhesion molecule E-cadherin, and depends on the presence of the EERLI motif (Klebes, 2000).

Data presented here suggest a model in which the Drosophila Crb protein organizes the assembly of an apically localized protein scaffold in epithelial cells that is required for the proper formation and localisation of the ZA. This scaffold includes the protein Dlt (now Patj) and probably other, as yet unidentified, proteins, its assembly depends on the carboxy-terminal segment of Crb. The model further suggests that the Crb-mediated control of DE-cadherin localization depends on interaction between the Crb cytoplasmic domain and the PDZ protein Dlt. Neither DE-cadherin nor Dlt are localized in crb mutant embryos, whereas both proteins are sequestered by mislocalized Crb. However Dlt remains apically localized after overexpression of DE-cadherin. The interaction of Crb with Dlt depends on Crb's carboxy-terminal motif, EERLI. This motif is also necessary for misdistribution of DE-cadherin upon Crb overexpression and for the rescue of crb mutant embryos. The presence of four PDZ domains in Dlt makes it an ideal partner for recruiting other proteins into a hypothetical Crb-dependent, membrane-associated protein network. PDZ domains have been shown to act as versatile organizers of multiprotein complexes. In many cases, the binding site of the interacting protein, often a transmembrane protein, is localized at its carboxyl terminus and ends with a hydrophobic amino-acid residue. Class I PDZ domains bind a conserved S/T-X-V motif (where X is any amino acid), whereas class II domains recognize ligands that carry a hydrophobic amino-acid residue at the -2 position. Since the Dlt-binding site in Crb differs from these motifs, the first PDZ domain of Dlt, which binds to Crb in vitro, may belong to a different class. The presence of the ERLI motif in both C. elegans homologs and the similarities between the phenotypes produced by overexpression of CD2-IntraWT and CD2-IntraCE in the Drosophila embryo suggest that this region might mediate comparable interactions in the nematode. Not surprisingly, a protein similar to Drosophila Dlt has also been detected in the C. elegans database, pointing to the possible conservation of additional components of the postulated protein network (Klebes, 2000).

Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome

Stem cell self-renewal can be specified by local signals from the surrounding microenvironment, or niche. However, the relation between the niche and the mechanisms that ensure the correct balance between stem cell self-renewal and differentiation is poorly understood. This study shows that dividing Drosophila male germline stem cells use intracellular mechanisms involving centrosome function and cortically localized Adenomatous Polyposis Coli tumor suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation (Yamashita, 2003).

Adult stem cells maintain populations of highly differentiated but short-lived cells such as skin, intestinal epithelium, or sperm through a critical balance between alternate fates: Daughter cells either maintain stem cell identity or initiate differentiation. In Drosophila testes, germline stem cells (GSCs) normally divide asymmetrically, giving rise to one stem cell and one gonialblast, which initiates differentiation starting with the spermatogonial transient amplifying divisions. The hub, a cluster of somatic cells at the testis apical tip, functions as a stem cell niche: Apical hub cells express the signaling ligand Unpaired (Upd), which activates the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway within GSCs to maintain stem cell identity (Yamashita, 2003).

Analysis of dividing male GSCs by expression of green fluorescent protein (GFP)-α-tubulin in early germ cells revealed that in 100% of the dividing stem cells observed, the mitotic spindle was oriented perpendicular to the hub-GSC interface throughout mitosis, with one spindle pole positioned within the crescent where the GSC contacted the hub. Stem cell division was rare, averaging one dividing stem cell observed per 5 to 10 testes (~2% of total stem cells) in 0- to 2-day-old adults. Spindles were not oriented toward the hub in gonialblasts (Yamashita, 2003).

Drosophila male GSCs maintained a fixed orientation toward the hub throughout the cell cycle, unlike Drosophila embryonic neuroblasts or the Caenorhabditis elegans P1 cell, in which spindle orientation is established during mitosis by a programmed rotation of the spindle. The single centrosome in early interphase GSCs was consistently located adjacent to the hub. After centrosome duplication, one centrosome remained adjacent to the hub, whereas the other migrated to the opposite side of the nucleus. The mechanisms responsible for Drosophila GSC spindle orientation may differ between sexes. In female GSCs, the spectrosome, a spherical intracellular membranous structure, remains localized next to the apical cap cells, where it may help anchor the spindle pole during mitosis. In interphase male GSCs, in contrast, the spectrosome was often located to the side, whereas at least one centrosome held the stereotyped position adjacent to the hub (Yamashita, 2003).

