InteractiveFly: GeneBrief

dachs: Biological Overview | References

Gene name - dachs

Synonyms -

Cytological map position - 29D1-29D1

Function - F-Actin-binding scaffolding protein

Keywords - Tissue polarity, Fat signaling pathway, gene expression, cell affinity and growth in imaginal discs

Symbol - d

FlyBase ID: FBgn0262029

Genetic map position - 2L:8,482,484..8,488,671 [+]

Classification - Myosin motor domain

Cellular location - cytoplasmic

NCBI link: EntrezGene

dachs orthologs: Biolitmine
Recent literature
Vrabioiu, A. M. and Struhl, G. (2015). Fat/Dachsous signaling promotes Drosophila wing growth by regulating the conformational state of the NDR kinase Warts. Dev Cell 35: 737-749. PubMed ID: 26702832

Nuclear Dbf2-related (NDR) kinases play a central role in limiting growth in most animals. Signals that promote growth do so in part by suppressing the activation of NDR kinases by STE20/Hippo kinases. This study identified another mechanism for downregulating NDR kinase activity. Specifically, activity of the Drosophila NDR kinase Warts in the developing wing was shown to depend on its transition from an inactive, 'closed' conformation to a potentially active, 'open' conformation mediated by Mats, a conserved Mps1-binder (Mob) protein. Further, this study shows that signaling interactions between the protocadherins Fat and Dachsous, organized by the morphogens Wingless and Decapentaplegic, suppress Warts by acting via the atypical myosin Dachs to inhibit or reverse this transition. The regulation of Warts conformation by Mats, Fat/Dachsous signaling, and Dachs appears independent of Warts phosphorylation by Hippo kinase, establishing a precedent for the control of NDR kinases, and hence growth, by distinct allosteric and phosphorylation mechanisms (Vrabioiu, 2015).

Misra, J. R. and Irvine, K. D. (2019). Early girl is a novel component of the Fat signaling pathway. PLoS Genet 15(1): e1007955. PubMed ID: 30699121
The Drosophila protocadherins Dachsous and Fat regulate growth and tissue polarity by modulating the levels, membrane localization and polarity of the atypical myosin Dachs. Localization to the apical junctional membrane is critical for Dachs function, and the adapter protein Vamana/Dlish and palmitoyl transferase Approximated are required for Dachs membrane localization. However, how Dachs levels are regulated is poorly understood. This study identified the early girl gene as playing an essential role in Fat signaling by limiting the levels of Dachs protein. early girl mutants display overgrowth of the wings and reduced cross vein spacing, hallmark features of mutations affecting Fat signaling. Genetic experiments reveal that it functions in parallel with Fat to regulate Dachs. early girl encodes an E3 ubiquitin ligase, physically interacts with Dachs, and regulates its protein stability. Concomitant loss of early girl and approximated results in accumulation of Dachs and Vamana in cytoplasmic punctae, suggesting that it also regulates their trafficking to the apical membrane. These findings establish a crucial role for early girl in Fat signaling, involving regulation of Dachs and Vamana, two key downstream effectors of this pathway.


The dachs gene was first identified almost a century ago based on its requirements for appendage growth. This paper describes the phenotypes of strong dachs mutations, reports the cloning of the dachs gene, characterizes the localization of Dachs protein, and investigates the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in Imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. These results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development (Mao, 2006).

The observation that a hypomorphic mutation of dachs could suppress the effects of fat mutations on wing growth and Wg expression in the proximal wing has led to the suggestion that dachs might act as a downstream component of a Fat signaling pathway. This study provides two types of evidence that confirm this suggestion. First, dachs is epistatic to fat for multiple phenotypes in multiple tissues, including gene expression, growth and cell affinity. Indeed, with the notable exception of the influence of fat on tissue polarity, all known fat mutant phenotypes are completely suppressed by mutation of dachs. Second, it was found that expression of regulators of Fat, Fj and Ds, or of Fat itself, influence the localization or stability of Dachs protein at the membrane, thus providing a molecular link from Fat to Dachs (Mao, 2006).

The predicted structure of Dachs is unique within the myosin superfamily, and places Dachs in a new class of unconventional myosins. It has most similarity to myosins V, VII, and X. This is intriguing, as a mammalian protocadherin, Cdh23, has been functionally linked to myosin VIIa during the development of sensory hair cells in the inner ear (Mao, 2006).

Within the myosin head region, the major conserved domains are all present, suggesting that Dachs functions as a motor protein. However, it is also possible that Dachs serves a structural or scaffolding role. For example, in the Hedgehog pathway, a kinesin-related protein, Costal2, is thought to function largely as a scaffold that brings together crucial kinases with their substrates (Mao, 2006).

The dGC2 mutation deletes part of the N terminal extension. As dGC2 mutants have relatively weak phenotypes, the N terminal extension might not be not essential for Dachs activity. Conversely, the severe phenotypes of alleles that truncate Dachs in the myosin head region imply that the myosin domain is essential. dGC13 in particular is predicted to eliminate almost all of the myosin head domain, and genetically it appears to act as a null allele (Mao, 2006).

Characterization of new dachs alleles has provided an opportunity to define more clearly the requirements for dachs. dachs is required for normal wing and leg growth, although some appendage growth is dachs independent. Importantly, the identification of dachs as a downstream component of a Fat signaling pathway that influences growth implies that the reduced growth in dachs mutants is reflective of a normal role for a Fat pathway in growth promotion. That is, while fat is a gene whose normal role can be thought of as to restrain growth, as mutant tissue overgrows, it is suggested that inhibition of Fat occurs during normal development, and that this inhibition contributes to normal appendage growth, as defined by the reduced growth of dachs mutants. Normal inhibition of Fat activity would presumably be effected by the two known regulators of Fat, Fj and Ds (Mao, 2006).

Whether available dachs mutations fully define the normal involvement of the Fat pathway in growth promotion is not yet clear. The possibility cannot be excluded that dachs is partially redundant with other proteins (e.g. other myosins), although this seems unlikely given the complete suppression of all non-polarity phenotypes of fat by dachs. It is also possible that dachs is required only for peak Fat signaling. This explanation is suggested by the observation that expression of the Fat target genes wg, Ser and fj is only partially or transiently lost in dachs mutants, yet the elevated or ectopic expression of these genes in fat mutants is completely eliminated by mutation of dachs (Mao, 2006).

The relatively mild tissue polarity phenotypes of dachs mutants, and the inability of dachs mutation to completely suppress the influence of fat on tissue polarity, contrast with the absolute dependence of fat gene expression, growth and affinity phenotypes on dachs. These observations suggest that there are two distinct Fat pathways. One, crucially dependent on Dachs, influences gene expression, growth and cell affinity, and another, partially independent of Dachs, influences tissue polarity. Studies of the atrophin protein Grunge also support the suggestion that there is a distinct Fat polarity pathway, as Grunge interacts with Fat and influences tissue polarity, but does not exhibit other phenotypes observed in fat mutants. Thus, Dachs might act redundantly with another protein in a polarity pathway, but non-redundantly in a pathway that influences gene expression. It should also be noted that effects of dachs on gene expression might contribute to the polarity phenotypes of dachs mutants. For example, fj is regulated by dachs, and fj has polarity phenotypes (Mao, 2006).

The asymmetric localization of Dachs observed in wild-type wings, and the influence of Fj and Ds on Dachs localization, have important implications for tissue polarity. (1) The asymmetric localization of Dachs is itself a form of polarity, and its detection in third instar imaginal discs emphasizes that these cells are polarized well before core polarity proteins such as Frizzled and Dishevelled become asymmetrically localization in pupal wings. A similar conclusion can be drawn from the recent observation that fat and ds influence the orientation of cell divisions in third instar discs. (2) The observations identify an ability to induce asymmetric protein localization as a mechanism through which the Fat pathway might influence tissue polarity. Dachs is one target, but the Fat polarity pathway might similarly involve asymmetric localization of other myosins, or of other types of proteins, to affect tissue polarity (Mao, 2006).

Mutation of fat is associated with elevated Dachs staining at the membrane, and overexpression of Fat decreases Dachs staining at the membrane. Although this negative effect of Fat on Dachs is subject to the caveat that only tagged overexpressed Dachs:V5 can be detected, this tagged protein rescues dachs mutants, and the effects of Fat on Dachs staining are consistent with their opposite phenotypes and the epistasis of dachs to fat. Manipulations of the expression of Fat regulators provide further evidence that Fat regulates Dachs levels at the membrane, and altogether these observations implicate Dachs as a crucial intracellular component of a Fat signaling pathway (Mao, 2006).

The concomitant elevation of Fat staining and loss of Dachs staining observed at the perimeter of Fj-expressing clones is consistent with the conclusion that Fat can antagonize the localization or stability of Dachs at the membrane. Because the elevation of Fat is limited to the periphery of Fj-expressing clones, it is hypothesized that it results from an influence of Fj on Fat-Ds interactions, rather than the expression of Fj per se. Tissue polarity studies have implied that Fj and Ds have opposite affects on Fat. Although it has not yet been determined whether Fj can directly modify Fat or Ds, the simplest explanation for the elevated Fat staining at the edge of Fj-expressing cells would be to propose that Fj modifies Ds to inhibit its interactions with Fat. In this case, Fat protein within Fj-expressing clones would be predicted to prefer to bind to Ds outside of the clone, and hence to accumulate at the clone perimeter, where it would then downregulate Dachs (Mao, 2006).

The interpretation of the elevated Dachs staining at the perimeter of Ds-expressing clones is more complex. Although Fat is elevated at the clone perimeter, the depletion of Fat from neighboring cells suggests that the elevated Fat staining largely reflects Fat outside of the clone, rather than in Ds-expressing cells. Given that dachs and fat influence transcriptional targets cell autonomously, and dachs acts genetically downstream of fat, the link between elevated Fat in one cell and elevated Dachs in a neighboring cells must be indirect. It might be that Ds can also influence Dachs localization, and does so in opposite fashion to Fat. According to this scenario, the elevated Fat staining in cells neighboring the clone would be reflective of high levels of Ds engaged by Fat at the clone perimeter, which would then recruit or stabilize Dachs at the membrane. However, mutation of ds did not result in any noticeable decrease of Dachs:V5 staining. Alternatively, it might be that Fat antagonizes the accumulation of Ds within the same cell. High Fat accumulation at the edge of one cell could then result in low Fat accumulation at the edge of its neighbor through this hypothesized downregulation of Ds. In this case, the elevated Dachs accumulation at the edge of Ds-expressing clones would be a consequence of low levels of Fat. This model would also imply that asymmetric localization of Fat could be propagated from cell to cell, which could have important consequences for Fat pathway regulation. However, there is as yet no evidence that Fat is asymmetrically localized at wild-type levels of Fj and Ds expression (Mao, 2006).

Vamana couples fat signaling to the Hippo pathway

The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. This study identified the vamana gene (CG10933; FlyBase name Dachs ligand with SH3s or Dlish) as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. These findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs (Misra, 2016).

Coordinated growth and morphogenesis is critical to the development of tissues of specific size and shape. Dachsous (Ds)-Fat signaling (henceforth, Fat signaling) controls both growth, through regulation of Hippo signaling, and morphogenesis, through regulation of planar cell polarity (PCP). Fat signaling regulates Hippo signaling and PCP by controlling the membrane localization of the atypical myosin protein Dachs. Many studies have provided important insights into both how Dachs influences Hippo signaling, and how it influences PCP. In contrast, the mechanism by which Fat signaling actually controls Dachs has remained less well understood (Misra, 2016).

Fat and Ds are atypical cadherins with novel intracellular domains (ICD), which localize to the plasma membrane just apical to the adherens junctions. Fat and Ds bind to each other in a heterophilic manner, and this interaction is modulated by the Golgi-resident kinase, Four-jointed (Fj), which phosphorylates their extracellular domains. This heterophilic binding, together with the graded expression of Ds and Fj, contribute to polarization of Ds and Fat localization within cells. Three different ways by which Fat signaling influences Hippo signaling have been described: Fat signaling influences the membrane localization of Expanded (Ex) , the levels of Wts protein, and the interaction of Wts with its cofactor Mats. Each of these effects on Hippo signaling depends upon Dachs. Fat signaling affects PCP in at least two ways: through an influence on junctional tension, and by regulating the Spiny-legs (Sple) isoform of the prickle locus. Both of these effects also involve Dachs (Misra, 2016).

Dachs was identified as a key downstream effector of Fat signaling because mutations in dachs completely suppress the overgrowth induced by fat mutations, and partially suppress the PCP defects induced by fat mutations. Dachs localizes to the cell membrane just apical to the adherens junction in a polarized manner; in the developing wing Dachs is localized to the distal sides of the cell, in response to the proximal-distal gradients of Ds and Fj expression. Dachs membrane localization requires a palmitoyltransferase encoded by approximated (app), but how App influences Dachs localization is unknown. In fat or ds mutants increased levels of Dachs are observed at the apical membrane and Dachs is no longer polarized. Forcing Dachs membrane localization by fusing it to Zyxin phenocopies fat mutants. Conversely, overexpression of full-length Fat or even just the Fat intracellular domain (ICD) displaces Dachs from the membrane into the cytoplasm. These and other observations have indicated that Fat regulates growth by modulating the levels of Dachs at apical membranes, and regulates Dachs-dependent PCP by directing Dachs asymmetry (Misra, 2016).

To understand how Fat functions, several studies have examined the roles of different regions of the Fat ICD. These studies identified two regions that mediate its growth-suppressive function. One, the D region, around amino acids 4,975 to 4,993, makes a modest contribution to Hippo pathway regulation, as when this region is deleted flies are viable but their wings are approximately 30% larger than normal, and also rounder than normal. The D region is required for interaction with the ubiquitin ligase, Fbxl7, which reduces Dachs membrane levels, and mutation of which results in phenotypes similar to deletion of the D region. A second region, which has been referred to as HM, Hpo, or H2, is defined by observations that deletions within this region block the ability of Fat to activate Hippo signaling. Two alleles of fat, fat61 and fatsum, have also been identified that harbor mutations within this region, and are associated with tissue overgrowth comparable with that caused by fat null mutations. However, the mechanism by which this region, which for simplicity is referred to as the H region, regulates the Hippo pathway, and whether it affects Dachs, are unknown (Misra, 2016).

This study reports the isolation and characterization of the Src homology 3 (SH3)-domain-containing protein encoded by vamana (vam). Loss of vam function decreases growth, whereas overexpression of vam promotes growth. These effects are mediated through regulation of the Hippo pathway, and vam functions genetically downstream of fat, as vam mutations can suppress both growth and PCP phenotypes of fat. Vam localizes to the apical region of epithelial cells in a polarized manner, co-localizing with Dachs, and is required for normal membrane localization of Dachs. Vam physically associates with the carboxy-terminal domain of Dachs and the ICDs of Ds and Fat, and is regulated by the H region of the Fat ICD. These observations identify Vam as a key mediator of signaling from Fat to Dachs (Misra, 2016).

These studies identified the C-terminal region of Dachs as sufficient to mediate its interaction with Vam. Interestingly, the original dachs allele, described almost a century ago by Bridges and Morgan (1919), is a hypomorphic allele associated with insertion of a blood transposon just upstream of the C-terminal region. Hence this allele likely encodes a truncated protein that lacks the Vam-interaction domain. Consistent with this inference, the vam null phenotype appears similar to the dachs1 phenotype. Thus, a requirement for interaction with Vam can explain the basis for the original identification of dachs (Misra, 2016).

Vam is evolutionarily conserved among insects but with no close homologs in vertebrates. This is consistent with the fact that Dachs is also only found in insects, and the sequence of the H region is not conserved in vertebrate Fat genes. Nonetheless, Vam is structurally related to a broad family of SH2- and SH3-domain-containing proteins exemplified by CRK, Grb2, Myd88, and NCK. These proteins are referred to as signal-transducing adapter proteins and facilitate formation of protein complexes that play key roles in signal transduction. Vam is composed of just three SH3 domains; this domain organization is most similar to that of the NCK family of adapters, which contain three SH3 domains along with one SH2 domain. The finding that Vam uses both SH3-1 and SH3-3 to interact with Fat and Ds is also reminiscent of NCK family adapters, as they engage effectors using multiple SH3 domains. The Drosophila ortholog of NCK, dreadlocks (dock), interacts with cell-adhesion molecules encoded by hibris, kirre, roughest, and sticks and stones (sns) to regulate actin polymerization and growth cone migration, and functional redundancy of SH3 domains has been observed for dock. Multiple SH3 domains are also commonly observed in proteins involved in vesicular trafficking. The observation that in vam mutants Dachs accumulates in cytoplasmic puncta that could be vesicular structures suggests that Vam might influence the trafficking of Dachs (Misra, 2016).

