Transcriptional Regulation

The gene spalt is expressed in the embryonic central nervous system of Drosophila but its function in this tissue is still unknown. To investigate this question, a combination of techniques was used to analyse spalt mutant embryos. Electron microscopy shows that in the absence of Spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy has revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments have each confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the ß-catenin Armadillo, its binding partner N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organization of the central nervous system (Cantera, 2002).

A possible interpretation of sal phenotype would be that components of cell adhesion are seriously compromised in the CNS of sal embryos during early stage 16. To test this hypothesis specific antibodies and laser confocal microscopy were used to survey the expression of molecules known to be important for cell adhesion in embryonic CNS at early stage 16. All the markers are detectably expressed in Df(2L)32FP-5;sal445 mutant embryos at both stages, and their spatial patterns of expression in the CNS are normal, showing that sal is not essential for any of these proteins to be expressed. However, the quantification of fluorescence intensity revealed that most markers were present in abnormally high or low levels. In transheterozygous Df(2L)32FP-5;sal445 mutants at early stage 16, when the strong transmission electron microscopy TEM phenotype is manifest, lower fluorescence levels were measured for Armadillo, N-Cadherin, Neuroglian, Fasciclin 2 and Fasciclin 3; higher fluorescence levels were measured for Notch; and levels similar to wild type for Neurotactin, Neurexin IV and Faint Sausage. Comparison between wild-type, heterozygous and null sal mutant embryos revealed a stepwise decrease in the fluorescence levels for Armadillo and N-Cadherin, indicating that the effect of the mutation is dominant (Cantera, 2002).

Fluorescence levels were measured at the stage when the TEM phenotype is reverted (stage 17). The wild-type fluorescence for the three markers studied in this regard (Armadillo, Fasciclin 2, Neuroglian) changes between early stage 16 and stage 17, indicating that during this short developmental interval the levels of cell adhesion proteins are regulated. Relative to these new wild-type levels, the three proteins that are not affected during the expression of the TEM phenotype (Neurotactin, Neurexin IV and Faint Sausage) remain normal in the mutant. The levels of Notch switch from abnormally high to slightly lower than normal. All other markers still exhibit lower-than-normal fluorescence levels, with the exception of N-Cadherin, which exhibits a partial recovery. Taken together, these data led to the conclusions that the expression of sal is necessary to maintain correct dynamic levels of several adhesion molecules in the CNS and that sal exerts this function in a persistent and dominant fashion (Cantera, 2002).

The rapid recovery of sal CNS during the course of stage 16 could be explained by the robustness inherent to a system in which adhesion is mediated by a combination of proteins and the possible compensatory effect mediated by upregulation of other members of the system. However, an alternative view is proposed. The ultrastructural recovery may as well reflect the normal dynamics of combinations of adhesion proteins expressed successively along embryonic development. From this point of view, the rapid recovery from the adhesion phenotype will reflect the normal transition between two particular combinations of adhesion proteins expressed at early or late stage 16. For this to be valid, the expression levels of several adhesion proteins must change along this interval during normal development. Interestingly, the data do support this possibility, since the fluorescence levels for Armadillo, Fasciclin 2 and Neuroglian change between stages 16 and 17 in wild-type CNS. Whether sal is required for the regulation of a combination of cell adhesion and cytoskeletal proteins at a particular developmental stage could be tested by deleting the expression of Sal exclusively in CNS tissue within short developmental intervals. This approach could now be possible using techniques based on combinations of the GAL4-UAS system and RNA interference (Cantera, 2002).