To investigate centrosome function in orientation of male GSCs, males were analyzed that were null mutant for the integral centrosome component centrosomin (cnn), which is required for normal astral microtubule function. In cnn mutant males, mitotic spindles were not oriented toward the hub in ~30% of the dividing GSCs examined. In an additional 10% to 20%, spindles were properly oriented, but the proximal spindle pole was no longer closely associated with the cell cortex at the hub-GSC interface and the entire spindle was displaced away from the hub. The frequency of spindle orientation defects was highest in metaphase. Loss of function of cnn also partially randomized the interphase centrosome positioning in male GSCs. In more than 35% of the cnn mutant GSCs with duplicated centrosomes that were scored, neither centrosome was positioned next to the hub (Yamashita, 2003).

The number of germ cells associated with the hub was increased 20% to 30% in cnn mutant males, from an average of 8.94 GSCs per hub in the wild type to 11.89 GSCs per hub in cnn^HK21/cnn^HK21 and 10.69 GSCs per hub in cnn^HK21/cnn^mfs3. Hub size was not significantly different in cnn compared with wild-type males. In cnn testes with many stem cells, GSCs appeared crowded around the hub and often seemed attached to the hub by only a small region of cell cortex. Finite available physical space around the hub may limit the increase in stem cell number in cnn mutant males (Yamashita, 2003).

As suggested by the increased stem cell number, there were several cases in both live and fixed samples from cnn males in which a stem cell that had recently divided with a mitotic spindle parallel to the hub-GSC interface produced two daughter cells that retained contact with the hub, a finding that was not observed in the wild type. GSCs were also observed dividing with a misoriented/detached spindle that lost attachment to the hub, probably explaining the mild increase in stem cell number relative to the frequency of misoriented spindles (Yamashita, 2003).

The normal close attachment of one spindle pole to a region of the GSC next to the hub and the effects of cnn mutants on centrosome and spindle orientation suggest that a specialized region of the GSC cell cortex touching the hub might provide a polarity cue toward which astral microtubules from the centrosome and spindle pole orient. High levels of DE-cadherin (fly epithelial cadherin) and Armadillo (Arm; fly ß-catenin) colocalized at the hub-GSC interface, as well as at the interface between adjacent hub cells, marked by high levels of Fas III. High levels of DE-cadherin and Arm were not detected around the rest of the GSC surface. Forced expression of DE-cadherin-GFP specifically in early germ cells confirmed that DE-cadherin in GSCs colocalized to the hub-GSC interface (Yamashita, 2003).

DE-cadherin and Armadillo at the hub-GSC interface may provide an anchoring platform for localized concentration of Apc2, one of two Drosophila homologs of the mammalian tumor suppressor gene Adenomatous Polyposis Coli (APC), which in turn may anchor astral microtubules to orient centrosomes and the spindle. Immunofluorescence analysis revealed Apc2 protein localized to the hub-GSC interface. In apc2 mutant males, GSCs were observed with mispositioned centrosomes, misoriented spindles, or detached spindles. Both the average number of stem cells and hub diameter increased in apc2 mutant males compared with that of the wild type. Unlike in cnn mutants, GSCs did not appear crowded around the hub in apc2 males, perhaps as a result of the enlarged hub (Yamashita, 2003).

The second Drosophila APC homolog, apc1, may also contribute to normal orientation of the interphase centrosome and mitotic spindle. Apc1 protein localized to centrosomes in GSCs and spermatogonia during late G₂/prophase, after centrosomes were fully separated but before nuclear envelope breakdown. Apc1 was not detected at centrosomes from prometaphase to telophase. Spindle orientation and centrosome position were perturbed in GSCs from apc1 males, and the number of stem cells per testis and the diameter of the hub both slightly increased in apc1 mutant testes compared with those of the wild type (Yamashita, 2003).

