Cell-culture studies indicate that tyrosine phosphorylation of the cadherin-catenin-complex (CCC) is one of the post-translational mechanisms regulating E-cadherin-mediated cell adhesion. In this investigation, controlled application of a tyrosine phosphatase inhibitor (orthovanadate) and tyrosine kinase inhibitor (tyrphostin) to early Drosophila embryos, followed by biochemical assays and phenotypic analysis, have been utilized to address the mechanism by which tyrosine phosphorylation regulates E-cadherin-mediated cell adhesion in vivo. The data suggest that, in the Drosophila embryo, ß-catenin (Drosophila Armadillo) is the primary tyrosine-phosphorylated protein in the CCC. The increase in tyrosine phosphorylation correlates with a loss of epithelial integrity and adherens junctions in the ectoderm of early embryos. Late application of the phosphatase inhibitor does not have this effect, presumably because of the formation of septate junctions in late embryos. Co-immunoprecipitation assays have demonstrated that tyrosine hyper-phosphorylation does not cause the dissociation of Drosophila (D)E-cadherin and alpha-catenin or Armadillo, suggesting that abrogation in adhesion is most likely attributable to the detachment of actin-associated proteins from the CCC. Finally, although the Drosophila epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is linked to the CCC and shows genetic interactions with DE-cadherin, it was found that a constitutively active Drosophila EGFR construct does not cause any detectable changes in the level of tyrosine phosphorylation of Armadillo or destabilization of the CCC (Wang, 2005).

Many studies in mammalian cells have established that E-cadherin-mediated cell adhesion is regulated by kinase activity. Investigations with cell lines have shown that the loss of cell adhesion, attributable to elevated kinase activity, results in an increase in cell motility. Several lines of evidence have demonstrated that both the receptor and non-receptor kinases are involved in the down regulation of E-cadherin-mediated adhesion. However, although the regulation of cell adhesion by kinase activity has been widely studied in cancer and epithelial cell lines, the question of whether this type of regulation exists in a physiological environment has not been addressed in much detail. Genetic evidence has been provided that DER plays a significant role in down-regulating DE-cadherin-mediated cell adhesion during the development of the Drosophila larval visual system. In the present study, biochemical methods were utilized to address the following questions. (1) Does tyrosine phosphorylation take a part in regulating cell adhesion in vivo? (2) What mechanism is involved in this post-translational regulation of cell adhesion? (3) Is DE-cadherin-mediated cell adhesion regulated by the activity of endogenous receptor tyrosine kinases, such as DER, in vivo (Wang, 2005)?

The tyrosine phosphorylation level was altered in living embryos by introducing the phosphatase inhibitor orthovanadate or a receptor tyrosine-kinase-specific inhibitor (tyrphostin). Phenotypic analysis showed that the orthovanadate-treated embryos displayed a loss-of-cell adhesion phenotype, including a reduction in adherens junctions, an absence of apical-basal polarity in the ectodermal epithelium and cell shape changes from cuboidal to spherical. Immunoprecipitation followed by Western blot of the orthovanadate treated embryos showed that Arm was the primary component in the CCC being tyrosine-phosphorylated, and this increase in tyrosine phosphorylation was correlated with the reduction in cell-cell adhesion. These findings are thus consistent with observations in mammalian cell-culture systems in which ß-catenin is hyper-phosphorylated at tyrosine residues when a global phosphatase inhibitor is applied to the cells (Wang, 2005).