Targets of Activity

Morphogenetic movements are closely regulated by the expression of developmental genes. This study examines whether developmental gene expression can in turn be mechanically regulated by morphogenetic movements. The effects of mechanical stress were examined on the expression of Twist, which is normally expressed only in the most ventral cells of the cellular blastoderm embryo under the control of the Dorsal morphogen gradient. At embryogenesis gastrulation (stage 7), Twist is also expressed in the anterior foregut and stomodeal primordia. Submitting the early Drosophila embryo to a transient 10% uniaxial lateral deformation induces the ectopic expression of Twist around the entire dorsal-ventral axis and results in the ventralization of the embryo. This induction is independent of the Dorsal gradient and is triggered by mechanically induced Armadillo nuclear translocation. Twist is not expressed in the anterior foregut and stomodeal primordia at stage 7 in mutants that block the morphogenetic movement of germ-band extension. The mutants can be rescued with gentle compression of these cells, the stomodeal-cell compression normally caused by the germ-band extension is interpretated as inducing the expression of Twist. Correspondingly, laser ablation of dorsal cells in wild-type embryos relaxes stomodeal cell compression and reduces Twist expression in the stomodeal primordium. The induction of Twist in these cells depends on the nuclear translocation of Armadillo. It is proposed that anterior-gut formation is mechanically induced by the movement of germ-band extension through the induction of Twist expression in stomodeal cells (Farge, 2003).

Therefore, lateral compression of the early embryo induces the ectopic expression of Twist around the entire dorsal-ventral axis and results in the ventralization of the embryo. Despite the probable variations in the direction and amplitude of the deformation of each cell as a function of its location in the embryo, all cells respond to this stress. This suggests that their transcriptional response is triggered by deformation per se and does not depend on the exact geometry and amplitude of the mechanical strain applied to each cell. However, it is unclear how the forces required to artificially deform the embryo lead to embryonic epithelium stresses and strains that are related to endogenous forces and deformations present in the embryo during development (Farge, 2003).

Importantly, the mechanical induction of Twist is independent of the maternal determinants of dorsal-ventral polarity. Instead, this induction depends on the nuclear translocation of Armadillo and its ability to activate transcription. The mechanism that triggers the nuclear translocation of Armadillo in response to mechanical stress is unknown. One possibility is that mechanical strain activates a noncanonical Wingless transduction pathway, which releases the cytoplasmic pool of Armadillo from Axin and allows it to enter the nucleus. Alternatively, mechanical strain might trigger the release and nuclear localization of the pool of Armadillo that is associated with Cadherin at the zonula-adherens. Indeed, this might provide a reason for dual function of Armadillo as an essential component of Cadherin adhesion complex and as a transcription factor (Farge, 2003).

It is interesting to note that the Armadillo homolog, beta-catenin, translocates into the nuclei at the dorsal pole of early frog and fish embryos, where it plays a role in determining dorsal-ventral polarity. Furthermore, the ectopic nuclear localization of beta-catenin induces the dorsalization of vertebrate embryos. Because the dorsal-ventral axis of invertebrates is inverted with respect to that of vertebrates, this corresponds well with the ventralization observed in Drosophila embryos upon the mechanical induction of Armadillo nuclear localization. Thus, mechanical compression may reactivate a conserved and ancient pathway for dorsal-ventral axis formation (Farge, 2003).

The results presented in this study suggest that the expression of Twist in foregut and stomodeal-primordia cells at the onset of gastrulation is mechanically induced by the compression caused by germ-band extension and that this is also mediated by the nuclear translocation of Armadillo. twist is involved in the differentiation and the formation of both the foregut and the anterior midgut. Interestingly, neither the anterior midgut nor the stomodeum invaginate in embryos that lack the mechanical compression and do not express Twist when epithelial dorsal cells have been photo-ablated. It is proposed that, through mechanical induction of twist, the anterior-gut formation is induced by stomodeal cell compression in response to germ-band extension (Farge, 2003).

In addition to its role in dorso-ventral axis formation, Armadillo is thought to induce the differentiation and invagination of the meso-endoderm cells that give rise to the gut in other vertebrate and nonvertebrate embryos. Although the maternal signals that induce the nuclear translocation of beta-catenin in zebrafish and Sea Urchins are not known, they have been shown to be independent of the classical determinant Wingless. These results in Drosophila raise the possibility that the nuclear translocation of Armadillo/beta-catenin in the gut primordia of these embryos might be mechanically induced by morphogenetic movements that are homologous to germ-band extension. Indeed, the nuclear translocation of beta-catenin and the formation of the meso-endodermal gut invagination/involution are concomitant with convergent extension, which tends to compress the meso-endoderm cells (Farge, 2003).