It is proposed that the reliably asymmetric outcome of male GSC divisions is controlled by the concerted action of (1) extrinsic factor(s) from the niche that specify stem cell identity, and (2) intrinsic cellular machinery acting at the centrosome and a specialized region of the GSC cortex located at the hub-GSC interface to orient the cell division plane with respect to the signaling microenvironment. Astral microtubules emanating from the centrosome may be captured by a localized protein complex including Apc2 at the GSC cortex where it interfaces with the hub, similar to the way in which cortical Apc2 may orient mitotic spindles in the syncytial embryo or epithelial cells (Yamashita, 2003).

Mechanisms that orient the mitotic spindle by attachment of astral microtubules to specific cortical sites may be evolutionally conserved. In budding yeast, spindle orientation is controlled by capture and tracking of cytoplasmic microtubules to the bud tip, dependent on Kar9, which has weak sequence similarity to APC proteins. Kar9 has been localized to the spindle pole body and the cell cortex of the bud tip, reminiscent of the localization of Drosophila Apc1 at centrosomes and Apc2 at the cell cortex (Yamashita, 2003).

Polarization of Drosophila male GSCs toward the hub could result simply from the geometry of cell-cell adhesion. GSCs appear to be anchored to the hub in part through localized adherens junctions. Homotypic interactions between DE-cadherin on the surface of hub cells and male GSCs could concentrate and stabilize a patch of DE-cadherin. The resulting localized DE-cadherin cytoplasmic domains could then provide localized binding sites for ß-catenin and Apc2 at the GSC cortex. Although binding of E-cadherin and APC to ß-catenin is thought to be mutually exclusive, APC could be anchored at the cortical patch through the actin cytoskeleton, which in turn could interact with ß-catenin/α-catenin (Yamashita, 2003).

Orientation of stem cells toward the niche appears to play a critical role in the mechanism that ensures a reliably asymmetric out-come of Drosophila male GSC divisions, consistently placing one daughter within the reach of short-range signals from the hub and positioning the other away from the niche. Oriented stem cell division may be a general feature of other stem cell systems, helping maintain the correct balance between stem cell self-renewal and initiation of differentiation throughout adult life (Yamashita, 2003).

Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila

Src42A is one of the two Src homologs in Drosophila. Src42A protein accumulates at sites of cell-cell or cell-matrix adhesion. Anti-Engrailed antibody staining of Src42A protein-null mutant embryos indicated that Src42A is essential for proper cell-cell matching during dorsal closure. Src42A, which is functionally redundant to Src64, was found to interact genetically with shotgun, a gene encoding E-cadherin, and armadillo, a Drosophila ß-catenin. Immunoprecipitation and a pull-down assay indicated that Src42A forms a ternary complex with E-cadherin and Armadillo, and that Src42A binds to Armadillo repeats via a 14 amino acid region, which contains the major autophosphorylation site. The leading edge of Src mutant embryos exhibiting the dorsal open phenotype is frequently kinked and associated with significant reduction in E-cadherin, Armadillo and F-actin accumulation. This phenotype suggests that not only Src signaling but also Src-dependent adherens-junction stabilization are essential for normal dorsal closure. Src42A and Src64 are required for Armadillo tyrosine residue phosphorylation but Src activity may not be directly involved in Armadillo tyrosine residue phosphorylation at the adherens junction (Takahashi, 2005).

In ectodermal cells, strong Src42A signals in apical or apicolateral regions were always associated with strong E-cad signals. E-cad is a core component of the adherens junction that is responsible for cell-cell adhesion and, hence, most, if not all, E-cad-associated membranous Src42A is probably related to adherens junction-dependent cell-cell adhesion (Takahashi, 2005).