Loss of cell adhesion, mediated by E-cadherin, upon ß-catenin hyper-phosphorylation at tyrosine residues has been proposed to result from phosphorylation of either ß-catenin or alpha-catenin, or both. Alternatively, in the mammary epithelial cell line MDM468, tyrosine phosphorylation of ß-catenin has been shown to cause the detachment of vinculin and actinin from the CCC, thus inducing the loss of cell adhesion. The data show that, despite the observed loss of adherens junctions, tyrosine hyper-phosphorylation of Arm does not cause a noticeable dissociation of Arm from DE-cadherin. Therefore, the remodeling of the CCC-actin interaction in the fly embryo is mediated by the detachment of alpha-catenin or an as yet unknown protein (or proteins) that forms a link between the alpha-catenin and the actin cytoskeleton. In vertebrate cell culture, vinculin and alpha-actinin both have been shown to interact with the CCC. Furthermore, vinculin has been suggested as the site of CCC detachment when the cellular tyrosine phosphorylation level is increased. Although homologs of vinculin and alpha-actinin have been isolated in Drosophila, the involvement of these two proteins in connecting the CCC to the actin cytoskeleton has not yet been studied. Genetic studies indicate that Drosophila vinculin and alpha-actinin, unlike DE-cadherin and Arm, are not essential for epithelial integrity and early morphogenetic movements in the Drosophila embryo. The exclusive expression of vinculin in the epidermis-muscle attachment sites in the Drosophila embryo suggests that this protein may be is not involved in connecting and regulating the CCC in epithelial cells (Wang, 2005).

The process of endocytosis could also explain the reduction in cell adhesion without immediate disassembly of the catenins from cadherin. When E-cadherin and catenin are tyrosine phosphorylated upon v-Src activation in MDCK cells, Haiki (an E3 ligase) is recruited to bind to E-cadherin; as a result, the CCC is subsequently internalized and ubiquitinated. Removal of the entire complex from the surface weakens cell adhesion, but the internalized CCC may remain intact, as shown biochemically. Further studies with immuno-electron microscopy may be required to determine DE-cadherin expression following orthovanadate treatment to ascertain the validity of this hypothesis (Wang, 2005).

Application of a specific receptor tyrosine kinase inhibitor or ectopic expression of the dominant negative DER construct did not show a detectable phenotype in embryos, and the state of tyrosine phosphorylation of the CCC appeared to remain unchanged. Ectopic expression of the activated DER construct also did not change the tyrosine phosphorylation of catenin assayed by immunoprecipitation. These two observations suggest that the phosphorylation level of the CCC is delicately maintained at equilibrium. Tyrosine phosphatases, such as low-molecular-weight protein tyrosine phosphatase, receptor tyrosine phosphatase, receptor protein kinase phosphatase, and PTP1B, are localized at the apical region of the epithelium or are even associated with the adherens junction proteins. These factors could be involved in counteracting the abnormal tyrosine phosphorylation of the CCC, such as the one following the expression of an activated form of DER. As a result, an in vivo decrease in tyrosine phosphorylation could not be detected, unlike the reported results obtained by using tissue cultures in which activation of EGFR gives rise to a detectable change in the tyrosine phosphorylation level in ß-catenin. Future experiments testing activated DER on a background of a phosphatase mutant may be more informative in elucidating in which DER regulates DE-cadherin-mediated cell adhesion in a biological system (Wang, 2005).

Regulation of Wnt transcriptional targets is thought to occur by a transcriptional switch. In the absence of Wnt signaling, sequence-specific DNA-binding proteins of the TCF family repress Wnt target genes. Upon Wnt stimulation, stabilized β-catenin binds to TCFs, converting them into transcriptional activators. C-terminal-binding protein (CtBP) is a transcriptional corepressor that has been reported to inhibit Wnt signaling by binding to TCFs or by preventing -catenin from binding to TCF. This study shows that CtBP is also required for the activation of some Wnt targets in Drosophila. CtBP is recruited to Wnt-regulated enhancers in a Wnt-dependent manner, where it augments Armadillo (the fly β-catenin) transcriptional activation. CtBP is required for repression of a subset of Wnt targets in the absence of Wnt stimulation, but in a manner distinct from previously reported mechanisms. CtBP binds to Wnt-regulated enhancers in a TCF-independent manner and represses target genes in parallel with TCF. The data indicate dual roles for CtBP as a gene-specific activator and repressor of Wnt target gene transcription (Fang, 2006).

CtBP has previously been identified as a repressor of Wnt signaling, as measured by TCF reporter genes in cultured cells. Consistent with this, CtBP was identified in an overexpression screen via its ability to suppress Wg and Arm action in the developing eye. In wing imaginal discs, CtBP overexpression also inhibited the Wg target Senseless (Sens). Consistent with this overexpression data, the reduction of CtBP in cultured cells via RNAi is also consistent with a role for CtBP in repressing some Wnt targets (Fang, 2006).