These parallels led to the speculation that mechanical induction may be an ancient mechanism for inducing gut formation. This could have evolved from a primitive reflex response to mechanical deformation. Such a response might have been the phagocytosis of particles in response to physical contact, which has been proposed to be the 'feeding-response' of the earliest organisms. The generation of a permanent gut might have then been stabilized by the Armadillo-induced expression of meso-endodermal genes in response to genetically controlled endogenous morphogenetic movements, such as cell intercalation generating convergent extension. These experiments may have reactivated the genetic pathway of such 'fossil sensorial behavior' in early Drosophila embryos (Farge, 2003).

Regulation of Armadillo Protein Stability

Members of the Hedgehog (Hh) and Wnt/Wingless (Wg) families of secreted proteins control many aspects of growth and patterning during animal development. Hh signal transduction leads to increased stability of the transcription factor Cubitus interruptus (Ci), whereas Wg signal transduction causes increased stability of Armadillo (Arm/beta-catenin), a possible co-factor for the transcriptional regulator Lef1/TCF. A new gene, slimb (for supernumerary limbs), is described which negatively regulates both of these signal transduction pathways. Loss of slimb function results in a cell-autonomous accumulation of high levels of both Ci and Arm, and the ectopic expression of both Hh- and Wg- responsive genes. Clones of slimb1 cells in the leg or wing disc ectopically express dpp or wg when they arise in the anterior (but not the posterior) compartments of these discs. Anterior clones reorganize normal limb pattern, creating supernumerary 'double-anterior' limbs. Slimb, like PKA, is a negative regulator that normally prevents activity of the Hh signal transduction pathway in the absence of ligand. slimb mutant cells that arise in the presumptive wing blade ectopically express Scute and differentiate ectopic sensory bristles instead of epidermal hairs on the surface of the wing blade. Both phenotypes are strictly autonomous to the mutant cells, as is the case when the Wg signal transduction pathway is constitutively activated, but not when Wg is ectopically expressed. The slimb gene encodes a conserved F-box/WD40-repeat protein related to Cdc4p, a protein in budding yeast that targets cell-cycle regulators for degradation by the ubiquitin/proteasome pathway. It is proposed that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression, at least in part, by inducing changes in Ci and Arm, which protect both Ci and Arm from Slimb-mediated proteolysis (Jiang, 1998).

The slimb phenotype differs from those of all previously known genes, in that it is the first gene found to deregulate both wg and dpp expression in the D/V axis. Disrupting components of the Hh signaling pathway deregulate wg and dpp only along the A/P axis. Thus, the control of wg and dpp expression in the D/V axis is not disrupted by disruption of the Hh pathway. The mechanism restricting wg and dpp in the D/V axis is not known. The mutant phenotype of slimb- clones in discs provides the first evidence that wg and dpp expression in the D/V axis is actively regulated during imaginal disc development, and is not solely defined during embryonic development. Since the Hh pathway regulates wg and dpp expression in the A/P axis, these results suggest that a pathway different from Hh may operate in imaginal discs to restrict their expression in the D/V axis. This pathway cannot be either the Wg or Dpp signaling pathway since inactivation of Wg or Dpp signaling is known to affect either dpp or wg expression, but not both. The slimb phenotypes described here were not observed in the previous study which used weak slimb alleles and revealed only A/P defects (Jiang, 1998). Jiang proposed that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression, at least in part, by inducing changes in Ci and Arm, which protect both Ci and Arm from Slimb-mediated proteolysis. The phenotypic differences probably reflect the fact that a null allele was used in the current study instead of hypermorphic alleles. In addition to D/V defects, slimb mutant clones also deregulate wg and dpp expression in the A/P axis. slimb is the first identified gene that regulates both wg and dpp expression in the A/P as well as D/V axes (Theodosiou, 1998).