A considerable fraction of ectodermal cells were also found associated with the second type of basal Src42A free of E-cad. E-cad-free Src42A is localized on the ectoderm/mesoderm interface and eliminated from ectodermal cells that have evaginated or invaginated without mesoderm association. The extracellular matrix (ECM) comprises several groups of secreted proteins such as integrin ligands. During embryogenesis, different cell layers become properly connected, most probably via cell adhesion to ECM. E-cad-free Src42A may thus be related to integrin-mediated cell-matrix adhesion. Cell-ECM adhesion may not be restricted to the interface between ectodermal and mesodermal cell layers. Strong Src42A signals have actually been found present on the interface between mesodermal and endodermal cell layers (Takahashi, 2005).

The current study shows that, as with JNK signaling genes, Src is required not only for thick F-actin accumulation at the leading edge but proper cell-cell matching along the midline seam as well. JNK signaling, which includes hemipterous (hep) and basket (bsk), is essential for dorsal closure of the embryonic epidermis in Drosophila. Based on examination of Tec29 Src42A mutant phenotypes, it has been suggested that Src42A acts upstream of bsk (Takahashi, 2005).

The adherens junction is necessary for cell-cell adhesion and thick F-actin accumulation occurs at the level of the adherens junction at the leading edge. Since E-cad and Arm signals along with actin signals are reduced significantly at the leading edge in Src42A^26-1;Src64^P1/+ embryos and the leading edge of the mutants is significantly kinked, the absence of Src protein from the adherens junction may possibly result in destruction of structural integrity, implying that the adherens junction is also involved in dorsal closure regulation in a structural way (Takahashi, 2005).

Dorsal closure and CNS defects similar to those in Src mutants have been observed in abl mutants. In vertebrates, Abl is tyrosine-phosphorylated with Src and is capable of interacting with delta-catenin, an E-cad-binding protein. Abl may thus function as well downstream of Src signaling in Drosophila. Germ-band retraction and possibly too, head involution, both of which require Src activity, may be regulated by the two above distinct Src functions. alpha1,2-laminin and alphaPS3ßPS integrin have clearly been shown to be essential for spreading a small group of amnioserosa epithelium cells over the tail end of the germ band during germ-band retraction. shg activity has also been shown to be essential for normal germ-band retraction and head involution (Takahashi, 2005).

Src-dependent dynamical regulation of E-cad-dependent cell-cell adhesion may also be necessary for visual system formation. E-cad overexpression or elimination of EGFR activity have been shown to render optic placode cells incapable of invaginating and prevent the separation of Bolwig's organ precursors from the optic lobe. Virtually identical phenotypes were induced by loss of Src activity, suggesting involvement of at least the adherens junction Src in larval visual system formation and that Src should function either upstream or downstream of EGFR signaling (Takahashi, 2005).

Regulatory mechanisms required for DE-cadherin function in cell migration and other types of adhesion

Cadherin-mediated adhesion can be regulated at many levels, as demonstrated by detailed analysis in cell lines. This study examines the requirements for Drosophila epithelial (DE) cadherin regulation in vivo. Investigating Drosophila oogenesis as a model system allowed the dissection of DE-cadherin function in several types of adhesion: cell sorting, cell positioning, epithelial integrity, and the cadherin-dependent process of border cell migration. Multiple fusions were generated between DE-cadherin and alpha-catenin as well as point-mutated ß-catenin and the ability of these fusion proteins to support these types of adhesion was analyzed. It was found that (1) although linking DE-cadherin to alpha-catenin is essential, regulation of the link is not required in any of these types of adhesion; (2) ß-catenin is required only to link DE-cadherin to alpha-catenin, and (3) the cytoplasmic domain of DE-cadherin has an additional specific function for the invasive migration of border cells, which is conserved to other cadherins. The nature of this additional function is discussed (Pacquelet, 2005).

Classic cadherin proteins have multiple essential roles during animal development both in keeping tissues/epithelia intact and in allowing dynamic cell rearrangements. One dramatic example of the latter is the invasive migration of border cells during oogenesis, for which DE-cadherin is essential. This study investigates which features of DE-cadherin are required for migration, and these features are compared with features that are required more generally for other adhesion functions. Cadherin proteins are well conserved from fly to man; the cytoplasmic domain, in particular, is well conserved, and it interacts with the cytoskeleton. Therefore, the in vivo genetic analyses focused on dissecting the functions of DE-cadherin cytoplasmic domain. In the type of in vivo replacement experiments performed, clear conclusions could be made about what is and is not required under physiological conditions. This is the strength of the analysis, and it is thought to be important to further the understanding of the much-studied cadherin molecules. Generally speaking, the idea cannot be excluded that a type of regulation that is not genetically required does, in fact, occur under normal conditions and contribute somewhat to regulation (e.g., to make the system more robust) (Pacquelet, 2005).