Fat and Ds proteins are conserved in vertebrates, where they play important roles in controlling PCP, and have also been proposed to influence Hippo signaling. In the absence of a Dachs homolog, however, it has been unclear how downstream signaling is mediated in vertebrate Ds-Fat pathways. The discovery that Vam links Ds and Fat to downstream signaling raises the possibility that a different member of the signal-transducing adapter proteins could mediate downstream Ds-Fat signaling in vertebrates (Misra, 2016).

The H region of the Fat ICD plays a crucial role in Hippo pathway regulation. This analysis of Fat ICD truncations revealed that the H region inhibits Vam and Dachs membrane accumulation, the influence of Fat ICD deletions on Hippo signaling correlates with their influence on Vam and Dachs membrane localization, and the H region of Fat can associate with Vam. Together with observations that Vam associates with and regulates Dachs, these observations lead to the inferrence that the H region normally functions to promote Hippo signaling through its association with, and regulation of, Vam. Fat also influences growth and Dachs accumulation through a second region of the ICD, the D region, which interacts with Fbxl7. Because mutation of the D region, or mutations in Fbxl7, have weaker phenotypes than mutations in the H region, the H region appears to play the larger role in Dachs regulation, but nonetheless it is expected that both regions normally act in parallel to regulate membrane levels of Dachs and thus, ultimately, Hippo signaling (Misra, 2016).

The localization of Vam in different genotypes, together with its physical interactions, suggests models for how Vam regulates Dachs localization. Since Vam and Dachs are reciprocally required for each other's membrane localization, it is inferred that a complex between these two proteins is required for their stable localization to apical junctions, where Dachs regulates PCP (via interactions with Sple) and Hippo signaling (via interactions with Zyxin and Warts). The observations that Fat promotes removal of Vam and Dachs from the subapical membrane, associates with Vam, yet does not visibly co-localize with Vam at apical junctions, suggests that Fat normally removes Vam-Dachs complexes from the subapical membrane. One mechanism by which this might occur is through binding of Fat to Vam, followed by endocytosis of Fat-Vam-Dachs complexes. Alternatively, Fat binding might disrupt Vam-Dachs binding, as these proteins normally do not localize to the membrane in isolation (Misra, 2016).

It was also observed that Vam can interact with the Ds ICD, and that it does so through the same SH3 domains as it uses to interact with the Fat ICD. This suggests that these interactions are likely to be competitive. In this case, interaction of Vam with the Ds ICD could promote Vam and Dachs membrane localization by opposing the influence of Fat on Vam. For example, by competing with Fat for binding to Vam, Ds could prevent Fat from disrupting Vam-Dachs interactions, or promoting endocytosis of a Vam-Dachs complex. Consistent with this suggestion that the Ds ICD stabilizes Vam and Dachs at apical junctions, Vam, Ds, and Dachs normally all co-localize in puncta on the distal side of wing cells. The ability of Vam to associate with the ICDs of both Fat and Ds could thus provide a simple mechanism explaining how the ICD of Ds seems to promote Dachs membrane localization, whereas the ICD of Fat inhibits it (Misra, 2016).

The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dach

Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, a structure-function analysis of Dachs was performed, and this was used to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein that has been named Dlish (Dachs ligand with SH3s). Dlish was found to be regulated by Fat and Dachs. Dlish also binds Fat and the Dachs regulator Approximated, and Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. The evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs (Zhang, 2016).

Heterophilic binding between the giant Drosophila protocadherins Fat and Dachsous (Ds) both limits organ growth, via regulation of the Hippo pathway, and orients planar cell polarity (PCP), through cell-by-cell polarization of Fat, Ds and their downstream effectors. Loss of Fat and, to a lesser extent, Ds, leads to the profound overgrowth of the Drosophila imaginal discs that give rise to adult appendages, and loss of either disorders the polarity of cell divisions, hairs and other morphological features in a variety of Drosophila tissues. But while players and pathways have been defined that are genetically downstream of Fat-Ds binding, only a little is known about the biochemical links between these and their most powerful regulator, the intracellular domain (ICD) of Fat (Zhang, 2016).

A good deal of the recent work on Fat effectors has focused on the regulation of unconventional type XX myosin Dachs. Dachs is critical first because it provides the only known marker specifically sensitive to changes in the Fat/Ds branches of both the Hippo and PCP pathways. Dachs is normally concentrated in the subapical cell cortex, overlapping subapically-concentrated Fat and Ds. Loss of Fat greatly increases subapical Dachs levels, and polarization of Fat and Ds to opposite cell faces can in turn polarize Dachs to the face with less Fat. Fat thus inhibits or destabilizes subapical Dachs, while Ds may do the opposite. Downstream changes in Hippo or PCP activities do not affect Dachs (Zhang, 2016).

Dachs changes are also critical because they play a major role downstream of Fat. Dachs binds to and inhibits the activity of the kinase Warts (the Drosophila Lats1/2 ortholog), both reducing Warts levels and changing its conformation. Warts is concentrated in the subapical cell cortex, and thus the increased cortical Dachs of fat mutants should reduce the phosphorylation of Yorkie by Warts, allowing Yorkie to move into the nucleus to drive the transcription of growth-promoting target genes. Indeed, Dachs is necessary for the overgrowth and increased Yorkie target gene expression of fat mutants. Dachs overexpression also causes overgrowth, although more weakly than the overgrowth caused by the loss of Fat, indicating that Dachs is partly sufficient (Zhang, 2016).

Dachs can also bind to the core PCP pathway component Spiny legs (Sple) and alter its localization, thus influencing PCP in the subset of tissues that rely on Sple. The increased levels of unpolarized Dachs in fat mutants may misdirect Sple, accounting for at least some of the PCP defects; fat mutant hair PCP defects are improved, although not eliminated, by loss of dachs (Zhang, 2016).

Dachs has not been shown to interact directly with Fat's ICD, and only three other proteins are known to affect Dachs accumulation in the subapical cell cortex, the casein kinase ε Discs overgrown (Dco), Approximated (App) and F-box-like 7 (Fbxl7). Dco may act through Fat itself: Dco binds and phosphorylates the Fat ICD, and loss of Dco function causes strong overgrowth and increases subapical Dachs, similar to loss of Fat (Zhang, 2016).

App suggests a mechanism in which Fat inhibits the tethering of Dachs to protein complexes in the subapical domain. app mutants decrease subapical Dachs levels and reduce Dachs activity. Thus, like dachs mutants, app mutants reverse the overgrowth and increased Yorkie target gene expression normally observed in fat mutants, and improve hair PCP. App is one of 20 Drosophila DHHC palmitoyltransferases, transmembrane proteins responsible for adding palmitates to cytoplasmic proteins and thereby anchoring them to cell membranes. App is also concentrated in the subapical cell membrane and can bind both Dachs and the Fat ICD. Thus, in the simplest model App palmitoylates or tethers Dachs, concentrating it in the cell cortex, and Fat works in part by sequestering or inhibiting App. However, Dachs is not detectably palmitoylated (Zhang, 2016).

This study describes the function of a novel Dachs binding protein, and shows that its effects provide strong evidence for both the palmitoylation-dependent and degradation-dependent regulation of Dachs. Structure-function analysis of Dachs found regions required for its normal subapical localization, and this information was used as the basis for a screen for novel Dachs binding partners. A direct binding partner was found for the Dachs C-terminus, the previously uncharacterized SH3 domain protein CG10933, which has been renamed Dachs ligand with SH3s, or Dlish. The activity and subapical concentration of Dlish are regulated by Fat, Dco and Dachs, and Dlish in turn is required for the subapical concentration and full activity of Dachs in both wild type and fat mutant cells. Dlish localization also depends on App; furthermore Dlish binds to and is palmitoylated by App, and palmitoylation can be suppressed by Fat. Loss of Dlish also increases the total levels of Dachs, likely by blocking Fat-mediated destabilization of Dachs. It is proposed that Dlish targets Dachs to subapical protein complexes in part via Fat-regulated, App-mediated palmitoylation. Dlish thereby concentrates Dachs where it can efficiently inhibit subapical Warts, and conversely links Dachs to the machinery for Fat-dependent destabilization (Zhang, 2016).

The unconventional myosin Dachs is an important effector Fat/Ds-regulated Hippo signaling, as its heightened subapical levels in fat mutants inhibit and destabilize Warts, freeing Yorkie to increase the expression of growth-promoting genes. A structure-function analysis of Dachs was used as a springboard to search for new binding partners that are critical for Dachs localization and function, and have found Dlish (CG10933), a novel SH3 domain protein. Dlish binds directly to the Dachs C-terminus; loss of Dlish disrupts Dachs localization, levels and function: subapical accumulation of Dachs is reduced and cytoplasmic and total levels increase, both in wild type and fat mutant tissue, while activity is lost. Importantly, Dlish is regulated by Fat, as loss of Fat greatly increases Dlish levels in the subapical cell cortex and, like Dachs, Dlish is needed for much of the fat mutant overgrowth (Zhang, 2016).

Dlish also binds the ICD of Fat and other Fat-binding proteins, including two that likely mediate part of its function: the palmitoyltransferase App and the F-box protein Fbxl7. Thus Dlish provides a new biochemical link from the Fat ICD to Dachs regulation. Evidence indicates that Dlish plays two different and opposing roles (see Models of Fat-mediated regulation of subapical Dachs by Fat-inhibited subapical tethering and Fat-stimulated destabilization). First, it helps tether Dachs in the subapical cell membrane, in part via Fat-regulated, App-dependent palmitoylation, so that Dachs can more efficiently inhibit Warts. Second, it links Dachs to Fat-organized machinery for Dachs destabilization, including Fbxl7, and thus helps reduce Dachs levels (Zhang, 2016).

Dlish and Dachs cooperate to target or tether the Dlish-Dachs complex, as each is necessary, and to a weaker extent sufficient, for the subapical concentration of the other. The Dachs contribution is likely through tethering the complex to the cortical cytoskeleton, as this study found that loss of the F-Actin-binding myosin head blocks the subapical localization of Dachs. This would agree with recent biochemical analyses that suggest that Dachs has no motor function, acting rather as an F-Actin-binding scaffolding protein (Zhang, 2016).

The Dlish contribution, on the other hand, depends at least in part on its ability to bind the transmembrane DHHC palmitoyltransferase App. Loss of App and Dlish have very similar effects on Dachs localization and activity. This study found that loss of App disrupts the subapical accumulation of Dlish in vivo and that App can stimulate palmitoylation of Dlish in vitro. Thus, palmitoylation of Dlish likely stimulates membrane association of both Dlish and its binding partner Dachs (Zhang, 2016).

App also has additional effects on Fat pathway activity. First, App has palmitoyltransferase-independent activity and can co-IP Dachs in vitro. Thus, while palmitoylation of Dlish may mediate some of App's activity, subapical App may simultaneously help localize the Dlish-Dachs complex by physical tethering. And while both palmitoylation and tethering of the Dlish-Dachs complex is likely critical for the fat mutant phenotype, App also has a function that depends on the presence of Fat, as App can bind, palmitoylate and inhibit the activity of Fat's ICD (Zhang, 2016).

An important question is whether the absence of Fat regulates the App-dependent tethering of the Dlish-Dachs complex. The Fat ICD can complex with both Dlish and App. Dlish and App can bind not only the C-terminal region of the Fat ICD where Fat is palmitoylated, but also the PH and Hippo domains which was shown to played the strongest role in Dachs regulation. An attractive mechanism is that Fat inhibits the interaction between App and Dlish, reducing App's ability to palmitoylate and tether the Dlish-Dachs complex. In the absence of Fat, App and Dlish are freed to tether Dachs, and Dachs now inhibits and destabilizes Warts, causing overgrowth. In support of this model, it was found that overexpression of Fat's ICD in vitro can reduce App-stimulated palmitoylation of Dlish (Zhang, 2016).

The evidence further indicates that Dlish targets Dachs for Fat-dependent destabilization. Loss of Fat increases not only subapical Dachs, but also total Dachs levels. In the presence of Dlish the increased Dachs remains subapical. Loss of Dlish also increases the total levels of Dachs, but now that increase is cytoplasmic, and much less effective at inhibiting Warts. These Dachs increases are unlikely to have independent causes, as they are not additive; total Dachs levels are similar after loss of Fat, Dlish or both (Zhang, 2016).

It is proposed that in wild type cells there is a flux of the Dachs-Dlish complex from the cytoplasm to the subapical cell cortex, where a Fat-dependent complex destabilizes Dachs. Normally the tethering effects of Dlish predominate over the Fat-dependent destabilization, and moderate levels of subapical Dachs are maintained. Destabilization is lost without Fat; this combines with Dlish-mediated tethering to increase subapical Dachs. Without Dlish the subapical tethering of Dachs is disrupted, access to Fat-dependent destabilization is lost and the now cytoplasmic Dachs increases. The model thus explains why the excess, largely cytoplasmic Dachs caused by reduced Dlish function is not greatly influenced by the presence or absence of Fat (Zhang, 2016).

In addition to any effects caused by changing the subcellular localization of Dachs, Dlish may also provide a direct link to the machinery for protein ubiquitination, as Dlish can co-IP with the E3 ubiquitin ligase Fbxl7, as well as the related F-box ubiquitin ligase Slimb. Fbxl7 is particularly intriguing, as it binds to and is regulated by Fat's ICD, and reduces subapical Dachs, perhaps via ubiquitination. Slimb can bind and ubiquitinate Expanded, a subapical regulator of Hippo signaling with links to Fat and Dachs function. But while it was found that loss of Fbxl7 or Slimb increases subapical Dachs and Dlish, these effects are weak, and the large increase in total Dachs levels caused by loss of Dlish or Fat must involve additional partners (Zhang, 2016).

Mutations in Fat’s closest mammalian homolog Fat4 (FatJ) and its Ds-like ligands strongly disrupt PCP-like processes, and have in humans been associated with the multisystem defects of Hennekam and Van Maldergem syndromes. There has been some debate, however, about whether the mammalian proteins retain direct regulation of Hippo activity. Nonetheless, Fat4 has been linked to Hippo changes in both normal development and tumors, mutations in Fat4 or Dachsous1 change the balance of precursors and mature neurons in the developing neuroepithelium of both humans and mice, and the mouse defect can be reversed by knockdown of the Yki homolog Yap. But the mechanisms underlying these effects are unknown, and Fat4 cannot regulate Hippo signaling in Drosophila (Zhang, 2016).

It is therefore important to note that while homologs of Dachs and Dlish are found throughout the animal kingdom, they are apparently absent from vertebrates. This suggests that the Dachs-Dlish branch of the Fat-Ds pathway, with its powerful effect on Warts activity, is also lacking. Nonetheless, it has been suggested that Drosophila Fat and Ds can affect Hippo pathway activity in a Dachs-independent manner. It is also clear that Drosophila Fat has Dachs-independent effects on PCP; indeed the N-terminal 'PCP' domain of the Fat ICD that did not affect Dachs in this study is sufficient to improve the PCP defects of fat mutants. These or alternative pathways may still be present in mammals (Zhang, 2016).

Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway

During animal development, several planar cell polarity (PCP) pathways control tissue shape by coordinating collective cell behavior. This study characterizes, by means of multiscale imaging, epithelium morphogenesis in the Drosophila dorsal thorax and shows how the Fat/Dachsous/Four-jointed PCP pathway controls morphogenesis. The proto-cadherin Dachsous is polarized within a domain of its tissue-wide expression gradient. Furthermore, Dachsous polarizes the myosin Dachs, which in turn promotes anisotropy of junction tension. By combining physical modeling with quantitative image analyses, it was determined that this tension anisotropy defines the pattern of local tissue contraction that contributes to shaping the epithelium mainly via oriented cell rearrangements. The results establish how tissue planar polarization coordinates the local changes of cell mechanical properties to control tissue morphogenesis (Bosveld, 2012).