Armadillo's role in signal transduction is normally negatively regulated by Shaggy/Zeste-white 3 kinase, which modulates Armadillo protein stability. Two sequences in the N-terminal domain of Armadillo are involved in its degradation. One is a consensus Shaggy/Zeste-white 3 phosphorylation site. The other is a sequence conserved between IkappaB and its fly homolog, cactus, surrounding the serines whose phosphorylation is thought to regulated ubiquitinization and control of protein stability. A mutant protein, Armadillo(S10), was generated with a 54 amino acid deletion in its N-terminal domain. Most of the wild-type Arm protein in an embryo is in adherens junctions, where it is highly phosphorylated; there is relatively little soluble Arm, which is less highly phosphorylated. In contrast, the least highly phosphorylated isoforms of ArmS10 predominate, resembling the pattern of accumulation of wild-type Arm in shaggy mutants. ArmS10 is constitutively active in Wingless signaling; its activity is independent of both Wingless signal and endogenous wild-type Armadillo. Armadillo(S10) is more stable than wild-type Armadillo, suggesting that it is less rapidly targeted for degradation. Armadillo(S10) is more stable and has escaped from negative regulation by Zeste white-3 kinase, and thus accumulates outside junctions even in the absence of Wingless signal. ArmS10 retains the Arm function in junctions even though it is constitutively active for Wg signaling. This suggests that the two Arm functions, the response to Wg signaling and acting as a structural protein in junctions, are independent. Even though overall levels of Arm phosphorylation are low in shaggy/zw3 mutants because the less phosphorylated isoform accumulates outside junctions, junctional Arm remains highly phosphorylated. It is concluded that kinases in addition to Zeste white-3 are implicated in Armadillo phosphorylation. Two models are discussed for the negative regulation of Armadillo in normal development. In one, the simple model, Shaggy/Zw3 negatively regulates Arm by direct phosphorylation within the N-terminus. Another model is suggested by the observation that other kinases besides Shaggy target Arm. An alternative direct target of Shaggy/Zw3 is the tumor suppressor APC, which is readily phosphorylated by GSK. This phosphorylation regulates APC binding to beta-catenin, reducing beta-catenin stability. In this model, Shaggy is not required for phosphorylation of Arm in adherens junctions, suggesting that this phosphorylation is mediated by other kinases. The effect of Zw3 inactivation on Arm phosporylation may be solely due to its effects on the stability of soluble Arm (Pai, 1997).

shotgun transcription level is regulated through the Wingless pathway. 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 Shotgun, which binds to Arm, three proteins (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 Shotgun 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 Shotgun protein levels, suggesting that Dsh-induced Shotgun elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative Zw-3-, and truncated Arm-induced accumulation of Shotgun protein is accompanied by a marked increase in the steady-state levels of Shotgun mRNA, suggesting that transcription of shotgun is activated by Wg signaling. In addition, overexpression of shotgun elevates Arm levels by stabilizing Arm at cell-cell junctions (Yanagawa, 1997).

Wnt signaling regulates ß-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by ß-catenin and the other by Jun kinase (JNK). A Dsh-associated kinase has been purified from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the ß-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signaling through ß-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the ß-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-ß-catenin signaling, indicating a link between two important developmental pathways (Sun, 2001).

To examine whether PAR-1 is required in the Wnt pathway, endogenous PAR-1 activity was suppressed by expressing a kinase-negative PAR-1 (hPAR-1Balpha KN). Chinese hamster ovary (CHO) cells were used because good expression from transfected DNA can be achieved and these cells have a well-characterized response to Wnt. Three hallmarks of Wnt activity were measured: Dsh phosphorylation, ß-catenin stabilization and transcriptional activation. Wnt treatment of CHO cells retards the mobility of Dvl proteins (mammalian homologs of Dsh) on SDS-PAGE, and phosphatase treatment increases the mobility of the Dvl band, thereby confirming that Dvl is phosphorylated in response to Wnt. The hPAR-1Balpha KN suppresses Wnt-mediated phosphorylation of endogenous Dvl proteins, as shown by the reduced amount of a retarded Dvl band. This result is consistent with the data that PAR-1 phosphorylates Dsh in vitro and in cells. Furthermore, both human and Drosophila PAR-1 KN strongly suppress Wnt-induced ß-catenin stabilization. The kinase-negative forms of hPAR-1A, hPAR-1B and hPAR-1C all strongly suppress Wnt-mediated transcriptional activation (measured by LEF1/TCF reporters) in a dose-dependent manner. Importantly, co-expression of wild-type hPAR1 can override the suppression mediated by hPAR-1 KN, indicating that hPAR-1 KN affects Wnt signaling by specifically blocking the effects of endogenous PAR-1 in cells. However, hPAR-1 KN is unable to inhibit the transcriptional activation induced by overexpression of ß-catenin, consistent with PAR-1's role in regulation of Dsh function upstream of ß-catenin (Sun, 2001).