Focus was initially placed on a conserved tyrosine of ß-catenin, the phosphorylation of which may induce ß-catenin to dissociate from cadherin, resulting in a decrease of adhesion. This conserved tyrosine (and, hence, its phosphorylation) is not essential even during border cell migration. The idea that phosphorylation of this tyrosine residue happens or that it may induce some dissociation of ß-catenin from DE-cadherin cannot be excluded. What these results show is that such phosphorylation is not an essential mechanism for adhesion regulation in any of the tested types of cadherin-dependent adhesion in vivo. Significant emphasis has been put in the literature on the putative regulatory role of this conserved tyrosine of ß-catenin. However, much of this emphasis is based on correlations between ß-catenin tyrosine phosphorylation and adhesion down-regulation. It is not clear whether ß-catenin phosphorylation is really the cause of adhesion down-regulation. In addition, the tyrosine kinase Src causes a decrease of adhesion in L cells expressing the fusion protein E-cadherin/alpha-catenin. Thus, Src-induced adhesion down-regulation can be independent of ß-catenin phosphorylation. Therefore, the ability to regulate adhesion without phosphorylating ß-catenin tyrosine may be more general (Pacquelet, 2005).

Next, it was found that neither the link between DE-cadherin and ß-catenin nor that between ß- and alpha-catenin need be regulated at all for DE-cadherin function in vivo. A fusion between DE-cadherin-FL and alpha-catenin fully substitutes for endogenous DE-cadherin during oogenesis even in the absence of endogenous ß-catenin. It was surprising to find that there is no need to regulate the link between DE-cadherin and alpha-catenin, since earlier studies using similar fusion proteins had concluded that regulation was required for mouse E-cadherin to support 'intercellular migration' (Nagafuchi, 1994). There are two main explainations for this discrepancy. (1) The previous study did not fuse alpha-catenin to E-cadherin-FL but fused to a truncated E-cadherin (analogous to DE-cadherinDeltaß/alpha-catenin). As was found in the current study, this not only affects the ability to regulate the link to alpha-catenin but also removes additional functionality from cadherin. It was not directly investigated in Nagafuchi (1994) whether the defects were caused by ß-catenin regulation as proposed. (2) Different cell types were analyzed; the previous study overexpressed E-cadherin in mouse fibroblasts that normally have very little of the protein, whereas this study investigated cells that normally depend on DE-cadherin for biological function(Pacquelet, 2005).

It is possible that the link between DE-cadherin and the actin cytoskeleton does need to be regulated but that it occurs downstream of alpha-catenin. More studies of alpha-catenin and of how its interactions are regulated will be of interest, in particular in a physiological context. Alternatively, regulation of adhesion may primarily occur by the turning over of DE-cadherin and/or DE-cadherin complexes via endocytosis. A Cbl-related E3 ligase called Hakai has been identified as a specific regulator of mammalian E-cadherin endocytosis (Fujita, 2002). It is recruited to specific phosphorylated tyrosines on E-cadherin. No evidence was found that the homologous D. melanogaster protein (CG10263) affects DE-cadherin or border cell migration, and the key docking tyrosines are not conserved. However, other regulators may play an analogous role. Finally, adhesive strength could be regulated by lateral clustering of cadherin complexes; for example, by the binding of additional regulatory proteins to the intracellular domain (Pacquelet, 2005).