Altogether this study found that Ds polarization promotes Dachs polarization within a domain of the opposing tissue-wide ds and fj gradients. Their local polarization produces an anisotropic distribution of junction tensions, which increases the contraction rates along the lines of Ds and Dachs planar polarization to shape the epithelial tissue mainly through oriented cell rearrangements. The Dachs myosin has the necessary domains to be an actin binding motor and, in complex with Dachsous, it may directly contribute to junction contractility, thereby favoring cell rearrangements. Since MyosinII also contributes to junction tension and cell rearrangements, future work should dissect the respective roles of Dachs and MyosinII in these processes. Morphogenesis is accomplished by the concerted activity of multiple signaling pathways. The subtractive method of tissue deformation rates is general enough to isolate the contribution of a given pathway to morphogenesis without making assumptions on its magnitude and its spatial dependence. Finally, given the multitude of cell shapes, cell sizes, and division patterns occurring in the thor ax epithelium,future work on this tissue should reveal how multiple signaling pathways are integrated to regulate proliferation, planar polarization, and morphogenesis (Bosveld, 2012).

Propagation of Dachsous-Fat planar cell polarity

The Fat pathway controls both planar cell polarity (PCP) and organ growth. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP, whereas their slope can influence growth. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling. This study shows that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, it was shown that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. A biochemical mechanism was identified that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat (Ambegaonkar, 2012).

The observation that differences in Fj or Ds expression can alter Fat PCP at a distance and that Ds, and to a lesser extent Fat, is polarized within the wing, together with other recent studies, imply that establishment of polarity in the Fat PCP system relies not just upon direct interpretation of Fj and Ds gradients but also upon amplification and propagation of PCP. To achieve this, PCP models incorporate both asymmetric intercellular signaling and antagonistic intracellular interactions between complexes that localize to distinct sides. Intercellular binding between Ds and Fat is well established, but on its own, this would not propagate polarity from cell to cell. However, incorporation of a local, intracellular antagonism of Ds by Fat activity could polarize Ds localization, which could then enable Fat-PCP to propagate. It is hypothesized that Fat regulates Ds by influencing production or stability of processed Ds isoforms (Ambegaonkar, 2012).

The propagation of polarity means that Fat-PCP is influenced not only by the local gradient but also by differential expression at a distance. Strong repolarization of Dachs was dependent upon having substantial differences in expression. Notably, strong differences in expression of both Fj and Ds normally occur in the proximal wing, and these differences have significant effects on Fat activity. Both measures of the range of Dachs repolarization and mathematical modeling suggest that the Fj/Ds expression boundary in the proximal wing would not be sufficient to direct Fat-PCP across 30 or more cells, as would be required at late third instar. However, at early third instar, when the developing wing is small, a mechanism that propagates PCP from an expression boundary for several cells could in principle be sufficient to establish PCP throughout the wing. Once established, the mechanisms that allow Fat-PCP to propagate could also help maintain Fat-PCP as the wing grows. In this case, the Fj and Ds boundaries at the edge of the developing wing would be the main drivers of polarity, rather than the shallow gradients of their expression within the wing itself (Ambegaonkar, 2012).

Signal transduction by the Fat cytoplasmic domain

The Drosophila protocadherin Fat (Ft) regulates growth, planar cell polarity (PCP) and proximodistal patterning. A key downstream component of Ft signaling is the atypical myosin Dachs (D). Multiple regions of the intracellular domain of Ft have been implicated in regulating growth and PCP but how Ft regulates D is not known. Mutations in Fbxl7 (CG4221), which encodes an F-box protein, result in tissue overgrowth and abnormalities in proximodistal patterning that phenocopy deleting a specific portion of the intracellular domain (ICD) of Ft that regulates both growth and PCP. Fbxl7 binds to this same portion of the Ft ICD, co-localizes with Ft to the proximal edge of cells and regulates the levels and asymmetry of D at the apical membrane. Fbxl7 can also regulate the trafficking of proteins between the apical membrane and intracellular vesicles. Thus Fbxl7 functions in a subset of pathways downstream of Ft and links Ft to D localization (Bosch, 2014).

The protocadherin Ft lies at the apex of multiple pathways that together regulate growth, several aspects of PCP, and proximodistal patterning. The mechanism by which Ft functions as a signaling molecule remains poorly understood. This study has identified the F-box protein Fbxl7 as an immediate effector of Ft, that functions to restrict the levels of the atypical myosin D at the apical membrane as well as its distribution around the perimeter of the cell. In addition, Fbxl7 can regulate levels of Ft at the apical membrane (Bosch, 2014).

Recent studies have revealed that Ft's effects on distinct pathways may be genetically separated, and that multiple effector domains can contribute to the same function. Indeed, the growth-suppressing function of Ft may occur via at least two regions of the Ft ICD. One or more regions between amino acids 4834 and 4899 in full-length Ft appear responsible for Ft's ability to regulate Hippo signaling. Several mutations within this region compromise this function of Ft and cause massive tissue overgrowth (Bossuyt, 2013). Intriguingly, an allele of ft, ft61, which harbors such a mutation, showed neither an effect on the recruitment of Fbxl7 to the apical membrane nor on the binding of Ft to Fbxl7. Thus, signaling via this region of the ICD appears to be independent of Fbxl7. A second, more C-terminal region of the Ft ICD (Region D) that extends between amino acids 4975 and 4993 of full-length Ft, is removed by the ftΔD deletion and also has a growth-suppressive function albeit weaker than that of HM. This second growth-suppressive pathway requires the function of Fbxl7, as the protein generated by the ftΔD allele cannot bind to Fbxl7 nor can it localize Fbxl7 to the apical membrane. Additionally, the phenotypic abnormalities of null alleles of ft rescued by ftΔD are very similar, if not identical to those of Fbxl7 mutants. Furthermore, like ftΔD, Fbxl7 mutations do not display overt abnormalities of hair orientation in the wingor abdomen (Bosch, 2014).

Hyperactivation of the 'weaker' Fbxl7-dependent pathway can overcome the absence of the ‘stronger' Fbxl7-independent pathway; overexpression of Fbxl7 can suppress the overgrowth of ft61. Thus, while these two pathways can be dissociated at the level of the Ft ICD, they nevertheless seem to converge further downstream. This point of convergence likely involves Dachs (D) since the overgrowth of ft mutant tissue can be suppressed completely by eliminating D function. Indeed, it has previously been suggested that Ft regulates growth by restricting the levels of apical D, and regulates PCP by influencing the planar asymmetry of apical D (Bosch, 2014).

Another key finding in these experiments is that Fbxl7 mutations perturb the distribution of D around the perimeter of the apical region of the cell. D is normally biased towards the distal edge of the cell; in Fbxl7 mutants, D is more evenly distributed around the cell perimeter. The asymmetric localization of D depends on at least two different regions of Ft (Pan, 2013). One is the region that binds to Fbxl7 (Region D) and the other is composed of the last three amino acids at the C-terminus of the protein (Region F), which is not necessary for Fbxl7 localization to the apical membrane. Thus, for the regulation of D asymmetry as well, there appears to be an Fbxl7-independent pathway. The existence of multiple downstream effector pathways that converge on common biological outcomes suggests that these pathways might function redundantly to some extent and thus provide robustness. This might also explain why the phenotypes elicited by overexpression of Fbxl7 are, in general, more severe than those observed in loss-of-function mutations (Bosch, 2014).

Previous observations of the localization of Ft, Ds, and D to vesicles are suggestive of trafficking events being involved in Ft signaling. It was therefore demonstrated that, in addition to the apical membrane, Fbxl7 localizes to vesicles. Moreover, FLAG-Fbxl7 vesicles can contain Ft, Ds and D, and these may be related to the apical puncta observed on cell edges. This localization is likely specific, since no Fbxl7 co-localization is seen with other cell surface proteins such as Crumbs, Notch, and E-cadherin. Currently very little is known about the role of each of these proteins in vesicles. However, there is an increasing appreciation that most transmembrane proteins, and even proteins that are associated with the inner leaflet of the cell membrane are maintained at the plasma membrane by a dynamic process involving endocytosis and vesicle recycling (Bosch, 2014).

Evidence is provided that Fbxl7 regulates Ft apical localization, but how this regulation relates to the Fbxl7 phenotypes is not clear. Since Fbxl7 overexpression increases Fat signaling, and rescues the overgrowth-inducing ft61 allele, perhaps this is due to the increased levels of Ft protein at the apical membrane. However, Ft levels are slightly elevated in Fbxl7 mutants, which display mild overgrowth. Therefore the mutant phenotype cannot be explained by the effect on Ft. Another known regulator of apical Ft levels is lowfat (lft). Fbxl7 and Lft appear to regulate Ft in different ways. Lft overexpression, like Fbxl7, increases Ft levels. However, while Ft levels are decreased in lft mutant cells, Ft levels are increased in Fbxl7 mutant cells, though less so compared to Fbxl7 overexpression. Interestingly, for many proteins that regulate cellular trafficking, similar phenotypic abnormalities are observed with gain-of-function and loss-of-function mutations, since the normal execution of the process requires the protein to shuttle efficiently between two states. Thus dynamic aspects of the localization of Ft, Ds and D clearly merit more attention (Bosch, 2014).

The interactions observed between Fbxl7 and the adapter protein Cindr may provide clues for how Fbxl7 regulates D localization. Fbxl7-associated vesicles show almost complete overlap with GFP-Cindr and Fbxl7 can re-localize Cindr from the apical membrane to the interior of the cell. This finding, together with the observed increase in basal levels of D upon Fbxl7 overexpression, suggests that Fbxl7 may function to regulate D trafficking in a similar manner. Cindr and its mammalian orthologues Cin85 and CD2AP are thought to regulate interactions between membrane proteins and actin cytoskeleton. D is an atypical myosin with a predicted actin binding domain in its conserved head domain. Therefore, the vesicles which Fbxl7 associates with D and Cindr may be linked to the actin cytoskeleton. In addition, the finding of partial colocalization of Fbxl7 with retromer components further supports the possibility that Fbxl7 may have a role in protein trafficking (Bosch, 2014).

Many F-box proteins associate with Skp1 and Cul1 to form an SCF E3 ubiquitin ligase complex. Recruitment of specific substrates results in their poly-ubiquitylation and degradation, or mono-ubiquitylation, which can have non-degradative signaling roles. In addition, some F-box proteins have SCF-independent roles. Fbxl proteins are thought to recruit substrates to the SCF complex through the interaction with their LRR domains, and substrates have been identified for several Fbxls such as Skp2 (Fbxl1), which degrades p27. However many, like Fbxl7, are still uncharacterized as 'orphan' F-box proteins with no known substrates (Bosch, 2014).

Since this study found that Fbxl7 associates with Skp1 and Cul1, its potential substrates may be involved in Ft signaling. Fbxl7 has one described substrate in mice, Aurora A. However it is not believed Aurora A is a relevant substrate in Drosophila, as no Ft signaling defects are observed when Aurora A is knocked down or overexpressed. The identification of F-box protein substrates has mainly been accomplished by unbiased approaches. Similarly, a combination of unbiased approaches, involving proteomics, genetic interaction screens, and identifying proteins that co-localize with Fbxl7 in vesicles could be used to identify Fbxl7 substrates (Bosch, 2014).

The ubiquitin ligase FbxL7 regulates the Dachsous-Fat-Dachs system in Drosophila

The atypical cadherins Dachsous (Ds) and Fat (Ft) are required to control the size and shape of tissues and organs in animals. In Drosophila, a key effector of Ds and Ft is the atypical myosin Dachs, which becomes planar polarised along the proximal-distal axis in developing epithelia to regulate tissue size via the Hippo pathway and tissue shape via modulating tension at junctions. How Ds and Ft control Dachs polarisation remains unclear. This study identified a ubiquitin ligase, FbxL7, as a novel component of the Ds-Ft-Dachs system that is required to control the level and localisation of Dachs. Loss of FbxL7 results in accumulation of Dachs, similar to loss of Ft. Overexpression of FbxL7 causes downregulation of Dachs, similar to overexpression of the Ft intracellular domain. In addition to regulating Dachs, FbxL7 also influences Ds in a similar manner. GFP-tagged FbxL7 localises to the plasma membrane in a Ft-dependent manner and is planar polarised. It is proposed that Ft recruits FbxL7 to the proximal side of the cell to help restrict Ds and Dachs to the distal side of the cell (Rodrigues-Campos, 2014).

How animal cells cooperate to build tissues of particular forms remains a fundamental unsolved problem in biology. One molecular system that controls tissue size and shape in animals is the Dachsous (Ds)-Fat (Ft) cadherin system. Ds and Ft encode large atypical cadherins that interact heterotypically to form cell-cell junctions in epithelia and are required to control tissue form in both Drosophila and mice. The Ds-Ft system is known to induce a molecular polarity in the plane of the epithelium, and this planar polarity has at least three distinct consequences, including control of tissue growth via regulation of the Hippo signalling pathway, control of tissue morphogenesis by modulating tension at cell-cell junctions, and control of the orientation of hairs, bristles and eye ommatidia in Drosophila, in part by modulating the Frizzled system of planar cell polarity (Rodrigues-Campos, 2014).

One important effector of Ds and Ft is the atypical myosin Dachs, which is thought to bind to the Ds intracellular domain and becomes planar polarised towards the distal side of each cell in the developing Drosophila wing or eye epithelium. Ds and Ft can also themselves become planar polarised, which may contribute to the polarisation of Dachs itself. Dachs then generates tension at distal cell-cell junctions to orient cell shapes, cell divisions or cell-cell rearrangements to drive tissue elongation along the proximal-distal axis of various fly epithelia. In addition, Dachs can signal to the nucleus via the Hippo pathway effector Yki (YAP/TAZ in mammals) to promote cell proliferation and tissue growth. Notably, Dachs appears to be dispensable for planar polarisation of the Frizzled system, and the ability of Ds and Ft to polarise hairs and bristles, a process that may instead depend on microtubules. This study focused on the Dachs-dependent roles of Ds and Ft in controlling tissue size and shape in Drosophila (Rodrigues-Campos, 2014).

The global cues that orient Dachs polarisation along the proximal-distal axis are known: Dachs localises distally in response to graded expression of the Ds cadherin and also of Four-jointed (Fj), a kinase that modulates Ds-Ft interactions. The gradients of Ds and Fj are opposing, such that Ds is highly expressed at the proximal end of the tissue and Fj is highly expressed at the distal end of the tissue. Yet, how epithelial cells read the slope of these gradients and translate this information into a planar polarised localisation of Dachs is still unknown. This study identified the ubiquitin ligase FbxL7 as a novel component of the Ds-Ft system that is crucial to control Dachs levels and localisation at apical cell-cell junctions (Rodrigues-Campos, 2014).

The results suggest a close relationship between the function of the Ft intracellular domain, Dco and FbxL7. Therefore whether phosphorylated Ft intracellular domain might recruit FbxL7 to the plasma membrane was tested. The localisation of GFP-tagged FbxL7 expressed in clones was examined and found that FbxL7-GFP localises to apical cell-cell junctions. By contrast, when FbxL7-GFP is expressed in ft mutant clones, it localises to the cytoplasm in a punctate pattern. A similar punctate pattern is observed when FbxL7-GFP is co-expressed with dominant-negative Dco3. Notably, the loss of Dachs that is normally induced by expression of FbxL7-GFP fails to occur when it is not recruited to the membrane by Ft and Dco. These findings support the notion that phosphorylated Ft recruits FbxL7 in order to downregulate Dachs. This model predicts that FbxL7 itself should be planar polarised to the proximal side of cells, where Ft is thought to be most concentrated and active, whereas Dachs localises to the distal side of cells away from FbxL7 and in a complex with Ds. Accordingly, low-level expression of FbxL7-GFP with ms1096.G4 reveals a planar polarised localisation, presumably to the proximal side of wing epithelial cells where Ft is known to concentrate (Rodrigues-Campos, 2014).

The above findings identify the FbxL7 ubiquitin ligase as a novel component of the Ds-Ft-Dachs system. FbxL7 is recruited to the membrane by Ft, where it then acts together with Ft and the Dco kinase to promote degradation or removal of both Dachs and Ds. The effect of FbxL7 loss and gain of function on Dachs levels are particularly strong and the phenotypic consequences in adult Drosophila closely resemble gain and loss of Dachs function, respectively. In vitro ubiquitylation assays suggest that FbxL7 can directly ubiquitylate Dachs, which is predicted to lead to its proteolytic degradation. In addition, it was observed that FbxL7 can also ubiquitylate the Ds intracellular domain in vitro and can modulate the level and localisation of Ds in vivo. It remains possible that FbxL7 acts indirectly by stabilising or activating Ft, which then acts via a different mechanism to degrade or remove Ds and Dachs proximally. The direct model is favored because of its simplicity and because ubiquitylation is generally thought to promote degradation, rather than stabilization, of proteins (Rodrigues-Campos, 2014).