The working model for CtBP repression of Wnt target gene expression holds that CtBP binds to the same area of the nkd and CG6234 loci as TCF, but this binding is TCF-independent. Consistent with this, knock down of CtBP and TCF or gro synergistically derepresses nkd expression. No synergism was seen with TCF/gro double depletions. The RNAi and ChIP data together favor a model where CtBP acts in parallel with TCF/Gro to repress nkd expression in the absence of Wg stimulation. Because CtBP has no detectable ability to bind nucleic acids, it is assumed that unknown DNA-binding protein(s) recruit CtBP to the WRE (Fang, 2006).

The existing models for CtBP antagonism of Wnt signaling cannot explain the data. TCF-independent recruitment of CtBP to WREs is not consistent with work suggesting direct binding of CtBP to TCF. The alternative mechanism, where a CtBP/APC complex diverts Arm/β-catenin away from TCF, also is inconsistent with the results. In this model, the activation of nkd expression after CtBP RNAi treatment would be dependent on TCF and arm. Because the derepression of nkd occurred when both CtBP and TCF were depleted and was not affected when arm was also inhibited, this model is not favored to explain the effects of CtBP depletion on nkd expression. These distinct mechanisms for CtBP repression are not mutually exclusive and may all occur in some contexts (Fang, 2006).

There is a qualitative difference in the amount of derepression found between the two Wg targets studied in Kc cells. Depletion of CtBP and TCF/gro causes a large (20- to 30-fold) increase in nkd basal expression, but has a much more modest (<3-fold) effect on CG6234. These differences may reflect a fundamental difference in the way TCF/Gro and CtBP act on various Wnt targets in unstimulated cells, but it is equally likely that the surrounding cis-elements in these targets have a strong influence on the degree of derepression that can be observed (Fang, 2006).

In addition to defining a novel mechanism for CtBP repression of Wg targets, strong evidence is provided for CtBP playing a role in Wg-mediated transcriptional activation. In the wing imaginal discs, loss of CtBP resulted in a lag in Wg-dependent activation of Sens and a reduction in Dll expression. In cultured Kc cells, CtBP depletion caused a two- to three-fold reduction in the ability of Wg to activate CG6234 expression. The ability of Gal4-Arm chimeras to activate a Gal4 reporter gene is also highly dependent on CtBP levels. In all these contexts, CtBP is not absolutely required for Wg signaling, but is necessary for maximal activation of Wg/Arm transcriptional activation (Fang, 2006).

The positive effect of CtBP on Wg signaling is direct, as judged by ChIP. Assuming that ChIP is measuring the degree of occupancy of CtBP on the chromatin, and not simply antigen accessibility, Wg stimulation promotes the association of CtBP with the CG6234 WRE. This increase in CtBP binding is not observed in TCF-depleted cells. Gal4-Arm recruits endogenous CtBP to a UASluc reporter. Taken together, these data support a model where TCF/Arm recruits CtBP to Wg targets. No binding between Arm and CtBP has been detected by co-immunoprecipitation, suggesting that another factor(s) may act as an adaptor between CtBP and the Arm bound to TCF (Fang, 2006).

Arm has transcriptional activation activity in both the N- and C-terminal portions of the protein. CtBP overexpression or RNAi depletion greatly effects the activity of the N-terminal half of Arm but has no effect on the C-terminal portion. Consistent with this, the N-terminal portion can recruit CtBP to a reporter gene, but not the C-terminus. Other factors that have been linked to the N-terminal portion of Arm include Lgs and Pygo and the ATPases Pontin and Reptin. It may be that CtBP acts in concert with one or more of these factors (Fang, 2006).

