Hippo/Warts pathway

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, wts^P2, 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).

Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules

Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathwayand hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), homologs of Drosophila Yorkie. However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. This study dissects how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, these results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation. (Yang, 2014).

This report addresses the effects of AJs and basolateral cell polarity determinants on the activity of the Hippo pathway in Drosophila imaginal discs. Knockdown of AJs and basolateral components both induced ectopic activation of Yki. However, knockdown of AJs and basolateral proteins had strikingly different effects on Yki. Disruption of the basolateral module induced mainly a cell-autonomous increase in Yki activity, whereas knockdown of AJs caused non-autonomous induction of Yki reporters. Therefore, these data identify and genetically uncouple multiple different molecular pathways from AJs and the basolateral module that regulate Yki activity (Yang, 2014).

These studies further show that knockdown of AJs induces cell-autonomous reduction of Yki activity and causes cell death and decreased size of Drosophila imaginal discs. Likewise, E-cad and :alpha;-cat mutant clones do not survive in imaginal discs. This effect may be mediated by LIM domain proteins of the Zyxin and Ajuba subfamilies, which regulate Hippo signaling by directly inhibiting Wts/Lats kinases and by interacting with Salvador (Sav), an adaptor protein that binds to the Hpo/MST kinases. A recent report shows that α-Cat recruits Ajuba and indirectly Wts to AJs and loss of Ajuba leads to activation of Wts and hence phosphorylation and inhibition of Yki and diminished tissue size. Thus, α-cat mutant cells may inactivate Yki because they lose Ajuba function (Yang, 2014).

In contrast, in mammalian systems, several in vivo and in vitro studies have shown the opposite effect on Hippo signaling upon AJ disruption; knockdown of E-cad or α-cat caused an increase in cell proliferation and nuclear accumulation of YAP, and conditional knockout of α-cat in mouse skin cells caused tumor formation and elevated nuclear YAP staining. This suggests that AJ components have a tumor suppressor function in mammals. The observation that Scrib is mislocalized upon disruption of AJs in several different mammalian cell lines suggested that YAP activation could be due to the concomitant disruption of the basolateral module. However, the finding that acute disruption of AJs can cause YAP activation without disrupting Scrib localization and vice versa indicates that AJs and the basolateral module also act independently on the Hippo pathway in mammalian cells. In mammalian cells, α-Cat forms a complex with YAP and 14-3-3 proteins, thereby sequestering phosphorylated YAP at the plasma membrane. However, α-Cat may function as a tumor suppressor only in epidermal stem cells, as conditional deletion of α-cat in differentiated cells only caused a mild phenotype with no overgrowth and tumor formation. Therefore, it is possible that the negative regulation of YAP by α-Cat is cell type-specific, although further testing is required to fully address this issue (Yang, 2014).

The non-cell-autonomous effect of AJ knockdown on the Hippo pathway is an intriguing phenomenon. Several groups reported non-autonomous effects on the Hippo pathway in Drosophila in other mutant conditions. Disrupting the expression gradients of the atypical Cadherin Dachsous or that of its regulator Four-jointed, clones of cells mutant for the tumor suppressor genes vps25 or hyperplastic discs (hyd) , clones of cells overexpressing Src64, or overexpression of the proapoptotic gene reaper or the JNK signaling ligand eiger all cause non-autonomous activation of Yki. This non-autonomous activation of Yki may be part of a regenerative response that stimulates cell proliferation in cells neighboring tissue defects. The signals that activate Yki in these situations are not known, nor is it known whether these mutant conditions activate the same or different signaling mechanisms. The non-autonomous activation of Yki around cells with AJ knockdown may be mediated by changes in mechanical forces. AJs are important for maintaining tension between cells across epithelia, and disruption of AJs leads to an imbalance of apical tension. Mechanical forces are known to regulate the Hippo pathway, and YAP/TAZ act as mediators of mechanical cues from the cellular microenvironment such as matrix stiffness. In particular, the Zyxin and Ajuba family LIM domain proteins can act as sensors of mechanical forces and may be involved in the non-autonomous activation of Yki. The effects on Hippo signaling of solely changing Zyxin and Ajuba may not be as strong as those described here, and these proteins may thus cooperate with other molecular conduits to regulate the activity of the Hippo pathway in response to changes in AJ strength. Unraveling these mechanisms will provide important new insights into understanding how cells interact with neighboring cells to regulate proliferation, apoptosis, and the Hippo pathway (Yang, 2014).