These observations suggest a model in which Ft, which has been reported to localise proximally, recruits FbxL7 to the proximal side of the cell to help restrict Dachs and Ds to the distal side of the cell. These results also suggest that polarised Ds may also promote degradation or removal of Ft on the distal side so that Ft concentrates proximally, thereby assisting polar Ds-Ft bridge formation. Thus, there appears to be mutual antagonism between Ds and Ft within the same cell, as well as heterotypic Ds-Ft bridge formation between neighbouring cells, an event that then leads to loss of Dachs proximally and recruitment of Dachs distally. Such a mechanism might explain how this system can become planar polarized; however, it is still unclear how the system is able to read the slope of the Ds and Fj gradients continuously, rather than switch to a more permanently polarised state (Rodrigues-Campos, 2014).

Notably, the degree of Dachs polarisation (and the strength of its effect on Hippo signalling and tissue growth) correlates with the steepness of the Ds and Fj gradients, indicating that cells can obtain both vectorial information and a measure of steepness at the same time from the Ds-Ft system. These features of the Ds-Ft system match very well with those proposed for the hypothetical gradients originally conceived following surgical manipulation of insect development and regeneration. This identification of FbxL7 as a key player in this system will help enable further work to understand how the system can translate the steepness of the gradient into the degree of Dachs polarisation (Rodrigues-Campos, 2014).

Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling

In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. dachs is identified as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. These observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway (Cho, 2004).

There is a progressive elaboration of patterning along the PD axis over the course of wing development. During the second larval instar, interactions among the Epidermal Growth Factor Receptor, Dpp and Wg signaling pathways divide the wing disc into a dorsal region, which will give rise to notum, and a ventral region, from which the wing will arise. An initial PD subdivision of the wing is then effected by signaling from the AP and DV compartment boundaries, which promotes the expression of two genes, scalloped and vestigial, that encode subunits of a heterodimeric transcription factor (Sd-Vg) in the center of the wing. This subdivides the wing into distal cells, which give rise to the wing blade, and surrounding cells, which give rise to proximal wing and wing hinge structures. The proximal wing is further subdivided into a series of molecularly distinct domains. Studies of Sd-Vg function in the wing led to the realization that the elaboration of this finer pattern depends in part upon signaling from the distal, Sd-Vg-expressing cells, to more proximal cells. Thus, mutation of vg leads to elimination, not only of the wing blade, where Vg is expressed, but also of more proximal tissue. Conversely, ectopic expression of Vg in the proximal wing reorganizes the patterning of surrounding cells (Cho, 2004 and references therein).

A key target of the distal signal is Wg, which during early third instar is expressed in a ring of cells that surround the SD-VG-expressing cells, and which later becomes expressed in a second, more proximal ring. Wg expression in the inner, distal ring within the proximal wing is regulated by an enhancer called spade-flag (spd-fg), after an allele of wg in which this enhancer is deleted (Neumann, 1996). Studies of this allele, together with ectopic expression experiments, have revealed that Wg is necessary and sufficient to promote growth of the proximal wing. Wg also plays a role in proximal wing patterning; it acts in a positive-feedback loop to maintain expression of Homothorax (Hth). The rotund (rn) gene has been identified as an additional target of distal signaling (Cho, 2004 and references therein).

This work identified Four-jointed (Fj), Dachsous (Ds), Fat and Dachs as proteins that influence signaling to proximal wing cells to regulate Wg and rn expression. Fj is a type II transmembrane protein, which is largely restricted to the Golgi. Null mutations in fj do not cause any obvious defects in the proximal wing. However, fj plays a role in the regulation of tissue polarity, yet acts redundantly with some other factor(s) in this process. Mutations in fat or ds can also influence tissue polarity. Although the molecular relationships among these proteins are not well understood, genetic studies suggest that fj and ds act via effects on fat, and both fj and ds can influence Fat localization in genetic mosaics (Cho, 2004 and references therein).

Interestingly, alleles of fj, ds and fat, as well as alleles of another gene, dachs, can result in similar defects in wing blade and leg growth. The similar requirements for these genes during both appendage growth and tissue polarity, together with the expression patterns of fj and ds in the developing wing, led to this investigation of their requirements for proximal wing development. All four genes influence the expression of Wg in the proximal wing, and genetic experiments suggest a pathway in which Fj and Ds act to modulate the activity of Fat, which then regulates transcription via a pathway that includes Dachs. These observations lend strong support to the hypothesis that Fj, Ds and Fat function as components of an intercellular signal transduction pathway, implicate Dachs as a key downstream component of this pathway, and identify a normal role for these genes in proximodistal patterning during Drosophila wing development (Cho, 2004).

The common feature of all of the manipulations of FJ and DS expression carried out in this study is that Wg expression, and by inference, Fat activity, can be altered when cells with different levels of Fj or Ds are juxtaposed. In the case of Fj, its normal expression pattern, and effects of mutant and ectopic expression clones are all consistent with the interpretation that juxtaposition of cells with different levels of Fj is associated with inhibition of Fat in the cells with less Fj and activation of Fat in the cells with more Fj. The influence of Ds, however, is more variable. Studies of tissue polarity in the eye suggest that Ds inhibits Fat activity in Ds-expressing cells, and/or promotes Fat activity in neighboring cells. The predominant effect of Ds during early wing development is consistent with this, but its effects in late discs are not. Studies of tissue polarity in the abdomen suggest that the Ds gradient might be interpreted differently by anterior versus posterior cells, and it is possible that a similar phenomena causes the effects of Ds to vary during wing development (Cho, 2004).

The influence of ds mutation on gene expression and growth in the wing is much weaker than that of fat. It has been suggested that Fj might influence Fat via effects on Ds, and fj mutant clones have been observed to influence Ds protein staining. The observations are consistent with the inference that both Ds and Fj can regulate Fat activity, but they do not directly address the question of whether Fj acts through Ds. They do, however, indicate that even the combined effects of Fj and Ds cannot account for FAT regulation, and, assuming that the strongest available alleles are null, other regulators of Fat activity must exist. It is presumably because of the counteracting influence of these other regulators that alterations in Fj and Ds expression have relatively weak effects. In addition, according to the hypothesis that Fat activity is influenced by relative rather than absolute levels of its regulators, the effects of Fj or Ds could be expected to vary depending upon their temporal and spatial profiles of expression, as well as on the precise shape and location of clones (Cho, 2004).

The observations imply the existence of at least two intracellular branches of the Fat signaling pathway. One branch involves the transcriptional repressor Grunge, influences tissue polarity, certain aspects of cell affinity, and fj expression, but does not influence growth or wg expression. An alternative branch does not require Grunge, but does require Dachs. Dachs is implicated as a downstream component of the Fat pathway, based on its cell autonomous influence on Fat-dependent processes, and by genetic epistasis. The determination that it encodes an unconventional myosin, and hence presumably a cytoplasmic protein, is consistent with this possibility. It also suggests that Dachs does not itself function as a transcription factor, and hence implies the existence of other components of this branch of the Fat pathway. This Grunge-independent branch influences Wg expression in the proximal wing and imaginal disc growth. However, further studies will be required to determine whether Dachs functions solely in Grunge-independent Fat signaling, or whether instead Dachs is required for all Fat signaling (Cho, 2004).

The observations that fj expression is regulated by Sd-Vg, and that fj is both necessary and sufficient to modulate the distal ring of Wg expression in the proximal wing, suggest that Fj influences the activity of a distal signal, which then acts to influence Fat activity. However, the relatively weak effects of fj indicate that other factors must also contribute to distal signaling, just as fj functions redundantly with other factors to influence tissue polarity. Since Ds expression is downregulated in a domain that is broader than the Vg expression domain, a direct influence of Vg on the Ds gradient is unlikely, and the essentially normal appearance of Wg expression in the proximal wing in fj ds double mutants implies that Ds is not a good candidate for the hypothetic factor Signal X. Rather, it is suggested that Ds acts in parallel to signaling from Vg-expressing cells to modulate Fat activity. This Vg-independent effect would account for the remnant of the distal ring that sometimes appears in vg null mutants. Importantly though, the observation that the phenotypes of hypomorphic dachs mutant clones on Wg expression are more severe than fj and ds suggests that the hypothesized additional factors also act via the Fat pathway. It is also noted that the limitation of Wg expression to the proximal wing even in fat mutant clones implies that Wg expression both requires Nubbin, and is actively repressed by distally-expressed genes (Cho, 2004).

The recovery of normal Wg expression by later stages in both fj and dachs mutant clones implies that the maintenance of Wg occurs by a distinct mechanism. Prior studies have identified a positive-feedback loop between Wg and Hth that is required to maintain their expression. It is suggested that once this feedback loop is initiated, Fat signaling is no longer required for Wg expression. Moreover, the recovery of normal levels of Wg at late stages suggests that this positive-feedback loop can amplify reduced levels of wg to near normal levels (Cho, 2004).

The distinct consequences of Vg expression and Fj expression in clones in the proximal wing suggest that another signal or signals, which are qualitatively distinct from the Fj-dependent signal, is also released from VG-expressing cells. When Vg is ectopically expressed, Wg is often induced in a ring of expression that completely encircles it. However, this is not the case for Fj-expressing clones. Both Vg- and Fj-expressing clones can activate rn and wg only within NUB-expressing cells, but Vg expression can result in non-autonomous expansion of the Nub domain, and this expansion presumably facilitates the expression of Wg by surrounding cells. Another striking difference between Vg- and Fj-expressing clones is that in the case of ectopic Fj, enhanced Wg expression is only in adjacent cells. By contrast, in the case of Vg, Wg expression initiates in neighboring cells, but often moves several cells away as the disc grows, resulting in a gap between Vg and Wg expression. This gap suggests that a repressor of Wg expression becomes expressed there, and recent studies have identified Defective proventriculus (Dve) as such a repressor (Cho, 2004).

In strong fat mutants, the wing discs become enlarged and have extra folds and outgrowths in the proximal wing. The disproportionate overgrowth of the proximal wing is due to upregulation of Wg in this region, as demonstrated by its suppression by wgspd-fg. At the same time, clones of cells mutant for fat overgrow in other imaginal cells, and fat wgspd-fg discs are still enlarged compared with wild-type discs. Thus, Fat appears to act both by regulating the expression of other signaling pathways (e.g. Wg), and via its own, novel growth pathway. The identification of additional components of this pathway will offer new approaches for investigating its profound influence on disc growth (Cho, 2004).

Delineation of a Fat tumor suppressor pathway

Recent studies in Drosophila of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. This study characterizes functional relationships among Drosophila tumor suppressors and identifies the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown (dco) upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded (ex), hippo, salvador (sav) and mob as tumor suppressor (mats) also share multiple Fat pathway phenotypes but regulate Warts activity independently. These results functionally link what had been four disparate groups of Drosophila tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals (Cho, 2006).

Since Dachs is required for loss of Wts protein in fat mutants, and Dachs encodes a large Myosin protein, a model was considered in which Dachs acts as a scaffold to link Wts to proteins that promote Wts proteolysis, analogous to the roles of Costal2 in Hedgehog signaling, or APC in Wnt signaling. This model predicts that Dachs should be able to bind to Wts. To evaluate this possibility, tagged forms of Dachs and Wts were coexpressed in cultured cells and assayed for coimmunoprecipitation. These experiments identified a specific and reproducible interaction between Dachs and Wts (Cho, 2006).

Recent studies have identified the transcriptional coactivator Yorkie (Yki) as a downstream component of the Hippo pathway and a substrate of Wts kinase activity. Phosphorylation of Yki by Wts inactivates Yki, and overexpression of Yki phenocopies wts mutation. The determination that the Fat tumor suppressor pathway acts through modulation of Wts thus predicts that Yki should also be involved in Fat signaling. When the influence of Yki overexpression was examined on Fat target genes, expression of Wg in the proximal wing, Ser in the proximal leg and fj in the wing and eye were each upregulated by Yki overexpression, consistent with the inference that Fat tumor suppressor pathway signaling acts through Yki (Cho, 2006).

In order to identify additional components of the Fat tumor suppressor pathway, advantage was taken of the observation that loss of fat in clones of cells is associated with an induction of Wingless (Wg) expression in cells just proximal to the normal ring of Wg expression in the proximal wing, reflective of its role in distal-to-proximal wing signaling. It was reasoned that this influence on Wg expression could be used to screen other Drosophila tumor suppressors for their potential to contribute to Fat signaling. Analysis of mutant clones in the proximal wing identified dco, ex, mats, sav, hpo and wts as candidate components of the Fat tumor suppressor pathway. As for fat, mutation of each of these genes is associated with induction of Wg expression specifically in the proximal wing, whereas Wg expression is not affected in more distal or more proximal wing cells. Although Wg expression often seems slightly elevated within its normal domain, the effect of these mutations is most obvious in the broadening of the Wg expression ring. The induction of Wg expression does not seem to be a nonspecific consequence of the altered growth or cell affinity associated with these mutations, since Wg expression is unaffected by expression of the growth-promoting microRNA gene bantam or by expression of genes that alter cell affinity in the proximal wing (Cho, 2006).

dco encodes D. melanogaster casein kinase I delta/epsilon. The overgrowth phenotype that gave the gene its name is observed in allelic combinations that include a hypomorphic allele, dco3, and it is this allele that is associated with induction of Wg. Null mutations of dco actually result in an 'opposite' phenotype: discs fail to grow, and clones of cells mutant for null alleles fail to proliferate. This is likely to reflect requirements for dco in multiple, distinct processes, as casein kinase I proteins phosphorylate many different substrates, and dco has been implicated in circadian rhythms, Wnt signaling and Hedgehog signaling (Cho, 2006).

Mer and ex encode two structurally related FERM domain-containing proteins. ex was first identified as a Drosophila tumor suppressor, whereas Drosophila Mer was first identified based on its structural similarity to human Merlin. Mutation of Mer alone causes only mild effects on imaginal disc growth, but Mer and ex are partially redundant, and double mutants show more severe overgrowth phenotypes than either single mutant. Consistent with this, elevation of Wg expression was observed in ex mutant clones (7/10 proximal wing clones induced Wg) and not in Mer mutant clones (0/8 clones), whereas Mer ex double mutant clones showed even more severe effects on Wg than ex single mutant clones. Because of the partial redundancy between Mer and ex, when possible, focus was placed for subsequent analysis on Mer ex double mutant clones (Cho, 2006).

Wts, Mats, Sav and Hpo interact biochemically, show similar overgrowth phenotypes and regulate common target genes. Mats, Sav and Hpo are all thought to act by regulating the phosphorylation state and thereby the activity of Wts. Mutation of any one of these genes is associated with upregulation of Wg in the proximal wing. The effects of sav (47/84 clones in the proximal wing induced Wg) and hpo (23/31 clones) were weaker than those of mats (19/19 clones) and wts (92/97 clones), but this might result from differences in perdurance or allele strength. Because sav, hpo and mats all act through Wts, focus for most of the subsequent analysis was placed on wts (Cho, 2006).

The observation that mutation of dco, Mer, ex, mats, sav, hpo or wts all share the distinctive upregulation of Wg expression in the proximal wing observed in fat mutants suggests that the functions of these genes are closely linked. To further investigate this, the effects of these tumor suppressors were characterized on other transcriptional targets of Fat signaling. Expression of the Notch ligand Ser is upregulated unevenly within fat mutant cells in the proximal region of the leg disc. A very similar upregulation occurred in dco3, Mer ex, and wts mutant clones. fj is a target of Fat signaling in both wing and eye imaginal discs, and fj expression was also upregulated in dco3, Mer ex, or wts mutant clones. The observation that these genes share multiple transcriptional targets in different Drosophila tissues implies that they act together in a common process (Cho, 2006).