CtBPs have strong sequence similarity with D2-hydroxyacid dehydrogenases. hCtBP1 is a functional dehydrogenase and point mutations blocking CtBP1 dehydrogenase activity inhibit its ability to interact with binding partners and act as a transcriptional corepressor. However, another group found that similar mutations had no effect on the ability of CtBP to repress transcription. In this report, mutation of two residues (D290A and H312T) predicted to be essential for catalytic activity had no effect on the ability of fly CtBP to potentiate Gal4-Arm transcriptional activation. Further complicating the issue is data from experiments expressing the fly CtBP fused to Gal4DBD in mammalian cells. In some cells, Gal4-CtBP activated a UAS reporter, while the same reporter was repressed in other cell lines. Interestingly, conversion of CtBP's catalytic histidine to glutamine abolished transcriptional activation, but not repression. The heterologous nature of these experiments and the differences in the assays employed may explain the discrepancy between these studies, and further experiments will be needed on endogenous targets to determine how much dehydrogenase activity of CtBP contributes to repression and activation of Wnt targets (Fang, 2006).

Although CtBP is required for maximal activation of CG6234 expression and a Gal4-Arm-dependent reporter gene, Wg activation of nkd does not appear to require CtBP. The basis for this gene-specific requirement for CtBP is not clear. CtBP is recruited to the nkd WRE in a Wg-dependent manner, similar to what was observed for CG6234. It may be that CtBP is required for nkd activation, but this is masked by its role in repressing nkd expression. This hypothesis could be tested if were possible to separate CtBP's activator and repressor activities (Fang, 2006).

The requirement for CtBP in Wnt transcriptional activation may have been previously overlooked due to its well-characterized role as a co-repressor. For example, mouse embryos that lack CtBP2 have axial truncations and reduced Brachyury (T) expression that is reminiscent of Wnt3a mutants. These results suggest that the activating role for CtBP in Wnt signaling that was identified is evolutionarily conserved (Fang, 2006).

Mechanical deformations associated with embryonic morphogenetic movements have been suggested to actively participate in the signaling cascades regulating developmental gene expression. This paper develops an appropriate experimental approach to ascertain the existence and the physiological relevance of this phenomenon. By combining the use of magnetic tweezers with in vivo laser ablation, physiologically relevant deformations were locally control in wild-type Drosophila embryonic tissues. The deformations caused by germ band extension upregulate Twist expression in the stomodeal primordium. Stomodeal compression triggers Src42A-dependent nuclear translocation of Armadillo/beta-catenin, which is required for Twist mechanical induction in the stomodeum. Finally, stomodeal-specific RNAi-mediated silencing of Twist during compression impairs the differentiation of midgut cells, resulting in larval lethality. These experiments show that mechanically induced Twist upregulation in stomodeal cells is necessary for subsequent midgut differentiation (Desprat, 2008).

Demonstrating the role of mechanical deformations in the regulation of developmental gene expression requires an ability to reproduce endogenous deformations by locally controlling tissue deformations within the living embryo. Although tools for measuring and applying global forces had been previously reported for studying Xenopus embryo tissue explants, approaches for locally manipulating tissues within developing embryos were still lacking. In this study, magnetized cells were remotely manipulated to produce a 60 ± 20 nN force necessary to generate deformations similar to those produced endogenously. The magnitude of this force is smaller by a factor of ~20 than the 1 μN force associated with the convergent extension movements in Xenopus explants measured using the deflection of an optical fiber. This is consistent with the fact that the Xenopus embryo is 10 times larger that the Drosophila embryo. This value is also consistent with the 13 nN force developed by a 20 MDCK cell assembly on a soft micropillar surface, noting that the cell colony is five times smaller than the Drosophila embryo length. Importantly, both magnetic and external uncontrolled forces rescued mechano-sensitive Twist expression in the stomodeum. This indicates that Twist expression might not be highly sensitive to the intensity or symmetry of tissue deformations (Desprat, 2008).

The remote manipulation of magnetized cells in the Drosophila embryo enabled demonstration that mechanical compression of stomodeal cells comparable to those induced by endogenous morphogenetic movements upregulates Twist expression in the stomodeal primordium. Arm nuclear translocation is a major instructive step in the mechanical-to-genetic transduction pathway, coupling the macroscopic events of morphogenetic shape changes to the molecular processes regulating developmental gene expression. Moreover, previous studies showed that Src family kinases are involved in mechano-transduction through two distinct modes: either though direct mechanical activation resulting in phosphorylation of Src (Wang, 2005), or through a permissive mode where a mechanically induced conformational change in a Src substrate makes its phosphorylation site accessible to the already activated p-Src (Sawada, 2006). This study found that Src42A acts in the permissive mode in the mechano-transduction pathway upstream of Arm. Because β-catenin is a substrate of Src in mammalian cells, one might speculate that the mechano-sensitive substrate of p-Src42A in Drosophila embryos may be junctional Arm. Further study will be necessary to determine whether this is the case, or if an unknown mechano-sensitive Src42A substrate controls Arm activation (Desprat, 2008).