It is currently unknown whether AJs also exert non-autonomous effects on the Hippo pathway in mammalian tissues. Amphiregulin, an EGF ligand, is a downstream target of YAP and can induce non-cell-autonomous cell proliferation through EGFR signaling. However, it is not known whether YAP itself is activated non-cell-autonomously to contribute to the hyper-proliferation phenotypes observed upon disruption of AJs in vivo and in vitro. It will be interesting to determine whether AJs and other cell-cell signaling mechanisms also have non-cell-autonomous effects on the activity of YAP in mammalian tissues, for example during regeneration (Yang, 2014).

Finally, the apical proteins aPKC and Crb modulate the activity of the Hippo pathway, and many Hippo pathway components are apically localized, which is important for their activity. The data presented in this study add to these findings. Disruption of AJs causes reduced Yki activity, despite the fact that Crb and Mer are mislocalized. Thus, AJs and cell polarity components regulate Yki activity through multiple, genetically separable inputs. It will be interesting to decipher all of the different underlying molecular mechanisms of how AJs and basolateral proteins regulate the Hippo pathway and how these mechanisms evolved in Drosophila and in mammals (Yang, 2014).

Localization of Hippo signalling complexes and Warts activation in vivo

Hippo signalling controls organ growth and cell fate by regulating the activity of the kinase Warts. Multiple Hippo pathway components localize to apical junctions in epithelial cells, but the spatial and functional relationships among components have not been clarified, nor is it known where Warts activation occurs. This study reports that Hippo pathway components in Drosophila wing imaginal discs are organized into distinct junctional complexes, including separate distributions for Salvador, Expanded, Warts and Hippo. These complexes are reorganized on Hippo pathway activation, when Warts shifts from associating with its inhibitor Ajuba LIM protein (Jub) to its activator Expanded, and Hippo concentrates at Salvador sites. This study identify mechanisms promoting Warts relocalization, and using a phospho-specific antisera and genetic manipulations, where Warts activation occurs was identified: at apical junctions where Expanded, Salvador, Hippo and Warts overlap. These observations define spatial relationships among Hippo signalling components and establish the functional importance of their localization to Warts activation (Sun, 2015).

Wts is a key control point within the Hippo pathway, where multiple upstream regulatory processes converge. A fundamental gap in understanding of Hippo signal transduction has been the cellular location of Wts activation. This study established that Wts activation in wing disc epithelial cells occurs at sub-apical junctions where Hpo, Sav, Ex and Wts overlap. Co-recruitment of Hpo and Wts kinases to a common scaffold is implicated as a central feature of Hippo pathway activation, and this helps to explain why genes required for apical junctions and apical-basal polarity promote Hippo signalling and can act as tumour suppressors (Sun, 2015).