The hypothesis that Fat pathway genes and Hippo pathway genes are linked predicts that not only should Fat target genes be regulated by Hippo pathway genes, but Hippo pathway target genes should also be regulated by Fat pathway genes. The cell cycle regulator CycE and the inhibitor of apoptosis Diap1 (encoded by thread) have been widely used as diagnostic downstream targets to assign genes to the Hippo pathway. Notably, then, clones of cells mutant for fat showed upregulation of both Diap1 and CycE protein expression. Genes whose expression is upregulated within fat mutant cells (such as wg, Ser and fj) have been shown previously to be induced along the borders of cells expressing either fj or dachsous (ds), and Diap1 is also upregulated around the borders of ds- or fj-expressing clones. That thread is affected by fat at a transcriptional level was confirmed by examining a thread-lacZ enhancer trap line. The regulation of Diap1 by the Hippo pathway is thought to be responsible for a characteristic eye phenotype in which an excess of interommatidial cells results from their failure to undergo apoptosis; an increase was also observed in interommatidial cells in fat mutant clones. Upregulation of both Diap1 and CycE is also observed in Mer ex double mutant clones. In dco3 mutant clones, consistent upregulation was detected only for Diap1, and CycE was upregulated only weakly and inconsistently. dco3 also has weaker effects on Wg and fj expression; the weaker effects of dco3 could result from its hypomorphic nature. ex has recently been characterized as another Hippo pathway target, and an ex-lacZ enhancer trap that is upregulated in wts or Mer ex mutant clones is also upregulated in fat or dco3 mutant clones. Analysis of ex transcription by in situ hybridization also indicated that ex is regulated by fat. Altogether, this analysis of Hippo pathway targets further supports the conclusion that the functions of the Fat pathway, the Hippo pathway and the tumor suppressors Mer, ex and dco are linked (Cho, 2006).

Genetic epistasis experiments provide a critical framework for evaluating the functional relationships among genes that act in a common pathway. The relationships was evaluated between each of the tumor suppressors linked to the Fat pathway and dachs, using both wing disc growth and proximal Wg expression as phenotypic assays. dachs is the only previously identified downstream component of the Fat tumor suppressor pathway. It acts oppositely to fat and is epistatic to fat in terms of both growth and gene expression phenotypes (Cho, 2006).

dachs is also epistatic to dco3 for overall wing disc growth and for proximal Wg expression. The epistasis of dachs to dco3 implies that the overgrowth phenotype of dco3 is specifically related to its influence on Fat signaling, as opposed to participation of dco in other pathways. By contrast to the epistasis of dachs to dco3, both wts and ex are epistatic to dachs for disc overgrowth phenotypes, and wts and Mer ex are epistatic to dachs in their influence on proximal Wg expression. Together, these epistasis experiments suggest that dco acts upstream of dachs, whereas Mer ex and wts act downstream of dachs (Cho, 2006).

Because wts and Mer ex have similar phenotypes, their epistatic relationship cannot be determined using loss-of-function alleles. However, overexpression of ex inhibits growth and promotes apoptosis, which suggests that ex overexpression affects ex gain-of-function. Clones of cells overexpressing ex are normally composed of only a few cells, and over time most are lost, but coexpression with the baculovirus apoptosis inhibitor p35 enabled recovery of ex-expressing clones. These ex- and p35-expressing clones were associated with repression of proximal Wg expression during early- to mid-third instar, as has been described for dachs2, consistent with ex overexpression acting as a gain-of-function allele in terms of its influence on Fat signaling. In epistasis experiments using overexpressed ex and mutation of wts, wts was epistatic; Wg was induced in the proximal wing. Additionally, when wts is mutant, coexpression with p35 was no longer needed to ensure the viability and growth of ex-expressing clones, indicating that wts is also epistatic to ex for growth and survival. Consistent with this conclusion, others have recently described phenotypic similarities between Mer ex and hpo pathway mutants and have reported that hpo is epistatic to Mer ex (Cho, 2006).

When Fat was overexpressed, a slight reduction was detected in Wg expression during early- to mid-third instar, suggesting that overexpression can result in a weak gain-of-function phenotype. Clones of cells overexpressing Fat but mutant for dco3 still showed reduced Wg levels, whereas clones of cells overexpressing Fat but mutant for warts showed increased Wg levels. Although experiments in which the epistatic mutation is not a null allele cannot be regarded as definitive, these results are consistent with the conclusion that wts acts downstream of fat and suggest that dco might act upstream of fat (Cho, 2006).

The epistasis results described above suggest an order of action for Fat tumor suppressor pathway genes in which dco acts upstream of fat, fat acts upstream of dachs, dachs acts upstream of Mer and ex, and Mer and ex act upstream of wts. However, the determination that one gene is epistatic to another does not prove that the epistatic gene is biochemically downstream, as it is also possible that they act in parallel but converge upon a common target. Thus, to better define the functional and hierarchical relationships among these genes, experiments were initiated to investigate the possibility that genetically upstream components influence the phosphorylation, stability or localization of genetically downstream (that is, epistatic) components. Focus in this study was placed on the most downstream of these components, Wts. As available antibodies did not specifically recognize Wts in imaginal discs, advantage was taken of the existence of functional, Myc-tagged Wts-expressing transgenes (Myc:Wts) to investigate potential influences of upstream Fat pathway genes on Wts protein. In wing imaginal discs, Myc:Wts staining outlines cells, suggesting that it is preferentially localized near the plasma membrane, and it was confirmed that expression of Myc:Wts under tub-Gal4 control can rescue wts mutation. Notably, mutation of fat results in a reduction of Myc:Wts staining. As Myc:Wts is expressed under the control of a heterologous promoter in these experiments, this must reflect a post-transcriptional influence on Wts protein. fat does not exert a general influence on the levels of Hippo pathway components; fat mutant clones had no detectable influence on the expression of hemagglutinin epitope-tagged Sav (HA:Sav) (Cho, 2006).

The decrease in Wts protein associated with mutation of fat contrasts with studies of the regulation of Wts activity by the Hippo pathway, which have identified changes in Wts activity due to changes in its phosphorylation state. To directly compare regulation of Wts by Fat with regulation of Wts by other upstream genes, Myc:Wts staining was examined in ex, sav and mats mutant clones. In each of these experiments, the levels and localization of Myc:Wts in mutant cells was indistinguishable from that in neighboring wild-type cells (Cho, 2006).

Since Myc:Wts appears preferentially localized near the plasma membrane, it was conceivable that the apparent decrease in staining reflected delocalization of Wts, rather than destabilization. To investigate this possibility, Wts levels were examined by protein blotting. Antisera against endogenous Wts recognized a band of the expected mobility in lysates of wing imaginal discs or cultured cells, and this band was enhanced when Wts was overexpressed. The intensity of this band was reproducibly diminished in fat or dco3 homozygous mutant animals but was not diminished in fat or dco3 heterozygotes or in ex mutants. Conversely, levels of Hpo, Sav, Mer or Mats were not noticeably affected by fat mutation (Cho, 2006).

The determination that Wts is affected by Fat, together with the genetic studies described above, place Wts within the Fat signaling pathway, as opposed to a parallel pathway that converges on common transcriptional targets. Indeed, given that even hypomorphic alleles of wts result in disc overgrowth, the evident reduction in Wts levels might suffice to explain the overgrowth of fat mutants. As a further test of this possibility, Wts levels were examined in fat dachs double mutants. As the influence of Fat on gene expression and growth is absolutely dependent upon Dachs, if Fat influences growth through modulation of Wts, its influence on Wts levels should be reversed by mutation of dachs. Examination of Myc:Wts staining in fat dachs clones and of Wts protein levels in fat dachs mutant discs confirmed this prediction (Cho, 2006).

Prior observations, including the influences of fat and ds on gene expression, and the ability of the Fat intracellular domain to rescue fat phenotypes, suggested that Fat functions as a signal-transducing receptor. By identifying kinases that act both upstream (Dco) and downstream (Wts) of the Fat effector Dachs and by linking Fat to the transcriptional coactivator Yki, these results have provided additional support for the conclusion that Fat functions as a component of a signaling pathway and have delineated core elements of this pathway from receptor to transcription factor. Fat activity is regulated, in ways yet to be defined, by Ds and Fj. The influences of Fat on gene expression, growth, and cell affinity, as well as on Wts stability, are completely dependent on Dachs, indicating that Dachs is a critical effector of Fat signaling. Since Dachs can associate with Wts or a Wts-containing complex, it is suggested that Dachs might act as a scaffold to assemble a Wts degradation complex. The observations that Fat, Ds and Fj modulate the subcellular localization of Dachs, that Wts is preferentially localized near the membrane and that Dachs accumulates at the membrane in the absence of Fat, suggest a simple model whereby Fat signaling regulates Wts stability by modulating the accumulation of Dachs at the membrane and thereby its access to Wts. The working model is that dco3 is defective in the phosphorylation of a substrate in the Fat pathway, but the recessive nature of dco3, the genetic epistasis experiments, and biochemical experiments argue that this substrate is not Wts, and further work is required to define the biochemical role of Dco in Fat signaling (Cho, 2006).

In addition to identifying core components of the Fat pathway, the results establish close functional links between the Fat pathway, the Hippo pathway and the FERM-domain tumor suppressors Mer and Ex. The common phenotypes observed among these tumor suppressors can be explained by their common ability to influence Wts. However, they seem to do this in distinct ways, acting in parallel pathways that converge on Wts rather than a single signal transduction pathway. The Fat pathway modulates levels of Wts, apparently by influencing Wts stability. By contrast, the Hippo pathway seems to regulate the activity of Wts by modulating its phosphorylation state. Thus, Wts seems to act as an integrator of distinct growth signals, which can be transmitted by both the Fat pathway and the Hippo pathway. It has been suggested that Mer and Ex also act through the Hippo pathway, although present experiments cannot exclude the possibility that Mer and Ex act in parallel to Hpo. Moreover, it should be noted that Mats might regulate Wts independently of Hpo and Sav and hence function within a distinct, parallel pathway. Although it is simplest to think of parallel pathways, there is also evidence for cross-talk. fj and ex are both components and targets of these pathways. Thus, they can be regarded as feedback targets within their respective pathways, but their regulation also constitutes a point of cross-talk between pathways. Another possible point of cross-talk is suggested by the observation that levels of Fat are elevated within Mer ex mutant clones. Although the potential for cross-talk complicates assessments of the relationships between tumor suppressors, the observations that fat, dco3 and dachs affect Warts protein levels in vivo, whereas ex, hippo, sav and mats do not, argues that there are at least two distinct pathways that converge on Warts. This conclusion is also consistent with the observations that ex, hippo, sav and mats can influence Wts phosphorylation in cultured cell assays, but Fat, Dachs and Dco do not (Cho, 2006).

Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, wtsP2, results in strong overgrowth phenotypes. The effects of Yki overexpression on growth and target gene expression can be even stronger than those of fat or wts mutations, which suggests that Yki levels become limiting when upstream tumor suppressors are mutant (Cho, 2006).

fat encodes a protocadherin, which in the past has led to speculation that its influences on growth and cell affinity might result from Fat acting as a cell adhesion molecule. However, all of the effects of fat on growth and affinity require dachs, which is also required for the effects of fat on transcription. Additionally, targets of Fat signaling include genes that can influence growth and affinity; recent studies identified an influence of fat on E-cadherin expression, and as describe in this study, Fat influences CycE and Diap1 expression. Thus, one can account for the influence of fat on growth and affinity by its ability to regulate gene expression. fat interacts genetically with other signaling pathways, including EGFR and Wnt, and in some cells Fat signaling also influences the expression of ligands (such as Wg and Ser) for other signaling pathways. Regulation of these ligands contributes to fat overgrowth phenotypes, but since clonal analysis indicates that fat is autonomously required for growth control in most imaginal cells, the principal mechanism by which fat influences growth presumably involves the regulation of general targets (Cho, 2006).

Normal tissue growth and patterning depend on a relatively small number of highly conserved intercellular signaling pathways. The Fat pathway is essential for the normal regulation of growth and PCP in most or all of the external tissues of the fly and also participates in local cell fate decisions. In this regard, its importance to fly development can be considered comparable to that of other major signaling pathways. Although the biological roles and even the existence of a Fat pathway in mammals remain to be demonstrated, there is clear evidence that the mammalian Warts homologs Lats1 and Lats2 act as tumor suppressors and that a mammalian Yorkie homolog, YAP, can act as an oncogene. Moreover, other genes in the Drosophila Fat pathway have apparent structural homologs in mammals. Thus, it is likely that mammals also have a Fat tumor suppressor pathway that functions in growth control (Cho, 2006).

Morphogen control of wing growth through the fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. The Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth (Rogulja, 2008).

Studies of regeneration first led to models that proposed that growth could be influenced by gradients of positional values, with steep gradients promoting growth and shallow gradients suppressing growth. Experimental manipulations of Dpp pathway activity in the Drosophila wing supported this concept, but have left unanswered the question of how differences in the levels of Dpp pathway activity perceived by neighboring cells are actually linked to growth. This study has established that the Fat signaling pathway provides this link. Dpp signaling influences the Fat pathway; the expression of upstream Fat pathway regulators, the subcellular localization of Fat pathway components, and downstream transcriptional outputs of Fat signaling are all affected by Dpp signaling. The effects that Tkv and Brk expression have on the expression of Fat target genes parallels their effects on BrdU labeling and depend genetically on Fat signaling (Rogulja, 2008).

Dpp signaling impinges on Fat signaling upstream of Fat, as the expression of both of its known regulators, Fj and Ds, is regulated by Dpp signaling. Although the Fat signaling pathway was only recently discovered, and understanding of Fat signaling and its regulation remains incomplete, the inference that Fat signaling is normally influenced by the Dpp morphogen gradient is supported by the polarized localization of Dachs in wild-type wing discs. Near the D-V compartment boundary, the vector of Dachs polarization parallels the vector of the Dpp morphogen gradient, and the consequences of altered Dpp pathway activity confirm that the correlation between them is reflective of a functional link. The expression of Fj and Ds and the localization of Dachs are also polarized along the D-V axis. The implication that signaling downstream of the D-V compartment boundary thus also impinges on Fat signaling, and indeed may also influence growth through this pathway, is consistent with the observation that normal wing growth requires both A-P and D-V compartment boundary signals, and is further supported here by the observation that Notch activation affects both fj expression and Dachs localization (Rogulja, 2008).

The results argue that Fat signaling is influenced by the graded expression of its regulators: uniform expression of Fj and Ds can activate Fat signaling and thereby inhibit growth, whereas juxtaposition of cells expressing different levels of either Fj or Ds can inhibit Fat signaling and thereby promote growth. Here, a model is proposed to explain how Fat signaling can be modulated by Fj and Ds gradients. Although aspects of the model remain speculative, it provides an explanation for a number of observations that would otherwise appear puzzling, and serves as a useful framework for future studies (Rogulja, 2008).

Central to the model is the inference that the interaction between Ds and Fat activates Fat. This inference is well supported by the observations that mutation or downregulation of ds results in overgrowth and upregulation of Diap1, whereas uniform overexpression of Ds inhibits growth and Diap1 expression. A second key aspect of the model is that once activated by Ds, Fat locally transmits a signal to a complex at the membrane. An important corollary to this is that if Fat and Ds are not engaged around the entire circumference of a cell, then there could be a region where Fat is locally inactive. This is hypothetical, but the Fat-dependent polarization of Dachs implies that there can be regional differences in Fat activity within a cell. Local Fat signaling is then proposed to locally promote Warts stability and activity, and thereby locally antagonize Yki activity. Conversely, a local absence of Fat signaling could result in a local failure to phosphorylate Yki, which could then transit to the nucleus, where it would promote the expression of downstream target genes. Formally, this model treats Fat signaling like a contact inhibition pathway: if Fat is engaged by Ds around the entire circumference of a cell, then Fat is active everywhere and downstream gene expression is off; however, if Fat is not active on even one side of a cell, then Yki-dependent gene expression can be turned on and growth can be promoted (Rogulja, 2008).

In this model, graded expression of Fat regulators, like Fj and Ds, could modulate Fat signaling by polarizing Fat activity within a cell. In theoretical models of PCP, even shallow gradients of polarizing activity can be converted to strong polarity responses through positive-feedback mechanisms. How this might be achieved in Fat signaling is not yet clear, but the polarized localization of Dachs implies that, at some level, Fat activity is normally polarized in wild-type animals, even where the Fj and Ds expression gradients appear relatively shallow. Importantly, this polarization hypothesis provides a solution to the puzzle of how Ds could act as a ligand to activate Fat, yet inhibit Fat along the edges of Ds-expressing clones. In this model, Ds overexpression in clones polarizes Fat activity, possibly through its ability to relocalize Fat. This would allow a strong derepression of Yki on the side of the cell opposite to where Ds and Fat are actually bound, resulting in the induction of Yki:Scalloped target gene expression and promotion of cell proliferation. Propagation of this polarization, e.g., through the influence of Fat-Ds binding on Fat and Ds localization, might explain the spread of effects beyond immediately neighboring cells. Conversely, uniform expression of Ds would generate cells presenting a ligand that activates Fat and dampens the relative difference in expression levels between neighboring cells. Yki would thus remain sequestered around the entire cell circumference, consistent with the reduced growth and Diap1 expression observed. A dampening of gradients could also explain why the induction of Fat/Hippo target gene expression or BrdU labeling associated with clones expressing Ds, Fj, or TkvQ-D is biased toward cells outside of clones (Rogulja, 2008).