At later stages of development during organogenesis, mechanical cues generated by organ functions were also suggested to shape the physiological function of specialized organs. For instance, embryonic muscle activity is involved in mouse bone development through β-catenin activation. This study has found that endogenous morphogenetic movements at early stages of development are able to control gene expression, thus identifying a feedback loop of the embryo morphological development onto the genome. Such mechanical cues may mediate long-range effects that coordinate and synchronize differentiation events throughout the whole embryo. Such effects may be especially important under conditions in which dynamical and complex topology prevents the establishment of the long-range morphogen gradients that are efficient at earlier stages, when cells are arranged in simpler, static geometrical patterns (Desprat, 2008).

To form a gonad, germ cells (GCs) and somatic gonadal precursor cells (SGPs) must migrate to the correct location in the developing embryo and establish the cell-cell interactions necessary to create proper gonad architecture. During gonad morphogenesis, SGPs send out cellular extensions to ensheath the individual GCs and promote their development. Mutations have been identified in the raw gene that result in a failure of the SGPs to ensheath the GCs, leading to defects in GC development. Using genetic analysis and gene expression studies, it was found that Raw negatively regulates JNK signaling during gonad morphogenesis, and increased JNK signaling is sufficient to cause ensheathment defects. In particular, Raw functions upstream of the Drosophila Jun-related transcription factor to regulate its subcellular localization. Since JNK signaling regulates cell adhesion during the morphogenesis of many tissues, the relationship was examined between raw and the genes encoding Drosophila E-cadherin and beta-catenin, which function together in cell adhesion. It was found that loss of DE-cadherin strongly enhances the raw mutant gonad phenotype, while increasing DE-cadherin function rescues this phenotype. Further, loss of raw results in mislocalization of beta-catenin away from the cell surface. Therefore, cadherin-based cell adhesion, likely at the level of beta-catenin, is a primary mechanism by which Raw regulates germline-soma interaction (Jemc, 2012).

raw is an important regulator of embryonic gonad morphogenesis and the establishment of proper gonad architecture. raw mutants exhibit a failure of SGPs to ensheath GCs in the gonad, resulting in defects in GC development. It was also found that raw affects gonad morphogenesis primarily by acting as a negative regulator of the JNK signaling pathway. Finally, it was found that raw mutants exhibit defects in cadherin-based cell adhesion, and that this is the primary cause of the failure of gonad morphogenesis. These results have clear implications for understanding of how important cell signaling pathways are regulated to control normal organogenesis and may be misregulated to cause disease (Jemc, 2012).

Previously raw has been proposed to be a negative regulator of the JNK pathway during closure of the dorsal epidermis, based on changes in JNK-dependent expression of target genes such as dpp and puc. Indeed, an increase was seen in puc expression in the region of the embryonic gonad, and more broadly throughout the embryo. Further, upregulation of a dedicated AP-1 reporter construct was observed, indicating that the changes in target gene expression are directly due to changes in AP-1 transcriptional activity regulated by the JNK pathway. When the JNK pathway was upregulated via independent means, similar defects were observed in gonad morphogenesis, indicating that the changes in the JNK pathway were the primary mechanism by which raw mutants cause gonad defects. Therefore, the results support and extend the previous observations that raw acts as a negative regulator of JNK pathway, both in the gonad and in other tissues in which raw mutants exhibit defects in morphogenesis (Jemc, 2012).