These studies indicate that a key step in Wts activation in disc epithelia is its relocalization from Jub to Ex. No special mechanism is needed to transport Wts from Jub to Ex, as Wts localization could simply be governed by equilibrium binding with a limited cytoplasmic pool. That is, if Wts normally binds relatively strongly to Jub, and relatively weakly to Ex, it could, depending on its concentration, accumulate at Jub sites but not at Ex sites. Expression of activated Yki induced a robust relocalization of Wts from Jub to Ex, and these studies identify three factors that contribute to the visible accumulation of Wts at Ex sites under these conditions. First, Yki activation appears to increase Hpo activity. It was also found that hpo RNAi suppresses the relocalization of Wts from Jub to Ex, and that increased Hpo activity promotes Ex-Wts binding, as assayed by co-immunoprecipitation experiments. These observations are consistent with the hypothesis that Wts shifts from Jub sites towards Ex sites due to an increased Ex-Wts binding affinity induced by Hpo. Second, Yki activation increases levels of Ex, which under equilibrium binding would also increase the recruitment of Wts to Ex sites. The relocalization of Wts back to adherens junctions in the absence of Ex indicates that the shift in Wts localization is Ex dependent, and implies that Jub and Ex can compete for association with Wts. A third factor that contributes to detection of Wts-Ex co-localization is the increase in Wts protein levels induced by activated Yki, which could lead to Wts concentrations high enough to bind even lower-affinity Ex sites, and indeed it was observed that simply overexpressing Wts was sufficient to induce Wts-Ex overlap, without removing Wts from adherens junctions where it co-localizes with Jub. It is suggested that an additional consequence of increased Wts levels that enables detection of Wts and pWts overlapping Ex could be a saturation of pWts removal. While at present this remains speculative, all signal transduction pathways require mechanisms to turn off after they have been activated, so there should exist mechanisms that either degrade or dephosphorylate pWts. Relatively low levels of pWts due to rapid turnover could also help explain why pWts was undetectable in wild-type wing discs (Sun, 2015).

The discovery of Ex-Wts binding, together with earlier studies that identified Ex-Hpo binding, implicate Ex as a scaffold that could promote Wts activation by co-localizing it with Hpo, and thus define a role for Ex distinct from previous suggestions that it functions as an activator of Hpo. Similarly, recent studies in cultured cell models showed that activated forms of Mer could bind Wts, and suggested a model in which Mer promotes Wts activation by recruiting it to membranes where it could be activated by Hpo. This suggests that in tissues where Mer, rather than Ex, plays key roles in Wts activation, such as glia, Mer, which can also associate with Hpo, through Sav, could play an analogous role in assembling a Wts activation complex. It is thus noteworthy that the best characterized upstream branches of Hippo signalling characterized in Drosophila (Fat, Ex and Mer) can all now be said to act principally at the levels of Wts regulation rather than Hpo regulation. Moreover, it is noted that Kibra, which has been suggested to act at a similar point in the Hippo pathway as Mer and Ex, has also been reported to be able to physically interact with both Hpo and Wts, and thus might also act principally as a scaffold that links them together rather than as a promoter of Hpo activation (Sun, 2015).

Indeed, external signals that impinge directly on Hpo activity have not yet been identified. The current discovery that Hpo localization to Sav is greatly increased by Yki activation reveals that regulators of Hpo localization exist, and implies that they are subject to negative feedback regulation downstream of Yki. As Hpo kinase activity can be promoted by Hpo dimerization, it is proposed that the increased recruitment of Hpo to Sav could elevate Hpo activity by increasing its local concentration, and thereby its dimerization. Relocalization of Hpo might also affect its interactions with kinases that can modulate Hpo activity. Recruitment of Hpo to Sav also concentrates Hpo near Ex, where it would more efficiently phosphorylate Ex-bound Wts. However, since most junctional Wts in disc epithelia is normally complexed with Jub rather than Ex, a mechanism-based solely on Hpo recruitment to apical junctions would not be expected to induce robust Wts activation. Importantly, then, these studies revealed that Hpo can increase Ex-Wts binding, possibly by phosphorylating Ex. Increased Ex-Wts binding would help recruit Wts to Ex, where it could then be phosphorylated by Hpo. Thus, it is now possible to suggest a sequential model for Hippo pathway activation in which Hpo is first recruited to membranes and activated, activated Hpo then phosphorylates Ex to recruit Wts and finally Hpo phosphorylates and activates Wts complexed with Ex. While further studies will be required to validate this model, it provides a framework that could guide future investigations, and these current studies clearly emphasize the importance of determining the in vivo localization of endogenous pathway components (Sun, 2015).

Impaired Hippo signaling promotes Rho1-JNK-dependent growth

The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. This study shows that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppressed Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promoted tissue growth when apoptosis was prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. These results identify a novel molecular link between the Hippo and JNK pathways and implicate the essential role of the JNK pathway in Hippo signaling-related tumorigenesis (Ma, 2005).