The hypothesis of Fat polarization and local signal transduction also suggests a solution to another puzzle. In terms of their effects on tissue polarity and Dachs localization, Fj and Ds always behave as though they have opposite effects on Fat. Conversely, in terms of their effects on cell proliferation and downstream gene expression, Fj and Ds behave as though they have identical effects on Fat. To explain this, it is proposed that Fj acts oppositely to Ds, by, for example, antagonizing Ds-Fat binding. The influence of Ds and Fj on polarity would be a function of the direction in which they polarize Fat activity, which, based on their effects on epitope-tagged protein Dachs:V5, is opposite. In contrast, their influence on downstream gene expression and growth would be a function of the degree to which they polarize Fat activity, which could be the same. In other words, their influence on polarity would be a function of the vector of their expression gradients, and their influence on growth would be a function of the slope. However, since Dachs:V5 generally appears to be strongly polarized, the actual interpretation of Fj and Ds gradients may involve feedback amplification and threshold responses rather than providing a continuous response proportional to the gradient slope (Rogulja, 2008).

The results have provided a molecular understanding of a how a gradient of positional values, established by the morphogen Dpp and reflected, at least in part, in the graded expression of Fj and Ds, can influence growth. However, it is clear that other mechanisms must also contribute to the regulation of wing growth. The relative contribution of Fat gradients to wing growth can be estimated by considering the size of the wing in dachs mutants, or when Fj and Ds are expressed ubiquitously, as, in either case, it would be expected that the derepression of Yki associated with normal Fat signaling gradients was abolished. In both cases, the wing is less than half its normal size. Fat signaling could thus be considered a major, but by no means the sole, mechanism for regulating wing growth. The determination that not all wing growth depends on the regulation of Fat activity fits with the observation that Dpp signaling promotes growth in at least two distinct ways, one dependent upon its gradient, and the other dependent upon its levels. Other models for wing growth, including a Vestigial-dependent recruitment of new cells into the wing, and an inhibition of Dpp-promoted wing growth by mechanical strain, have also been proposed. It is emphasized that these models are not incompatible with the conclusion that a Fat gradient influences growth. Rather, it is plausible, and even likely, that multiple mechanisms contribute to the appropriate regulation of wing growth. Indeed, it is expected that a critical challenge for the future will be to define not only the respective contributions of these or other mechanisms to growth control, but also to understand feedback and crosstalk processes that influence how these different mechanisms interact with each other (Rogulja, 2008).

The Salvador/Warts/Hippo pathway controls regenerative tissue growth in Drosophila melanogaster

During tissue regeneration, cell proliferation replaces missing structures to restore organ function. Regenerative potential differs greatly between organs and organisms; for example some amphibians can regrow entire limbs whereas mammals cannot. The process of regeneration relies on several signaling pathways that control developmental tissue growth, and implies the existence of organ size-control checkpoints that regulate both developmental, and regenerative, growth. This study explored the role of one such checkpoint, the Salvador-Warts-Hippo (SWH) pathway, in tissue regeneration. The Salvador-Warts-Hippo pathway limits tissue growth by repressing the Yorkie transcriptional co-activator. Several proteins serve as upstream modulators of this pathway including the atypical cadherins, Dachsous and Fat, while the atypical myosin, Dachs, functions downstream of Fat to activate Yorkie. Using Drosophila imaginal discs this study showed that Salvador-Warts-Hippo pathway activity is repressed in regenerating tissue and that Yorkie is rate-limiting for regeneration of the developing wing. Regeneration is compromised in dachs mutant wing discs, but proteins in addition to Fat and Dachs are likely to modulate Yorkie activity in regenerating cells. In conclusion these data reveal the importance of Yorkie hyperactivation for tissue regeneration and suggest that multiple upstream inputs, including Fat-Dachsous signaling, sense tissue damage and regulate Yorkie activity during regeneration of epithelial tissues (Grusche, 2011).

This study has shown that the SWH pathway regulates regenerative growth of Drosophila imaginal discs. Hyperactivation of Yki activity was observed in regenerating cells following tissue disruption using a range of genetic and surgical assays, and full Yki activity was essential for efficient wing regeneration. These findings imply that the important role that the SWH pathway has in specification of organ size during development involves an ability to control proliferation in response to tissue damage. These results extend recent data describing a role for the SWH pathway in regeneration of the cricket leg (Bando, 2009). Interestingly, while this manuscript was under review, the SWH pathway was found to modulate regeneration of other epithelial tissues such as the adult Drosophila gut and the murine small intestine in response to chemical and bacterial insults (Cai, 2010; Karpowicz, 2010; Shaw, 2010; Staley, 2010). Coupled with the data in this study, these findings suggest that the SWH pathway is a regulator of 'epithelial fitness', i.e. it is primed to sense damage in epithelial tissues, and to coordinate a robust repair mechanism in response to damage stimuli (Grusche, 2011).

Other growth-regulatory proteins such as Wg and Myc are important for D. melanogaster imaginal disc regeneration, as is the JNK pathway. Interestingly however, regeneration of wing imaginal discs does not appear to be modulated by all growth pathways. For example, another study provided evidence that the target of rapamycin pathway does not modulate regenerative growth of the wing imaginal disc. This lends weight to the hypothesis that the SWH pathway has a specific role in regulating regeneration, rather than regenerative tissue growth being controlled by all growth-regulatory pathways (Grusche, 2011).

What then are the upstream signaling mechanisms that, in response to tissue damage, permit Yki hyperactivation in regenerating cells? Given that a reduction in Wts-dependent phosphorylation of Yki was observed in regenerating tissue, obvious candidate signaling inputs are the three major classes of upstream regulatory proteins of the SWH pathway; the Ft–Ds branch, the KEM complex and the apicobasal polarity proteins, Lgl, Crb and aPKC. The Ft and Ds cadherins limit cell proliferation by engaging in physical interactions with each other on neighbouring cells and repressing the Yki activator, Dachs. Ft and Ds are also required for oriented mitoses of cells neighbouring apoptotic cells. Given this, it is hypothesized that in a damaged tissue, cell–cell contacts would be broken, causing Ft–Ds signaling to be abrogated and a resultant elevation in Dachs, and hence Yki, activity. dachs mutant wing imaginal discs displayed impaired regenerative capacity following γ-irradiation-induced apoptosis, thus implicating the Ft–Ds signaling module in the control of Yki-dependent regeneration. However, genetic wounding and tissue ablation assays of this study have suggested that Yki hyperactivation in regenerating cells involves signaling inputs in addition to the Ft–Ds branch of the SWH pathway, since Yki activation was still observed in regenerative tissue in the absence of dachs or ft (Grusche, 2011).

What are possible reasons for this apparent contradiction? Firstly, there could be subtle differences in Yki activation in wild type versus dachs cells that were not observed in this system, since lacZ enhancer trap lines offer poorly quantitative data, especially when comparing expression levels between independent tissues. Secondly, there is evidence that Ds controls SWH pathway activity by functioning not only as a ligand for Ft but also as a receptor. Whether Ds signaling to downstream SWH pathway proteins requires Dachs, has not yet been determined. Thirdly, Ft and Ds signaling might influence SWH pathway activity independent of Dachs in regenerating cells. For example Ft has been proposed to control Yki activity by influencing the subcellular localization and levels of the Ex protein. However this is is thought to be unlikely given that Yki hyperactivation in regenerative tissue was maintained in ex and ft mutant backgrounds. A more likely scenario is that upstream SWH pathway proteins in addition to Ft, Ds and Dachs participate in control of Yki activity in regenerative tissue growth. Candidates include the KEM complex components, Mer and Kibra, as well as the apicobasal polarity proteins, Crumbs and atypical Protein Kinase C. Another possibility is that the altered physical status of cells that surround tissue wounds might regulate SWH pathway activity via a mechanism such as tension, which has been hypothesized to influence the specification of Drosophila wing size. It is also plausible that signaling pathways that are known to control regeneration, such as the JNK pathway, regulate the SWH pathway during tissue regeneration (Grusche, 2011).

Regenerating cells have been postulated to revert to a more primitive differentiation state. Recently the murine Yki orthologue, YAP, was found to promote stem cell pluripotency along with other transcriptional regulatory proteins including Myc. Significantly, Myc has previously been shown to be induced in regenerating Drosophila tissues, and to be a potent driver of the regenerative response. Therefore a plausible hypothesis is that Yki hyperactivation promotes regeneration by altering the cell's transcriptional program, and thus cellular plasticity, in conjunction with Myc (Grusche, 2011).

Regeneration has often been used as a paradigm to describe the idea of an organ size-checkpoint that limits the size of tissues during both development and adult homeostasis in metazoans. The current findings suggest that the SWH pathway, which is a crucial regulator of organ size, also controls regeneration, providing evidence for a molecular link between the two processes. In the future, it will be interesting to determine whether the SWH pathway controls regeneration in animals with robust regenerative capacity such as axolotls, hydra and planaria. It will also be important to determine whether the SWH pathway controls tissue regeneration in mammals; if this proves to be the case, modulation of SWH pathway activity might be a powerful approach to modulate tissue regeneration post trauma or surgery. Finally, aberrant tissue regeneration upon chronic injury or inflammation has been proposed to contribute to tumour formation. Since increasing evidence points to a role of aberrant SWH pathway signaling in cancer, the current findings provide a potential molecular link between regeneration and tumorigenesis (Grusche, 2011).

Planar polarization of the atypical myosin Dachs orients cell divisions in Drosophila

Tissues can grow in a particular direction by controlling the orientation of cell divisions. This phenomenon is evident in the developing Drosophila wing epithelium, where the tissue becomes elongated along the proximal-distal axis. Orientation of cell divisions in the wing requires planar polarization of an atypical myosin, Dachs. Evidence suggests that Dachs constricts cell-cell junctions to alter the geometry of cell shapes at the apical surface and that cell shape then determines the orientation of the mitotic spindle. Using a computational model of a growing epithelium, it was shown that polarized cell tension is sufficient to orient cell shapes, cell divisions, and tissue growth. Planar polarization of Dachs is ultimately oriented by long-range gradients emanating from compartment boundaries, and is therefore a mechanism linking these gradients with the control of tissue shape (Mao, 2011).

Recently, Dachs was found to be localized in a planar-polarized manner along the P-D axis in response to the Dachsous gradient. This study observed that the localization of Dachs correlates with the orientation of cell divisions and tissue growth in the developing fly wing. Dachs localizes to the distal side of each cell's apical surface, and clones tend to grow preferentially along the P-D axis to form elongated shapes. This intriguing correlation has led to a speculation that Dachs might mediate the orientation of cell divisions and tissue growth in the fly wing (Mao, 2011).

To test this hypothesis, the behavior of marked clones of cells was examined in wing discs lacking functional Dachs protein (dachs mutant), and wing discs in which Dachs is abnormally localized around the entire apical cell surface (fat mutant). The normal elongation of clone shapes along the P-D axis is completely disrupted in either dachs mutant discs, with clones tending to be rounded and misoriented. Orientation of mitotic spindles is also disrupted in these mutants. This failure to orient cell divisions in dachs and fat mutants results in abnormally shaped adult wings that are reduced in the P-D axis relative to wild-type controls. This shape change is evident despite opposite effects of the two mutations on size. These results indicate that planar polarization of Dachs is essential for orienting cell divisions and tissue growth (Mao, 2011).

Whether reorientation of Dachs is sufficient to reorient cell divisions and tissue growth was examined. Dachsous was expressed with the dpp.Gal4 driver, which is expressed in a gradient along the A-P axis in the anterior compartment. This ectopic gradient of Dachsous runs perpendicular to the endogenous gradient and repolarizes Dachs. As a result, clones are reoriented perpendicular to the P-D axis, as are mitotic spindles. In adult wings, the ectopic Dachsous gradient drives elongation of the wing perpendicular to the P-D axis, in the anterior compartment (Mao, 2011).

How might Dachs control the orientation of the mitotic spindle? Dachs might directly orient the mitotic spindle by tethering. Alternatively, since Dachs is a myosin, it might indirectly orient the spindle by controlling cell shape. Existing evidence supports the latter view: Mitotic spindles align along the long axis of the cell prior to division in both yeast and mammalian cells. In the case of epithelial cells, spindles are restricted to the plane of the epithelium and their orientation may be affected by apical surface geometry. Apical cell shape and the orientation of cell division were examined in live wing discs in culture; cells were found to divide along their long apical axis. This correlation persists even in cases where divisions are not oriented along the P-D axis. These results indicate that the geometry of apical cell-cell junctions determines the orientation of cell division (Mao, 2011).

To test whether Dachs controls cell shape at the apical surface, clones of dachs or fat mutant cells were examined in an otherwise wild-type disc. Cells mutant for dachs are, on average, 60% more dilated in their apical surface area than wild type. Cells mutant for fat are, on average, 40% more constricted than neighboring wild-type cells. When fat mutant cells are also mutant for dachs, their apical surface area reverts to that of single dachs mutants. Overexpression of Dachs enhances constriction of fat mutant cells. These results show that Dachs exerts a contractile force on apical cell junctions. Since Dachs is normally planar-polarized, it would be predicted to constrict cell-cell junctions at the distal end of each cell and the proximal end of its neighbor. Measurement of tension indicates that distal (and proximal) cell-cell junctions are under more tension than others. Consequently, the cell must grow by lengthening its other cell-cell junctions, resulting in cell shape elongation in the P-D axis prior to division, which then orients the mitotic spindle. This force-driven model is supported by the fact that clones of cells mutant for dachs show normally oriented growth when surrounded by wild-type cells that are capable of nonautonomously exerting force on the mutant clone. Thus, Dachs appears to act by exerting mechanical force because its loss can be compensated by restoring these forces (Mao, 2011).

To further test the proposal, a computational model of a growing epithelial tissue was built that was adapted from a previous model. It was found that, compared with isometric apical tension, polarized apical tension is sufficient to orient cell divisions and tissue growth. As in vivo, cell divisions are only imperfectly correlated with the P-D axis in this model; this emergent behavior is in fact important to generate the type of clone shapes observed in the developing wing. If cell divisions are forcibly oriented in the P-D axis, abnormal clone shapes result and the link between cell shape and orientation of division is broken, indicating that Dachs is unlikely to directly orient the spindle in vivo. This model therefore supports the idea that Dachs indirectly orients the mitotic spindle by polarizing apical constriction to promote cell shape elongation in the P-D axis (Mao, 2011).

This model also explains an apparent paradox: Despite the presence of planar-polarized Dachs, the apical geometry of most wing cells is not elongated in the P-D axis. Indeed, elongation of cells in the P-D axis is commonly observed only in those cells about to undergo division. The simulations show that this is because elongated cells divide to produce daughter cells of more rounded or random shapes. The polarized tension exerted by Dachs is of moderate strength, and hence takes time to promote elongation in the P-D axis. Thus, only older cells ready to divide once more tend to show elongated forms. Furthermore, some cells never manage to elongate along the P-D axis, explaining why not all cell divisions occur in the P-D axis. The model shows how this complex behavior of cells in a tissue emerges simply from the mechanical properties of a dividing epithelium (Mao, 2011).

It is interesting to compare the function of the Dachs myosin in the proliferating wing epithelium with the function of myosin-II in convergent extension movements of the quiescent embryonic epithelium. Both myosins appear to generate anisotropic junctional tension, but at different strengths. In embryos, myosin-II is planar-polarized at gastrulation and exerts very strong tension, collapsing cell-cell junctions entirely such that cells rearrange their positions and intercalate. In the wing disc, Dachs appears much weaker than myosin-II because convergent extension movements are not obvious in live-imaged wing disc epithelia. Thus, orientation of cell divisions appears to require much more subtle polarization of cell tension than convergent extension, and this may explain why an atypical myosin, rather than myosin-II, orients cell divisions. It will be interesting to investigate the role of Dachs in other oriented cell division scenarios, such as wound healing (Mao, 2011).

In conclusion, planar polarization of Dachs links long-range gradients of secreted morphogens - known to be the fundamental organizers of tissue pattern, size, and shape -- with the orientation of cell divisions and tissue growth during development. The mechanism that are proposed (a gradient directing the planar polarization of a molecule, which then orients cell behavior) may prove to be a widespread mechanism for organizing tissue development (Mao, 2011).