How might raw be regulating the JNK pathway? The evidence indicates that raw regulates the JNK pathway at the level of transcription factor JRA. It was found that the nuclear localization of JRA, but not FOS, was altered in raw mutants. JRA was more strongly concentrated in the nucleus in a variety of cell types in raw mutants, whereas no changes were observed in the global levels of JRA protein. These observations are consistent with previous genetic epistasis experiments that indicated that raw acts at the level of JRA, rather than further upstream in the pathway. It has been proposed that raw acts as a general negative regulator of the JNK pathway to suppress basal activity and perhaps establish a threshold for pathway activation. The data are consistent with this hypothesis, as a general nuclear accumulation of JRA was seen in a variety of cell types in the embryo, along with generalized activation of the transcriptional reporter for AP-1 activity. Presumably, not all of these different cells are normally exposed to activators of the JNK pathway at this time, indicating that the pathway may be activated in cells in which the pathway would normally be turned off. Thus, rather than being just a modulator of the level of signal a cell might receive under conditions of JNK pathway activation, raw is likely also responsible for ensuring that the pathway remains inactive in cells that are not experiencing pathway activation (Jemc, 2012).

It is difficult to predict exactly how Raw may be regulating JNK signaling, as the Raw protein has no readily identifiable protein domains and exhibits only limited homology to proteins of other species. It may be the case that similar JNK pathway regulators are present in other species and have structural and/or functional conservation with Raw, but are difficult to identify based on primary sequence homology. Studies examining the subcellular localization of Raw in cultured mammalian or Drosophila cells indicate that it is primarily found in the cytoplasm. One attractive hypothesis is that Raw directly binds to JRA to block its nuclear translocation and sequester JRA in the cytoplasm. Unfortunately, efforts to identify a direct, physical interaction between Raw and JRA have so far been unsuccessful (Jemc, 2012).

The JNK pathway is subject to negative regulation at several levels. Most familiar are the MAP kinase phosphatases (MKPs, a subfamily of Dual-specificity phosphatases), like Drosophila Puckered, that provide negative feedback by dephosphorylating activated MAP kinases such as JNK. Additional modes of regulation include nuclear repressors of AP-1 target genes (e.g., Anterior open) and a secreted protease that acts in negative feedback on the JNK pathway (Scarface). Raw appears to represent a distinct mode of regulation, acting on the ability of JRA/JUN to translocate to the nucleus. Regulation of the subcellular localization of transcription factors and cofactors is a strategy that is commonly deployed to regulate signaling pathway activity, and many transcription factors are sequestered in the cytoplasm as a mechanism for negatively regulating their activity. It is proposed that JRA is subject to such regulation as a means to repress its activity in cells that are not experiencing sufficient levels of JNK pathway activity (Jemc, 2012).

Further studies of Raw are necessary to determine how Raw functions at a molecular level to regulate JRA subcellular localization (Jemc, 2012).

This study has found that raw mutants also exhibit defects in cadherin-based cell adhesion, which is known to be important for proper gonad morphogenesis and GC ensheathment by SGPs. Loss of DE-cad function exacerbates the gonad defects observed in raw mutants while increasing DE-cad function strikingly rescues these defects. It is likely that the increase in JNK pathway activity in raw mutants leads to defects in DE-cad-based adhesion and that this is the primary cause of the gonad morphogenesis defects. This is in contrast to the role of raw and the JNK pathway in the closure of the dorsal epidermis, which is largely thought to be due to regulation of dpp expression. Consistent with this, less up-regulation of dpp was observed in the region of the gonad, relative to the overall activation of the AP-1 transcriptional reporter (Jemc, 2012).

Previous studies in mammalian cells have implicated JNK signaling in negative regulation of cadherin-based cell adhesion, while in other contexts the JNK pathway has also been observed to upregulate DE-cad. The current results favor a repressive role for the JNK pathway on DE-cad in the gonad. It is also known that cadherins can act upstream of the JNK pathway, and that loss of cadherin can lead to an increase in c-Jun protein levels. However, the current results are consistent with DE-cad acting downstream of the JNK pathway, since DE-cadherin expression could rescue gonad morphogenesis independently of rescuing JRA localization. It is concluded that during gonad morphogenesis, raw acts as a negative regulator of the JNK pathway, and increased JNK pathway activity observed in raw mutants leads to a downregulation of DE-cadherin based cell adhesion and a failure of proper ensheathment of the GCs by the somatic gonad (Jemc, 2012).