Recent studies have revealed a complex interaction network between Hippo and other key signaling pathways, including TGF- β /SMAD and Wnt/β-catenin pathways, whereas its communication with JNK signaling remains elusive. This study provides genetic evidences that impaired Hippo signaling promotes overgrowth through Rho1-JNK signaling in Drosophila. First, loss of Hippo signaling induces JNK activation and its target gene expression. Second, Yki-induced overgrowth is suppressed by blocking Rho1-JNK signaling. Third, ectopic Rho1 expression phenocopies Yki-triggered overgrowth and proliferation when cell death is compromised (Ma, 2005).

Yki/YAP's ability in promoting tissue growth depends on transcription factors, including Sd/TEADs and SMADs. Consistent with this notion, this study found Sd, but not Mad, is essential for Yki-induced JNK activation, whereas ectopic Sd expression is sufficient to activate JNK signaling by itself. The Rho1 GTPase was further implicated as the critical factor that bridges the interaction between Hippo and JNK signaling. Rho1 not only mediates Yki-induced JNK activation and overgrowth, but also serves as a direct transcriptional target of Yki/Sd complex. Intriguingly, Rho1 activation was also found to promote nuclear translocation of Yki in wing discs, and reducing Yki activity significantly impeded Rho1 induced growth, implying the existence of a potential positive feedback loop to amplify Yki-induced overgrowth and to help maintain signaling in a steady state. Consistent with thi observation, recent studies reported that GPCRs could activate YAP/TAZ through RhoA in mammals, whereas elevated JNK signaling in Drosophila could stimulate Yki nuclear translocation during regeneration and tissue growth. Thus, these results provide the other side of the story about a novel cross-talk between Hippo and JNK signaling (Ma, 2005).

Intriguingly, it was found that ectopic Yki expression driven by ptc-Gal4 induced MMP1 activation, puc-LacZ expression, rho1 transcription, and Yki target gene transcription predominantly in the proximal region of wing disk, but not that of the dorsal/ventral boundary. This is consistent with a recently published paper showing that tension in the center region of Drosophila wing disk is lower than that in the periphery, which correlates with lower Yki activity. It is also worth noting that despite the requirement of JNK signaling in Yki-induced wing overgrowth, JNK was not activated strictly in an autonomous manner upon Yki overexpression. This could be caused by supercompetitive activity of Yki expression clones, or, alternatively, through a propagation of JNK signal into neighboring cells, which would be very interesting to study further (Ma, 2005).

Apart from its role in growth control, the Hippo pathway also regulates tumor invasion and metastasis. Similarly, JNK signaling plays a major role in modulating metastasis in both flies and mammals. Rho1 was also reported to cooperate with oncogenic Ras to induce large invasive tumors. Hence, it is likely that Rho1 also acts as the molecular link between Yki and JNK signaling in modulating metastasis as well (Ma, 2005).

Cabut/dTIEG associates with the transcription factor Yorkie for growth control

The Drosophila transcription factor Cabut/dTIEG (Cbt) is a growth regulator, whose expression is modulated by different stimuli. This study determined Cbt association with chromatin and identified Yorkie (Yki), the transcriptional co-activator of the Hippo (Hpo) pathway as its partner. Cbt and yki co-localize on common gene promoters, and the expression of target genes varies according to changes in Cbt levels. Down-regulation of Cbt suppresses the overgrowth phenotypes caused by mutations in expanded (ex) and yki over-expression, whereas its up-regulation promotes cell proliferation. These results imply that Cbt is a novel partner of yki that is required as a transcriptional co-activator in growth control (Ruiz-Romero, 2015).

Gene expression is regulated through the integrated action of, among others, many cis-regulatory elements, including core promoters and enhancers located at greater distances from transcription start sites (TSS). The combinatorial binding of transcription factors (TF) to these elements can lead to diverse types of transcriptional output, and an understanding of this mechanism is crucial, for example, in the context of development. In fact, the final size and shape of an organism require a complex genetic network of signaling molecules, the final outcome of which must be finely regulated in space and time to ensure a proper response (Ruiz-Romero, 2015).