The DHHC palmitoyltransferase approximated regulates Fat signaling and Dachs localization and activity

Signaling via the large protocadherin Fat (Ft), regulated in part by its binding partner Dachsous (Ds) and the Golgi-resident kinase Four-jointed (Fj), is required for a variety of developmental functions in Drosophila. Ft and, to a lesser extent, Ds suppress overgrowth of the imaginal discs from which appendages develop and regulate the Hippo pathway. Ft, Ds, and Fj are also required for normal planar cell polarity (PCP) in the wing, abdomen, and eye and for the normal patterning of appendages, including the spacing of crossveins in the wing and the segmentation of the leg tarsus. Ft signaling has been shown to be negatively regulated by the atypical myosin Dachs. This study identifies an additional negative regulator of Ft signaling in growth control, PCP, and appendage patterning, the Approximated (App) protein. App encodes a member of the DHHC family, responsible for the palmitoylation of selected cytoplasmic proteins. Evidence is provided that App acts by controlling the normal subcellular localization and activity of Dachs (Matakatsu, 2008).

Crossvein spacing and tarsal leg segmentation are extremely sensitive to changes in Fat (Ft) activity; they are disrupted in weak Ft-pathway mutants that have no obvious growth or planar cell polarity (PCP) defects. Similar disruption occurs in app1 homozygotes: The distance between the anterior crossvein (ACV) and posterior crossvein (PCV) is reduced, and one or more tarsal leg joints are lost or reduced. app1 hemizygote wings also have weak PCP defects. Ethyl methanesulfonate was used to generate additional mutations that failed to complement app1. Of these, appe6 was semilethal in homozygotes and hemizygotes, and escaper adults had more extensive wing PCP defects, both proximally and in a distal region between the third and fourth longitudinal veins. They also had abdominal PCP defects: In the anterior compartment, most hairs point in the normal posterior direction, but polarity was disturbed around the anterior-posterior (A/P) boundary and extensively reversed in the posterior compartment. appe6 appears amorphic, given that the defects were not noticeably stronger in hemizygotes (Matakatsu, 2008).

The development of PCP in the pupal wing is accompanied by the polarized redistribution of the 'core' planar-polarity proteins to the proximal, distal, or proximal and distal faces of single wing cells. PCP mutants can be separated according to their effects on this polarization. Mutations in the core PCP proteins reduce the levels and block the polarization of the other core PCP proteins, whereas changes in ft, ds, or fj expression can reorient core PCP protein polarization along inappropriate axes. The levels of the core PCP protein Flamingo (Fmi, also known as Starry night) were not reduced in appe6 mutant clones (more than 30 examined), and Fmi polarization was in some cases reoriented. This further supports App's involvement in Ft signaling (Matakatsu, 2008).

app was mapped to a portion of 69A2-A4 containing seven known or predicted genes. app1, appe1, appe3, and appe6 all contained mutations in the 5' coding exons of one of these, the CG5620 Flybase gene prediction. A UAS-driven RNAi transgene was constructed corresponding to the 5' end of CG5620; it was expressed in developing dorsal wings by using ap-gal4; this produced app-like wing and PCP defects on the dorsal surface (Matakatsu, 2008).

However, the 3' end of the CG5620 coding prediction is in error. Products obtained by using 3' RACE contained instead the 3' exon of the adjacent CG17144 prediction, as did a BDGP EST. This transcript was called app-RA, and it was shown that the corresponding App-PA protein is produced in flies. Another EST predicts a shorter transcript called app-RB. No transcripts were found covering the entire final coding exon of CG5620 in embryonic or larval cDNA libraries or by RACE. However, the full CG5620 prediction is conserved in Drosophila pseudoobscura, suggesting that it might be utilized; this was termed app-RC (Matakatsu, 2008).

The wing and leg defects of app homozygotes were rescued by expressing UAS-app-RA, UAS-app-RB, or UAS-app-RC with either act-gal4 or en-gal4. Overexpression of higher levels of UAS-app with strong drivers such as ap-gal4 or tub-gal4 also disrupted PCP in the proximal wing and abdomen (Matakatsu, 2008).

app encodes a member of the DHHC protein family responsible for adding palmitates to cytoplasmic proteins. Eukaryotes contain multiple members of the DHHC family, with 8 predicted in yeast, 23 in mammals, and 20 in Drosophila. The region common to all predicted App isoforms contains four predicted transmembrane domains, with a DHHC cysteine-rich domain (DHHC-CRD) located between the second and third transmembrane domains. It is likely that the DHHC-CRD is located on the cytoplasmic side of the membrane, as in the yeast DHHC protein AKR1 (Matakatsu, 2008 and references therein).

Alignments using the region common to the App isoforms indicate that App is in the same subfamily as the human ZDHHCs 9, 14, and 18 and is the Drosophila protein most similar to yeast ERF2. The similarity between App and these proteins drops off at the C-terminal end of the common region. The divergent C termini of App-PA and App-PC have no significant similarity to each other or to other proteins in the database outside the drosophilids, except for a short region at the C terminus of App-PA that is similar to predicted App homologs from the insects Tribolium and Apis. App-PB has a much shorter C terminus. Because any of the putative app transcripts rescued the app leg and wing phenotypes, the different C termini are dispensable for these phenotypes (Matakatsu, 2008).

app1 contains a missense mutation N-terminal to the DHHC-CRD domain and an aberration that introduces a frameshift predicted to truncate the C-terminal end of App-PA. appe3 contains a missense mutation that changes a conserved cysteine in the DHHC-CRD. Because the DHHC-CRD is required for palmitoyltransferase activity, this supports a role for palmitoylation in Ft signaling. appe6 and appe1 contain nonsense mutations predicted to truncate the protein prior to or toward the end of the DHHC-CRD, respectively, and therefore appe6 is likely null for App function (Matakatsu, 2008).

Two antisera were generated, one specific for App-PA and one for the common region. Both antisera uniformly stained embryos, imaginal discs, and pupal wings, and staining was lost from mitotic homozygous appe6 clones, confirming the expression of the App-PA isoform; similar results with anti-App-common. There is no obvious asymmetric distribution of the App protein along the proximodistal or anterior-posterior axes of imaginal discs or pupal wings. However, staining was especially strong in the apical cell cortex, and this concentration did not extend more basally to the adherens junction marker DE-cadherin. This is similar to the distribution of Ft and Ds, and there is overlap between the regions where App, Ds, and Ft are concentrated. This result is surprising because human ZDHHC9, 14, and 18 and yeast ERF2 are concentrated in the Golgi or ER; only a few, less similar ZDHHCs have been detected at the plasma membrane. No significant overlap was observed between App and Golgi or ER markers in wing discs. Although App must traffic through the ER and Golgi, these results suggest that App is active in the plasma membrane, in or near the apical region where Ft and Ds are concentrated (Matakatsu, 2008).

However, App does not detectably regulate Ft and Ds levels or their binding. The binding between Ft and Ds stabilizes both proteins at the cell surface in wing discs, but appe6 clones did not affect Ft or Ds levels or distribution. Creating artificial boundaries of ft or ds expression also strongly polarizes wing PCP, as do boundaries of the Fj kinase that phosphorylates Ft and Ds and modulates their levels. If App affected Ft-Ds levels or binding, App boundaries would be expected to affect PCP. However, small to moderately sized appe6 clones did not affect PCP, whereas large clones only affected PCP in the regions of the wing where defects were observed in appe6 homozygotes. There was no tendency to reorient hairs at clone boundaries, and only rarely did regions with altered PCP affect PCP in adjacent wild-type cells; these findings may be due to altered cell interactions mediated by the core polarity proteins. Sharp boundaries of UAS-app-RA misexpression (e.g., driven with the posterior driver en-gal4) also had no effect on PCP. Anti-App staining was not altered in wing discs by ft or ds clones. Thus, despite their colocalization, there is no evidence that App physically interacts for Ft or Ds (Matakatsu, 2008).

PCP defects, reduced crossvein spacing, and lost tarsal leg joints can be caused by either gains or losses in Ft signaling, so the phenotypes of app ft double mutants were examined. ftfd and ftG-rv are likely null alleles predicted to truncate Ft N-terminal to its transmembrane region. ftfd homozygotes and ftfd/ftG-rv die during late pupal stages with strongly overgrown imaginal discs and disc-derived tissues; late pupal abdomens are not overgrown but have strong PCP defects. These phenotypes were partially rescued in ftfd; appe6 and ftfd/ftG-rv; appe6 flies: Overgrowth and extra folding of imaginal discs were suppressed and lethality and abdominal PCP defects were reduced. PCP was still defective in proximal portions of the wing but was normal in the distal wing, in contrast to the distal defects in viable ft18 wings. Thus, App acts genetically downstream of and in opposition to Ft in both growth control and PCP (Matakatsu, 2008).

The effects of app mutations on Ft signaling are strikingly similar to those caused by reducing the function of the atypical myosin Dachs). Like app mutations, dachs (d) mutations partially suppress the overgrowth and PCP defects caused by loss of Ft. The adult phenotypes are also similar, although the defects in d null mutants are stronger than those of appe6. Null dGC13 hemizygotes and hypomorphic d1 homozygotes reduce tarsal leg segmentation and the distance between the ACV and PCV and cause mild wing PCP defects that are quite similar to those observed in null app mutants. Like appe6 clones, dGC13 clones had PCP defects when in regions of the wing where defects occur in d homozygotes, and Fmi polarization was reoriented in d1 pupal wings. d mutants also had abdominal PCP defects similar to those in app mutants: Polarity was almost normal in the anterior compartment, but abnormal near the A/P compartment boundary and reversed in the posterior compartment (Matakatsu, 2008).

Therefore the effects of App on the levels and distribution of a V5-tagged Dachs protein were examined. Dachs:V5 normally accumulates at higher levels in the apical cell cortex in wing imaginal discs, overlapping the region of high anti-App staining. Apical Dachs accumulation was greatly reduced, although not completely eliminated, in appe6 clones. No changes were detected in the levels of basolateral or cytoplasmic Dachs:V5. Although App affects Dachs accumulation at the apical cell cortex, anti-App staining was normal in d mutant clones (Matakatsu, 2008).

Co-overexpression of App and Dachs:V5 greatly increased the accumulation of Dachs at the cell cortex compared with the expression of Dachs:V5 alone. Coexpression of App also increased the efficacy of Dachs in growth and PCP. Even though overexpression of App-RA did not obviously increase growth, coexpression of App-RA and Dachs caused greater overgrowth than did the expression of Dachs alone. Co-overexpression of Dachs and App caused more extreme PCP defects in the wing and abdomen than did the overexpression of App alone, whereas overexpression of Dachs:V5 alone did not affect PCP (Matakatsu, 2008).

It is likely that much or all of the app mutant phenotype is mediated by the reduction of effective Dachs at the apical cell cortex. The effects of app and d mutants are not additive: Double mutants for null app and d alleles resembled the stronger d null phenotype, as expected if App works by controlling Dachs activity. Because App affects Dachs post-transcriptionally, it is unlikely that overexpressed Dachs would fully rescue the app null. Nonetheless, overexpression of UAS-d with ap-gal4 or en-gal4 rescued the wing PCP defects normally found in the distal wing of appe6 mutants and partly rescued the crossvein spacing and leg-joint defects of appe6. That Dachs retains some activity in the absence of App is consistent with the low but significant levels of Dachs that remain at the apical cell cortex in appe6 clones. Different DHHC proteins can palmitoylate the same target, so other Drosophila DHHC proteins may be supplying residual activity in the absence of App (Matakatsu, 2008).

It is concluded that App palmitoyltransferase acts in opposition to the Ft pathway, largely or wholly by controlling the apical-cell-cortex localization and the activity of the atypical myosin Dachs. This localization is probably required for full Dachs activity. For growth control, this localization would place Dachs near not only Ft, but also the Hippo pathway member Warts. Warts is concentrated near the cell cortex, with an apical bias that overlaps the region of strong App and Dachs accumulation. Dachs binds Warts and may thereby regulate the Hippo pathway, accounting for its effects on Ft-dependent growth control (Matakatsu, 2008).

Dachs also modulates the effects of Ft signaling on PCP. It is not clear whether this modulation is also mediated through changes in Warts activity. Warts is thought to act by changing the activity of the transcription factor Yorkie, an effect that would not directly confer polarity. Moreover, Dachs was reported to accumulate preferentially on the distal faces of some wing-disc cells, suggesting that Dachs is involved in cell polarization independent on any effect on transcription, probably via as-yet-unknown binding partners (Matakatsu, 2008).

It remains possible that Dachs is palmitoylated by App. However, there is no precedent for palmitoylation of a myosin, nor does Dachs score highly when an algorithm is used that detects palmitoylation sites. Nor has Dachs palmitoylation been detected by using the acylbiotin-exchange technique. The effect of App may thus be less direct, palmitoylating a binding partner or regulator of Dachs. Although Dachs is a myosin, portions of Dachs are unique and lack known protein-interaction motifs. Warts is the only proven binding partner for Dachs, but appe6 clones did not affect the levels or cell-cortex localization of Myc-tagged Warts in wing discs (Matakatsu, 2008).

Given that the human and yeast DHHC proteins that App most resembles palmitoylate many targets, the adult phenotypes of app mutants are surprisingly specific to the Ft pathway. One known target of ERF2 and ZDHHC9 is Ras, whose activity relies on membrane localization through both farnesylation and palmitoylation. Intriguingly, the Ras and MAPK pathways interact with the Ft pathway in growth control. However, reducing Ras activity causes loss of wing veins, a phenotype not observed in app mutations, and does not cause the PCP and appendage-patterning defects of app and d mutants. Moreover, reducing Ras activity via expression of a dominant negative EGF receptor did not affect the levels of Dachs:V5 in wing discs. The different subcellular distributions of App, to the cell cortex, and ZDHHC9 and ERF2, to endomembranes, suggests they have different roles and targets, despite their strong similarity at the amino acid level (Matakatsu, 2008).

four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg

The molecular basis of segmentation and regional growth during morphogenesis of Drosophila legs is poorly understood. four-jointed is not only required for these processes, but also can direct ectopic growth and joint initiation when its normal pattern of expression is disturbed. These effects are non-autonomous, consistent with the demonstration of both transmembrane and secreted forms of the protein in vivo. The similarities between four-jointed and Notch phenotypes led to an investigation of the relationships between these pathways. Surprisingly, it was found that although four-jointed expression is regulated downstream of Notch activation, four-jointed can induce expression of the Notch ligands, Serrate and Delta, and may thereby participate in a feedback loop with the Notch signaling pathway. four-jointed interacts with abelson, enabled and dachs, which suggests that one target of four-jointed signaling is the actin cytoskeleton. Thus, four-jointed may bridge the gap between the signals that direct morphogenesis and those that carry it out (Buckles, 2001).

To understand Fj function, whether Fj exists in vivo as a secreted or a transmembrane protein was investigated. Drosophila S2 cell lines containing the fj cDNA under the control of a heat-shock promoter were generated (S2:fj). Homogenates from these cells and from the parent S2 line were analyzed on Western blots using antibodies generated against Fj. Antisera detected three major bands that were present in the S2:fj cells after heat-shock but were absent from the S2 parent line. In cell fractionation experiments of the S2:fj cells, the two more slowly migrating forms behaved as integral membrane proteins, since they segregate with both the heavy and the light membrane fractions and can not be washed off the membranes by alkaline treatment. Their sizes are consistent with the full-length protein predicted from sequence analysis. By contrast, the smallest polypeptide is secreted into the medium and co-migrates on SDS-PAGE with the intact C-terminal domain (Buckles, 2001).

To detect Fj reliably in larval tissue, glycoproteins were first concentrated by precipitation with ConA Sepharose beads before loading on the gel for Western blot analysis. Under these conditions, three polypeptides of the same size seen in cultured cells were also detected in homogenates from wild-type third instar larvae but were absent from homogenates produced from larvae homozygous for mutant alleles of fj. Just as the in vitro forms were shown to be altered by endoglycosidase H treatment and therefore N-glycosylated, so all three larval forms can be bound by ConA and therefore contain asparagine-linked core glycosylation. While the relative abundance of the three forms is somewhat variable, under all conditions the largest transmembrane form is present in equal or up to five-fold excess of the secreted form. Thus a significant fraction of Fj protein remains membrane-bound, although some protein molecules are cleaved and secreted (Buckles, 2001).

Fj is expressed in a series of concentric rings in the developing leg and its expression in the tarsus is tightly associated with forming segment boundaries. Fj is necessary for the formation of the T2/3 joint and is required for growth of the femur, tibia and tarsal segments 1-3. If Fj is a key regulator of these morphogenetic processes, it might be expected that Fj would also be sufficient to initiate joint formation and growth in the leg. Furthermore, since fj is expressed in only a subset of the cells within each leg segment, whether distinct domains of fj expression are indeed important for proper leg development was investigated (Buckles, 2001).