All three human PAR-1 homologs strongly potentiated the responses to Wnt or Dvl3 in CHO cells. The hPAR-1 proteins alone do not activate the signaling pathway but require co-expression of either Wnt or Dvl, indicating that there is synergy between hPAR-1 and other components of the Wnt pathway. The specificity of these responses was verified by their dependency on the co-expression of the LEF1 transcription factor, which is required for Wnt signaling. Furthermore, the effects of hPAR-1 were suppressed by Axin, a negative regulator of the Wnt pathway that acts downstream of Dsh. As predicted, hPAR-1 overexpression does not alter the gene response induced by overexpression of ß-catenin, consistent with the idea that PAR-1 regulates Wnt signaling at a step upstream of Axin and ß-catenin (Sun, 2001).

Wnt regulation of ß-catenin degradation is essential for development and carcinogenesis. ß-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adenomatous polyposis coli (APC). Another Axin-associated kinase is described, whose phosphorylation of ß-catenin precedes and is required for subsequent GSK-3 phosphorylation of ß-catenin. This 'priming' kinase is casein kinase Ialpha (CKIalpha). CKIalpha phosphorylation of ß-catenin precedes and is obligatory for subsequent GSK-3 phosphorylation of ß-catenin. Depletion of CKIalpha inhibits ß-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/ß-catenin signaling. This study uncovers distinct roles and steps of ß-catenin phosphorylation, and identifies CKIalpha as a component in Wnt/ß-catenin signaling (Liu, 2002).

The level of cytosolic ß-catenin determines the activation of Wnt responsive genes. Without Wnt stimulation, ß-catenin is constantly degraded by the proteosome. This degradation strictly depends upon ß-catenin phosphorylation, which occurs in a multiprotein complex composed of the following tumor suppressor proteins: adnomatous polyposis coli (APC), Axin, and glycogen synthase kinase-3 (GSK-3). It is believed that in this complex assembled by Axin, GSK-3 phosphorylates the ß-catenin amino-terminal region, thereby earmarking ß-catenin for ubiquitination-dependent proteolysis. Wnt signaling is suggested to inhibit ß-catenin phosphorylation, thus inducing the accumulation of cytosolic ß-catenin, which associates with the TCF/LEF (T cell factor/lymphocyte enhancer factor) family of transcription factors to activate Wnt/ß-catenin-responsive genes. Thus, ß-catenin phosphorylation controls ß-catenin protein level and Wnt signaling (Liu, 2002 and references therein).

Four serine (S)/threonine (T) residues (S33, S37, T41, and S45) at the amino-terminal region of ß-catenin are conserved from Drosophila to human and conform to the consensus GSK-3 phosphorylation site. Indeed, ß-catenin can be phosphorylated by GSK-3 in vitro, and these phospho-S/T residues are critical for ß-catenin recognition by the F box protein ß-Trcp (homolog of Drosophila Slimb), which is the specificity component of a ubiquitination apparatus. The significance of S33, S37, T41, and S45 phosphorylation in ß-catenin degradation is underscored by the observation that mutations at these S/T residues frequently occur in human colorectal cancer and several other malignancies, which are associated with and most likely caused by deregulated accumulation of ß-catenin (Liu, 2002 and references therein).

Whether CKIalpha regulates degradation of Armadillo (Arm), the Drosophila ortholog of ß-catenin, was investigated. Strikingly, RNAi depletion of Drosophila CKIalpha results in a dramatic increase of Arm protein in S2 cells. Furthermore, RNAi depletion of CKIalpha in Drosophila embryos generates a naked cuticle phenotype and a strong expansion of the expression domain of Wingless, which itself is an Arm target gene. This is reminiscent of the phenotype caused by loss-of-function mutations in Drosophila Axin or GSK-3 (zeste-white 3/shaggy) gene. Therefore, the Arm protein accumulation in S2 cells and the segment polarity phenotype in embryos resulting from CKIalpha RNAi together suggest that CKIalpha function is conserved and essential for ß-catenin degradation in both Drosophila and human (Liu, 2002).