While no change was observed in DE-cad localization in the gonad, the localization of ARM/ß-catenin was dramatically altered. Since ARM is essential for proper DE-cad function in cell adhesion, this indicates that DE-cadherin-based adhesion is strongly affected in raw mutants. It has been shown that JNK can directly phosphorylate ß-catenin and negatively regulate its activity. Consistent with this, a modest increase was observed in the relevant phospho-form of ARM/ß-catenin in raw mutants. Thus, this may represent one aspect of how the JNK pathway regulates DE-cad based adhesion in the gonad. However, the change in ARM/ß-catenin phosphorylation observed is unlikely to account for the more dramatic change in ARM/ß-catenin immunostaining observed in the gonad. Considering that a strong increase was also observed in transcriptional activation by AP-1 in raw mutants, and that mutations in the JRA transcription factor can partially suppress the gonad morphogenesis defects observed in raw mutants, it is concluded that at least some of the JNK pathway effect on DE-cad function and ß-catenin localization is likely to depend on changes in gene expression mediated by AP-1. Since no overall changes were observed in protein levels for DE-cad or ARM/ß-catenin in raw mutants, the changes in gene expression may reflect changes in other regulators of DE-cad based cell adhesion. Interestingly, previous work identified a zinc transporter, Fear of intimacy, that also affects gonad morphogenesis and GC ensheathment by regulating DE-cad. Regardless of whether there is an interesting connection between zinc transport and the JNK pathway, or these represent independent pathways, they highlight the importance of careful regulation of cadherin-based cell adhesion in controlling morphogenesis (Jemc, 2012).

Previous work has indicated that GC ensheathment requires preferential adhesion between SGPs and GCs, such that SGP-GC adhesion is favored over GC-GC or SGP-SGP adhesion. Indeed, just increasing the adhesion between GCs via DE-cadherin expression in these cells is sufficient to prevent ensheathment of the GCs by SGPs. In raw mutants, changes were primarily observed in the JNK pathway in SGPs and surrounding somatic cells. In addition, expression of DE-cad in the soma, but not the germline, is sufficient to rescue the ensheathment defects in raw mutants. Together, these data indicate that raw mutants likely affect gonad ensheathment by decreasing DE-cad function in the SGPs, which decreases SGP-GC adhesion relative to GC-GC adhesion. While it is possible that effects of raw on somatic cells outside of the gonad affect ensheathment within the gonad, it is less easy to imagine how decreasing DE-cad activity in these cells would influence ensheathment (Jemc, 2012).

The JNK pathway has been implicated in many diseases, including birth defects, neurodegeneration, inflammatory diseases, and cancer. Signaling pathways must be tightly regulated both positively, to ensure rapid and robust signaling responses, and negatively, to terminate signaling events and prevent inappropriate signaling. As a negative regulator of JNK pathway signaling, raw represents the type of gene that might be mutated or misregulated in diseases caused by altered JNK pathway activity. This idea is supported by the strong developmental phenotypes associated with mutations in negative regulators of the JNK pathway in Drosophila and mice (Jemc, 2012).

One disease where the JNK pathway has been particularly well studied is cancer. The JNK pathway's role in cancer is complex, however, and the pathway can act in tumor suppression or oncogenesis, depending on the context. In mouse and Drosophila models of cancer due to activated Ras, upregulation of the JNK pathway is required for tumor formation and disease progression. Interestingly, downregulation of E-cadherin is also associated with cancer progression, including in the models of activated Ras where the JNK pathway is involved. Thus, a similar link between the JNK pathway and cadherin regulation that was observed in morphogenesis of the gonad during development may play a role in oncogenesis. Since upregulation of the JNK pathway promotes cancer in these examples, negative regulators of the pathway such as the MAPK phosphatases or Raw would act as tumor suppressors whose mutation could contribute to disease progression. A better understanding of how the JNK pathway is regulated, and how Raw contributes to this regulation, is essential for understanding the normal roles of the JNK pathway in development and homeostasis, and how it is misregulated to cause disease (Jemc, 2012).