Ubiquitous expression of fj in the developing legs was accomplished by either inducing HS-fj or by driving UAS-fj with 69B-Gal4. Ectopic fj expression is capable of inducing the formation of ectopic joint-like structures, which resemble the partial joints seen at T2/3 in fj hypomorphic alleles. In flies homozygous for the HS-fj insertion, 80.5% of legs contained such ectopic partial joints. These appeared as donut-shaped invaginations in T3 or, much less frequently, in T2 and T4, usually centered approximately equidistant from the flanking joints. These invaginations resemble the ball-and-socket structure of a normal tarsal joint and, like normal joints, contain only bare cuticle. In the HS-fj flies, these ectopic structures occur with minimal loss of growth in the segment and no loss of endogenous joints. These results suggest that fj is sufficient to initiate joint formation in the tarsus and that this capacity is largely independent of growth control in the segment. The UAS-fj driven expression causes more widespread ectopic joint-like structures in the tarsus, additional loss of PD growth within the tarsal segments, and loss of the T2/3 joint (Buckles, 2001).

Patterned misexpression of fj across multiple segment boundaries causes more dramatic results. Expression of fj along the AP compartment boundary using UAS-fj and ptc-Gal4 results in legs with reduced leg growth and segmental fusions. The effects are particularly dramatic in the tarsus. A similar effect is seen when dpp-Gal4 is used as the driver. In addition, these animals display occasional outgrowths from the leg. Some of these outgrowths appear to be segmented and most contain at least some bristles. Similar outgrowths and truncations are seen when expression is driven with en-Gal4, which is expressed within the posterior compartment of the leg disc. Together, these results suggest that the endogenous pattern of fj expression is critical to its function in both growth control and segmentation of the leg (Buckles, 2001).

Smaller, randomly positioned clones ectopically expressing fj were examined to address whether fj affects leg growth and segmentation non-autonomously, as would be predicted if Fj acts as a signaling molecule. Smaller patches of fj-expressing cells were produced using the flip-out actin-Gal4 technique, and such clones were marked by the cuticular marker yellow. Clones expressing fj that span a segment border result in the fusion of most segments. Effects are most dramatic in the tarsus, with a fusion of tarsal segments and accompanying reduced growth. Although in all examples an autonomous influence of fj was observed, in many instances apparent non-autonomy, in which joint structures were lost both within and adjacent to the clone, were observed (Buckles, 2001).

In addition to the disruptions in leg segmentation and growth observed with larger clones, occasional outgrowths from the leg were found. Importantly, these outgrowths were entirely composed of wild-type tissue, while the fj-expressing clone neighbored the outgrowth. Together, these results strongly argue that fj is a key regulator of leg segmentation and growth, and that fj can function non-autonomously in these processes (Buckles, 2001).

Fj is regulated downstream of N signaling and many of the phenotypes observed with ectopic fj expression are similar to those seen upon ectopic activation of N. It is possible that these similarities might derive from a common molecular cause. For example, deregulation of N signaling may cause a deregulation of fj expression, which would then disrupt normal morphogenesis. Alternatively, since fj is known to have a transcriptional feedback on its own expression, perhaps it also participates in a feedback loop onto the N pathway such that misexpression of fj actually results in misexpression of activated N. The most likely target for such feedback would be the N ligands, since N is expressed widely in the disc but only becomes activated at the restricted positions of ligand expression (Buckles, 2001).

To investigate whether Fj feeds back onto the N signaling pathway, the expression of the N ligands Ser and Dl was examined in leg discs in which fj was ectopically expressed along the AP axis using ptc-Gal4-driven expression of UAS-fj. Such misexpression of fj results in severe truncation of the tarsus. ptc is expressed at highest levels along the AP boundary, with graded expression in the anterior compartment of the disc. Ectopic fj expression induces the expression of both Ser and Dl along the posterior edge of the fj-expressing stripe, and does so largely non-autonomously. The non-autonomy is consistent with biochemical data, and provides further evidence that Fj acts as a signaling molecule. Furthermore, the ectopic expression of Ser and Dl, leading to ectopic activation of N, could account for some of the observed effects of ectopic fj expression on leg development (Buckles, 2001).

The asymmetry of induction only along the border of highest expression raised the possibility that induction might only occur at sharp boundaries of expression, such as that on the posterior edge of the ptc domain. To test this, UAS-fj was expressed with two additional drivers, dpp-Gal4 and en-Gal4, which are both expressed at somewhat lower levels than ptc-Gal4. dpp-Gal4 is expressed within the anterior compartment of the leg disc, while en-Gal4 is expressed in the posterior compartment with a sharp boundary of expression along the AP border. Misexpression of fj under either driver produces truncations of the tarsus as well as apparent outgrowths and/or bifurcations of the distal leg. As with ptc-Gal4, both dpp- and en-Gal4-driven expression of fj induces expression of Ser in cells neighboring those expressing high levels of fj: at the posterior edge of the dpp domain and at the anterior edge of the en domain. Similar non-autonomous induction of Dl is observed with these drivers (Buckles, 2001).

Whether fj is required for normal Ser expression was investigated. Ser expression was examined in pupal leg discs homozygous mutant for fj. Expression of Ser is unaffected in all leg segments except for one: Ser expression is significantly reduced in the second tarsal segment. This finding is consistent with the observation that fj mutants have a partial or complete lack of the joint between the second and third tarsal segments and reduced growth within the fused segment (Buckles, 2001).

Since fj induces Ser expression non-autonomously, it was of interest to examine their endogenous expression patterns during development of the leg. Consistent with the inductive behavior observed, fj and Ser appear to be expressed in adjacent but largely non-overlapping stripes in tarsal segments 2-4 in the developing leg disc (Buckles, 2001).

Together, these results suggest that there is a feedback loop between N ligand expression and the N target gene fj. Fj appears to be necessary for the initiation, upregulation, or maintenance of Ser expression. Although fj is expressed in every tarsal segment, Ser expression is only affected by loss of fj in tarsal segment 2 (Buckles, 2001).

To begin to understand how Fj signaling affects such diverse processes as leg segmentation and growth, ommatidial rotation, and epithelial planar polarity, attempts were made to identify other genes with which Fj interacts. Some of the effects of Fj are likely to be due to its feedback onto the N signaling pathway, and this would presumably require a Fj signal transduction pathway. In addition, it is likely that Fj also functions independently of its regulation of Ser and Dl, since the loss-of-function and gain-of-function phenotypes of N ligands and Fj are not identical (Buckles, 2001).

The predicted molecular structure of Fj suggests that it is a type II transmembrane protein, with two potential signal peptidase cleavage sites near the end of the transmembrane domain whose use would be consistent with the size of the secreted product. However, not all Fj is secreted, since two integral membrane forms remain present in all preparations. While there are many examples of growth factors that have both transmembrane and secreted forms, all of these appear to undergo cleavage at or near the cell surface, including N and its ligand Dl. If signal peptidase is indeed responsible for Fj cleavage (and this seems likely, as Fj is cleaved even in an in vitro microsomal preparation), it is unusually inefficient. Alternatively, it is possible that, in vivo, Fj is instead cleaved by a protease later in the secretory pathway or in response to signaling. This will need to be resolved in future experiments (Buckles, 2001).

It still remains to be determined which forms of Fj have functional significance. Certainly, the non-autonomous effects of Fj in the wing, eye and abdomen, all implicate secreted Fj as biologically relevant. Indeed, in vertebrates Fj appears to be produced as a wholly secreted protein with the transmembrane domain serving as an N-terminal signal sequence. In the gain-of-function clones described here, the induction of outgrowths composed of wild-type tissue similarly supports a non-autonomous role for Fj in the leg, although the non-autonomous influence may be indirect, for example by the early induction of a second growth factor. The failure of joint formation by wild-type tissue adjacent to these clones may also provide examples of non-autonomy. Alternatively, however, it is possible that an inhibition of joint formation within the clone mechanically inhibits nearby cells from forming joints. A similar local inhibitory influence has been observed on heterozygous cells adjacent to loss-of-function fj clones. Interestingly, the opposite is not observed: wild-type tissue is never seen to rescue joint formation within mutant clones. Again, this may represent the competing influences of induction from outside the clone and mechanical inhibition from within the clone. Thus the cooperative nature of joint development makes any determination of local non-autonomy ambiguous (Buckles, 2001).

Local suppression of joint formation adjacent to clones may also explain why ectopic joints are not seen along the borders of the flip-out clones, which produce sharp boundaries of expression that might be expected to resemble the normal patterning of fj expression. By contrast, ectopic joints are produced when fj is uniformly elevated in a wild-type background. While the significance of this remains unclear, it is noted that these ectopic structures tend to form in the center of the segment, where endogenous activity (and thus presumably additive activity) is lowest. Thus, in addition to the patterning of fj expression, the absolute level of Fj may determine whether joint initiation is permissible in any region of the disc (Buckles, 2001).

Similar mutant phenotypes may indicate that the genes causing them may act in the same molecular pathway. dachs and abl mutant phenotypes imitate those of fj, thus both of these genes are attractive candidates for the fj signaling pathway. A major substrate for Abl kinase activity is the Ena gene product. Ena homozygotes are embryonic lethal and imaginal phenotypes are not known. However, Abl and Ena appear to function in the same pathway in Drosophila. Finally, given the molecular epistatic interactions observed between fj and Ser, whether fj and Ser interact genetically was tested (Buckles, 2001).

To test the relationships of these genes, two hypomorphic alleles of fj, fj4 and fjN7 were used. The majority of legs of fjN7 flies retain partial joints of a ball and socket morphology at the juncture between T2 and T3, while fj4 produces larger partial joints or complete joints at the T2/3 boundary. Introduction of one mutant copy of dachs, abl, ena, or Ser into these backgrounds significantly increases the severity of the hypomorphic phenotypes, while each of these genes by itself is wholly recessive in the leg. Thus, dachs, abl, ena, and Ser act as dominant enhancers of fj, suggesting these genes may be part of a common pathway or network (Buckles, 2001).

Loss-of-function abl alleles are recessive, and heterozygous abl flies have normal leg morphology. However, when one copy of abl was removed in a strong fj background, the complete or partial loss of an additional tarsal joint at T1/2 on one or more legs was observed in one third of the animals. A wild-type abl transgene (Tnabl+) can restore this joint, confirming that abl is indeed responsible for the interaction. To test if kinase activity is required for abl activity at this joint, a transgene with an inactive kinase (TnablK-N) was inserted into the same genetic background. This transgene is unable to rescue the interaction, suggesting that abl kinase activity is indeed required. These results suggest that abl and fj participate in redundant pathways in the leg. Moreover, they demonstrate that fj is required at segment boundaries other than T2/3, which is the only boundary lost in fj null mutants. More widespread activity is consistent with the expression of fj at additional segment boundaries in the leg. These results reveal a concealed function for abl in leg morphogenesis (Buckles, 2001).

Abl kinase function partially masks a requirement for fj at the T1/2 segment boundary. While fj is expressed at all tarsal segment boundaries, it appears to be required for segmentation only at T2/3, although rare instances of partial loss of T1/2 have been observed. However, in the absence of one copy of abl, additional loss of the T1/2 boundary is seen in a third of all animals. This is interpreted to mean that additional information, modified by or working through abl, acts together with fj to elaborate that boundary. Most abl homozygous flies have normal legs, although occasional severe truncations of the legs in individual abl flies are observed. In addition, over-expression of abl causes tarsal truncations and segment fusions. A likely target of Abl activity is Ena. However, Abl is not the only tyrosine kinase that phosphorylates Ena, and this multiple regulation may in part explain the variability of abl loss-of-function phenotypes. A critical test of whether Ena is indeed part of a common pathway for the morphogenetic work of segmentation at all leg segment boundaries will be to examine loss-of-function ena clones in the leg. Alternatively, the common pathway at each segment boundary may be the actin cytoskeleton itself, with multiple inputs providing redundancy, and overlapping position-specific regulators competing or cooperating to regulate the state of actin dynamics in each cell. Indeed, in the larger sense, Fj itself may serve to coordinate information provided by multiple signaling pathways (Notch, Jak/Stat, and Wingless, which all regulate fj) with alterations in the actin cytoskeleton that ultimately have morphological consequences (Buckles, 2001).


Search PubMed for articles about Drosophila Dachs

Ambegaonkar, A. A., Pan, G., Mani, M., Feng, Y. and Irvine, K. D. (2012). Propagation of Dachsous-Fat planar cell polarity. Curr Biol 22: 1302-1308. PubMed ID: 22727698

Bando, T., et al. (2009). Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration. Development 136: 2235-2245. PubMed ID: 19474149

Bossuyt, W., Chen, C. L., Chen, Q., Sudol, M., McNeill, H., Pan, D., Kopp, A. and Halder, G. (2014). An evolutionary shift in the regulation of the Hippo pathway between mice and flies. Oncogene 33: 1218-1228. PubMed ID: 23563179

Bosch, J. A., Sumabat, T. M., Hafezi, Y., Pellock, B. J., Gandhi, K. D. and Hariharan, I. K. (2014). The Drosophila F-box protein Fbxl7 binds to the protocadherin Fat and regulates Dachs localization and Hippo signaling. Elife 3: e03383. PubMed ID: 25107277

Bosveld, F.,et al. (2012). Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway. Science 336(6082): 724-7. PubMed ID: 22499807

Buckles, G. R., et al. (2001). four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg. Development 128: 3533-3542. 11566858

Cai, et al. (2010). The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program. Genes Dev. 24: 2383-2388. PubMed ID: 21041407

Cho, E. and Irvine, K. D. (2004). Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling. Development 131: 4489-4500. 15342474

Cho, E., Feng, Y., Rauskolb, C., Maitra, S., Fehon, R. and Irvine, K. D. (2006). Delineation of a Fat tumor suppressor pathway. Nat. Genet. 38(10): 1142-50. 16980976

Grusche, F. A., Degoutin, J. L., Richardson, H. E. and Harvey, K. F. (2011). The Salvador/Warts/Hippo pathway controls regenerative tissue growth in Drosophila melanogaster. Dev. Biol. 350(2): 255-66. PubMed ID: 21111727

Karpowicz, P., et al. (2010) The Hippo tumor suppressor pathway regulates intestinal stem cell regeneration. Development 137: 4135-4145. PubMed ID: 21098564

Mao, Y., et al. (2006). Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila. Development 133(13): 2539-51. PubMed ID: 16735478

Mao, Y., et al. (2011). Planar polarization of the atypical myosin Dachs orients cell divisions in Drosophila. Genes Dev. 25(2): 131-6. PubMed ID: 21245166

Misra, J.R. and Irvine, K.D. (2016). Vamana couples fat signaling to the Hippo pathway. Dev Cell 39(2):254-266. PubMed ID: 27746048

Matakatsu, H. and Blair, S. S. (2008). The DHHC palmitoyltransferase approximated regulates Fat signaling and Dachs localization and activity. Curr. Biol. 18(18): 1390-5. PubMed ID: 18804377

Neumann, C. J. and Cohen, S. M. (1996). Distinct mitogenic and cell fate specification functions of wingless in different regions of the wing. Development 122(6): 1781-9. PubMed ID: 8674417

Pan, G., Feng, Y., Ambegaonkar, A. A., Sun, G., Huff, M., Rauskolb, C. and Irvine, K. D. (2013). Signal transduction by the Fat cytoplasmic domain. Development 140: 831-842. PubMed ID: 23318637

Rodrigues-Campos, M. and Thompson, B. J. (2014). The ubiquitin ligase FbxL7 regulates the Dachsous-Fat-Dachs system in Drosophila. Development 141: 4098-4103. PubMed ID: 25256343

Rogulja, D., Rauskolb, C. and Irvine, K. D. (2008). Morphogen control of wing growth through the fat signaling pathway. Dev. Cell 15: 309-321. PubMed ID: 18694569

Shaw, R. L., et al. (2010). The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration. Development 137: 4147-4158. PubMed ID: 21068063

Staley, B. K. and Irvine, K. D. (2010). Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation. Curr. Biol. 20: 1580-1587. PubMed ID: 20727758

Zhang, Y., Wang, X., Matakatsu, H., Fehon, R. and Blair, S. S. (2016). The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dachs. Elife 5. PubMed ID: 27692068

Biological Overview

date revised: 10 December 2017

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