Thus ß-catenin phosphorylation in vivo is sequentially carried out by two distinct kinases, CKIalpha and GSK-3. CKIalpha phosphorylation of S45 proceeds and is required for subsequent GSK-3 phosphorylation of T41, S37, and S33. These findings identify CKIalpha as an essential component that controls ß-catenin phosphorylation degradation. This understanding of ß-catenin phosphorylation at a single-residue resolution enables an examination of how ß-catenin mutations found in human cancers disrupt distinct steps in ß-catenin degradation. Thus, mutations surrounding S33 and S37 abolish ß-catenin recognition by ß-Trcp and the ubiquitination of ß-catenin; mutations at T41 prevent GSK-3 phosphorylation of S37 and S33 and thus ß-Trcp recognition; and mutations at S45 block the priming phosphorylation by CKIalpha and consequently all phosphorylation events by GSK-3. Each of these mutations causes ß-catenin to escape recognition by ß-Trcp and subsequent degradation (Liu, 2002).

CKIalpha was among the first protein kinase activities to be discovered, yet its function and regulation remain poorly understood. Like GSK-3, CKIalpha is expressed ubiquitously and appears to be constitutively active, consistent with its role in ß-catenin degradation. The finding that ß-catenin is a CKIalpha substrate in vivo therefore identifies CKIalpha as a central player in cell fate determination and growth control. This study shows that CKIalpha controls segment polarity during Drosophila embryogenesis. Interestingly, ß-catenin phosphorylation by CKIalpha and by GSK-3 are both stimulated by Axin. In fact, CKIalpha and GSK-3 bind to different regions of Axin such that they 'sandwich' ß-catenin in the Axin complex, thereby promoting effective ß-catenin phosphorylation. Since Wnt signaling inhibits only GSK-3 but not CKIalpha phosphorylation of ß-catenin, CKIalpha may represent a node at which other signaling pathways regulate ß-catenin protein level. Since depletion of CKIalpha causes ß-catenin accumulation in a manner similar to a lack of function of GSK-3, APC, or Axin, CKIalpha is a candidate tumor suppressor (Liu, 2002).

Inactivation of the Adenomatous Polyposis Coli tumor suppressor triggers the development of most colorectal carcinomas. APC is required for targeted degradation of ß-catenin, the central transcriptional activator in the Wnt/Wingless (Wg) signal transduction pathway; however, the precise biochemical functions of APC remain uncertain. The two Drosophila homologs of APC (Apc1 and Apc2) appear to have predominantly different tissue distributions, different subcellular localizations and mutually exclusive phenotypes upon inactivation. Unexpectedly, despite these differences, simultaneous reduction in both Drosophila Apc proteins results in the global nuclear accumulation of ß-catenin and the constitutive activation of Wg transduction throughout development. This redundancy extends even to functions previously thought to be specific to the individual Apc homologs. Together, these results reveal that the combined activity of Apc1 and Apc2 allows a tight regulation of transcriptional activation by ß-catenin and suggest that APC proteins are required for the regulation of Wnt transduction in all cells (Ahmed, 2002).

The in vivo analyses of loss-of-function mutations in the two Drosophila homologs of Apc have been crucial in providing conclusive evidence that transcriptional transactivation by ß-catenin can in fact be negatively regulated by APC. However, previous studies using loss-of-function mutations in either of the two Drosophila Apc genes have failed to establish an absolute requirement for Apc in regulating Wg signaling throughout development, since many Wg transduction events proceed normally, particularly during post-embryonic stages. These findings raised questions as to whether Apc is required to prevent the constitutive activation of Wg transduction in only a subset of cells, and whether Apc function could be compensated for by other mechanisms elsewhere. Simultaneously reducing the activities of both Drosophila Apc proteins is reported in this study. An absolute requirement is found for Apc proteins in preventing the constitutive activation of Wg signaling in many epithelial cells throughout development. In those limited situations for which the inactivation of one of the two Drosophila Apc proteins does lead to hyperactivation of transcriptional activation by Arm, the other Apc protein can functionally substitute if provided in sufficient quantity. This result argues against a specific function for either Apc protein in regulating Wg transduction (Ahmed, 2002).