The transcription factor Cabut/dTIEG (Cbt) is the fly ortholog of TGF-β-inducible early genes 1 and 2 (TIEG1 and TIEG2) in mammals, which belong to the evolutionary conserved TIEG family. TIEGs are zinc finger proteins of the Krüppel-like factor (KLF) family that can function as either activators or repressors depending on the cellular context, the promoter to which they bind or the interacting partners. TIEG proteins participate in a wide variety of cellular processes, from development to cancer, and regulate genes that control proliferation, apoptosis, regeneration or differentiation (Ruiz-Romero, 2015).

Drosophila cbt was identified and characterized from an overexpression screen of EP lines conducted to determine genes involved in establishing epithelial planar cell polarity. This TF is ubiquitously expressed in the wing disk, and its expression increases in response to metabolic, hormonal and stress signals. Cbt levels rise upon inhibition of TOR signaling, and it is among the most highly Mlx-regulated genes. Among its functions, it is known that Cbt is required during dorsal closure downstream of JNK signaling, that it is a modulator of different signaling pathways involved in wing patterning and proliferation and that it promotes ectopic cell cycling when overexpressed. Moreover, Cbt is a crucial downstream mediator gene of the JNK signaling required during wing disk regeneration. In spite of this, little is known about its downstream target genes or its precise mechanism of action. This study reports a novel function for Cbt as a partner of yki (Yorkie). yki is the key effector of growth control and the downstream element of the highly conserved Hpo (Hippo) signaling pathway. The Hpo pathway limits organ size by phosphorylating and inhibiting Yki, a key regulator of proliferation and apoptosis. yki can also act as an oncogene, since it has potent growth-promoting activity. The results show a role for Cbt as a transcriptional activator with the capacity to modulate yki growth response (Ruiz-Romero, 2015).

To characterize Cbt target genes, chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) were performed from third instar larval wing imaginal disks. Analysis of Cbt-bound regions in the entire genome revealed that approximately 70% of its peaks were located in proximal promoters or introns, consistent with its role as a transcriptional regulator. Thus, 2,060 putative target genes were identified in the wing disk. Gene Ontology (GO) analysis indicated that this subset of genes was enriched in transcriptional activity, cell migration, mitotic cell cycle and signaling pathways known to play a role in imaginal disk development. As expected, among Cbt targets previously described genes were found such as salm (spalt major) or cbt itself, but also several unidentified target genes such as wg (wingless) or vg (vestigial) (Ruiz-Romero, 2015).

Cbt association around the TSS may be an indication of its function as a primary regulatory element, but does not provide any information about its role as an activator or a repressor. To elucidate this question, published data on chromatin modifications as well as recently obtained RNA-Seq data from the wing disk were examined and Cbt targets were ranked according to their expression level. Although at different levels, target genes are mostly expressed in the wing disk. This positive correlation with gene expression was also detected in the extensive overlap between Cbt occupancy and trimethylated histone 3 lysine 4 (H3K4me3). In contrast, only 13% of Cbt target genes correlated with the repressive chromatin mark H3K27me3. Although 200 Cbt targets seemed to present both modifications, these may be coupled to the differential expression pattern of several genes in the wing disk (Ruiz-Romero, 2015).

To clarify whether Cbt binds to active or inactive genes, Cbt occupancy was examined of genes known to be differentially expressed in a subpopulation of cells within the wing disk tissue. The gene nub (nubbin) is expressed in the wing primordium. GFP expression in the wing pouch was examined using a nub-GAL4 driver and ChIP assays followed by quantitative PCR (qPCR) were performed in sorted cells. In the vicinity of the TSS of genes expressed in the wing pouch, such as rn (rotund) and nub, Cbt was only found in GFP-positive cells. Cbt was also present in the promoter of cycA (cyclin A), both in GFP-positive and GFP-negative cells, in accordance with its expression throughout the entire wing disk (wing pouch and notum). These observations indicate that Cbt might act as a positive activator of transcription in this tissue. To further confirm this, the expression of selected targets was examined after cbt overexpression. Induction of cbt in the dorsal domain of the wing using an ap-GAL4 (apterous) driver led to a clear increase in the expression levels of target genes, as detected by qPCR. cbt was also ectopically expressed in the ptc (patched) domain of the wing disk using the ptc-GAL4 driver, and the pattern of Wg (normally restricted to cells adjacent to the D/V boundary in the wing blade and to two rings in the hinge region) and Vg (expressed throughout the wing blade) was examined by immunostaining. After cbt induction, spread staining of Wg was observed in the boundary and ring regions. Likewise, analysis of Vg revealed increased protein levels in the region where cbt was upregulated. No ectopic expression of Wg or Vg was detected in regions far from where they are normally expressed, suggesting that cbt alone is not sufficient to ectopically activate transcription of these genes but can modulate or cooperate with factors that promote their basal expression. Taken together, these results suggest that Cbt functions as a transcriptional activator in the wing disk. Nevertheless, its contribution to repression in some contexts or through binding to different partners cannot be disregarded, as previous experiments have demonstrated the relevance of the Sin3A interaction domain for Cbt's repressive role (Ruiz-Romero, 2015).