The full functionality of DE-cadherin-FL/alpha-catenin in the absence of ß-catenin also indicates that ß-catenin has no essential adhesive function other than linking DE-cadherin to alpha-catenin. Based on the abnormal localization of various DE-cadherin mutants, it had been proposed that ß-catenin was required for proper translocation of cadherin to the plasma membrane. However, the relatively normal subcellular localization of DE-cadherin-FL/alpha-catenin that was observed in the absence of ß-catenin suggests that this is not generally the case. It remains possible that ß-catenin also contributes to modifying cadherin localization in D. melanogaster cells, but in a more subtle, nonessential way. This study suggests that parts of the cadherin tail that bind ß-catenin may also have ß-catenin-independent functions. This would complicate the interpretation of how modified cadherin molecules behave unless it is also investigated by ß-catenin loss-of-function experiments (Pacquelet, 2005).

In contrast with DE-cadherin-FL/alpha-catenin, a fusion protein between DE-cadherin and alpha-catenin lacking the DE-cadherin cytoplasmic tail (DE-cadherinDeltaCyt/alpha-catenin) could not substitute for DE-cadherin during border cell migration. It was targeted to the cell surface and was functional in all other contexts. This indicates that the DE-cadherin cytoplasmic tail has a specific function during invasive migration in addition to the basic ß-catenin/alpha-catenin linkage. The function could not be provided by an unrelated cytoplasmic linker (CD2) but could be provided by the corresponding region from mouse E-cadherin or D. melanogaster N-cadherin. Most likely, one or more interactions that are specific to cadherin tails have a critical function in this context. These results raise two questions: (1) why is DE-cadherin tail specifically important for border cell migration and (2) what is the molecular nature of the required function (Pacquelet, 2005)?

With regard to the specific requirement in border cells, the role of DE-cadherin in their migration needs to be considered. Given the absolute requirement for this particular cell-cell interaction to achieve invasive border cell movement, it is likely to be force bearing. DE-cadherin-mediated adhesion between the front of border cells and the attachment point on nurse cells needs to be strong enough to allow border cells to pull themselves into the compact germ line tissue. As the border cell cluster initiates migration using a long, slender cellular extension, the local force application at the tip may be quite high. As an illustration of the forces involved, it was found that mutant border cells with impaired cortical cytoskeleton will break apart when they attempt to invade, whereas other follicle cells (including centripetal cells) with the same defect appear to be relatively normal. It is suggested that the DE-cadherin tail may be required to allow a build-up of sufficiently strong adhesion to withstand forces that are involved in migration (Pacquelet, 2005).

Another important aspect of adhesion during cell movement is that it may need to be effectively down-regulated at the rear of the cells to allow cell translocation along the substrate. Experiments indicated that the primary defect for DE-cadherinDeltaCyt/alpha-catenin in border cells is not a lack of down-regulation; in other words, it is not caused by an excess of adhesion. However, an inability of DE-cadherinDeltaCyt/alpha-catenin to provide sufficient adhesion for migration as discussed above could mask possible additional (migration specific) defects of the fusion protein such as the ability to be down-regulated (Pacquelet, 2005).

The molecular nature of the DE-cadherin tail requirement in migration is in need of further investigation. The function does not simply map to any previously known signal or interaction, suggesting involvement of a novel interaction and/or a redundancy of interactions. The DE-cadherinDeltaß/alpha-catenin fusion results indicate that the most COOH-terminal domain contributes to DE-cadherin function in border cells independently of ß-catenin binding. However, this domain is not essential on its own nor when coexpressed with p120 catenin RNA interference constructs, indicating that additional important signals are located in the more proximal region of the DE-cadherin cytoplasmic domain. A mutant form of Xenopus laevis C-cadherin lacking the 94 proximal amino acids of its cytoplasmic domain can mediate some adhesion but is unable to support strong adhesion. This seems to be caused by its inability to form lateral clusters. Similarly, an absence of the proximal region in DE-cadherinDelta-Cyt/alpha-catenin could prevent its clustering and, thereby, prevent adhesion strengthening (Pacquelet, 2005).

In conclusion, this structure/function analysis of DE-cadherin in different types of cell adhesion has given new information about cadherin regulation in vivo. Several previously defined potential points of regulation that were established through detailed work in tissue culture were found not to be essential for functionality in vivo. The cytoplasmic tail of cadherin was found to have a unique role in the demanding process of invasive cell migration, possibly through a novel interaction (Pacquelet, 2005).

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