Wnt signaling causes changes in gene transcription that are pivotal for normal and malignant development. A key effector of the canonical Wnt pathway is ß-catenin, or Drosophila Armadillo. In the absence of Wnt ligand, ß-catenin is phosphorylated by the Axin complex, which earmarks it for rapid degradation by the ubiquitin system. Axin acts as a scaffold in this complex, to assemble ß-catenin substrate and kinases (casein kinase I [CKI] and glycogen synthase kinase 3ß [GSK3]). The Adenomatous polyposis coli (APC) tumor suppressor also binds to the Axin complex, thereby promoting the degradation of ß-catenin. In Wnt signaling, this complex is inhibited; as a consequence, ß-catenin accumulates and binds to TCF proteins to stimulate the transcription of Wnt target genes. Wnt-induced inhibition of the Axin complex depends on Dishevelled (Dsh), a cytoplasmic protein that can bind to Axin, but the mechanism of this inhibition is not understood. This study shows that Wingless signaling causes a striking relocation of Drosophila Axin from the cytoplasm to the plasma membrane. This relocation depends on Dsh. It may permit the subsequent inactivation of the Axin complex by Wingless signaling (Cliffe, 2003).

Membrane bound forms of activated Armadillo ('Arm*', i.e., forms lacking their N termini) show significantly more signaling activity than Arm* without a membrane-targeting domain; this finding led to the suggestion that Armadillo exerts its signaling function in the cytoplasm rather than in the nucleus. However, overexpression of membrane-targeted Arm* causes a dramatic relocation of Axin-GFP, and of E-APC, to the plasma membrane throughout the embryonic epidermis, presumably by direct binding. This mimics the Wingless-induced membrane relocation of Axin-GFP, except that the membrane-targeted Arm* relocates Axin-GFP and E-APC to the entire lateral membrane where it itself is localized. No such relocation is seen under conditions of ubiquitous high levels of untargeted Arm*. The striking relocation of Axin-GFP to the plasma membrane by the membrane-targeted Arm* may cause its inactivation even in cells that are only weakly stimulated by Wingless; thus, this finding provides an alternative explanation for the increased activity of membrane bound Armadillo (Cliffe, 2003).

This work provides evidence that the assembly of Axin complex in the cytoplasm depends on a membrane-targeting function of E-APC. This function may also affect targeting to internal membranes, or vesicles, suggesting that the Axin complex may be associated with vesicles. In support of this, overexpressed Axin is associated with vesicles in Xenopus embryos. Furthermore, Dsh (which is required for the Wingless-induced membrane relocation of Axin) is also associated with vesicles, and to some extent with the plasma membrane, in vertebrate and Drosophila cells. Indeed, Axin and Dsh colocalize after overexpression in vertebrate cells. Notably, the DIX domain of the mammalian Dsh protein Dvl-2 contains a phospholipid binding motif that is conserved in the DIX domain of Axin, and targeting of Dvl-2 to vesicles by this motif is essential for its function in controlling the degradation of β-catenin (Cliffe, 2003).

Therefore, a possible model is that the Axin complex and Dsh are associated with the same vesicles, which may be recycling endocytic vesicles. Dsh may target these vesicles constitutively to the plasma membrane, where the Axin complex can interact potentially with Wnt receptors. This complex may be retained at the plasma membrane as a result of a Wnt-induced interaction between Axin and LRP/Arrow, and this retention may allow its subsequent inactivation. It is noted that LRPs are thought to recycle to the plasma membrane through endocytic vesicles, like their rapidly recycling LDL receptor relative. Recycling vesicles may thus provide a platform for APC-mediated assembly of the Axin complex and may convey this complex to the plasma membrane for inactivation by Wnt receptors (Cliffe, 2003).

Regulation of cell adhesion in the Drosophila embryo by phosphorylation of the Cadherin-Catenin-complex

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).

C-terminal-binding protein directly activates and represses Wnt transcriptional targets in Drosophila: CtBP and Arm function

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).

Tissue deformation modulates twist expression to determine anterior midgut differentiation in Drosophila embryos: Stomodeal compression triggers Src42A-dependent nuclear translocation of Armadillo/beta-catenin

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).

raw Functions through JNK signaling and cadherin-based adhesion to regulate Drosophila gonad morphogenesis

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).

armadillo continued: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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