TIEG factors contain three conserved C-terminal zinc finger motifs that seem to bind to GC-rich sequences in vertebrates. To characterize the set of motifs enriched within Cbt binding sites, different pattern discovery methods were used. Among others, GC sequences and the Sp1 motif, were detected as expected for a TIEG family member, but in addition, one of the most enriched motifs comprised GAGA-binding sequences. No enrichment of the proposed consensus TIEG motif 5'GGTGTG3' was found, which suggests that Cbt binds to degenerated or alternative motifs or may function through its interaction with other TFs. A recent study identified a novel Mad-like motif in promoters of Cbt-regulated genes. However, this new motif does not coincide with previously reported Cbt binding data (Ruiz-Romero, 2015).

Many studies have emphasized the complexity of yki and its mammalian homologs YAP and TAZ regulation, including multiple combinations with associate proteins in distinct target genes. Besides DNA-binding partners such Sd (Scalloped) and Hth (Homothorax) in Drosophila, yki can cooperate with other factors directly on target promoters, such as the cell cycle-related gene dE2F1. Remarkably, a recent report shows that Cbt and dE2F1 regulate an overlapping set of cell cycle genes. In the Dpp pathway, Mad (Mothers against decapentaplegic) and yki interact to form a transcription complex to activate their common targets. This association is conserved through evolution, as YAP and TAZ interact with Smad proteins to potentiate transcriptional activity. Recent studies have also identified Mask (Multiple ankyrin repeats single KH domain) as a novel cofactor for Yki/YAP, required to induce target gene expression. The results highlight the role of Cbt as a new yki partner involved in the activation of some yki target gene expression. This function of Cbt may occur in part through association with GAF as well as chromatin remodeler complexes (Ruiz-Romero, 2015)

Since overexpression of cbt results in an increase in proliferation as well as wing size, it was hypothesized that Cbt's role in size control could be mediated through its association with Yki. To address this question, cbt levels were depleted, and the effect on the growth of ex mutant clones and in clones overexpressing yki in wing and eye-antenna imaginal disks was examined. The yki target gene ex acts as an upstream positive modulator of the Hpo pathway, and in accordance with its role as a tumor suppressor, its loss-of-function mutation results in large clones. Expression of cbt RNAi in this mutant background caused a clear reduction in the clone size. In the same direction, the overgrowth known to occur by overexpression of a yki-activated form is prevented in a mutant cbt background as well as expressing cbt RNAi. Moreover, impaired growth caused by yki depletion could not be rescued increasing cbt levels and overexpression of yki and cbt triggered massive growth in imaginal tissues. Finally, depletion of cbt in adult organs (wings and eyes) also reduced Yki-mediated overgrowth, indicating a general function for Cbt in the regulation of Hippo pathway-mediated tissue growth (Ruiz-Romero, 2015).

In addition to its role during development, it has been shown that Cbt expression is highly regulated by stress and metabolic conditions. Cbt has also been identified as a JNK-inducible gene during dorsal closure, and this study has shown that JNK and tissue damage trigger cbt transient overexpression to promote wing disk regeneration, indicating that its levels must be finely controlled during regenerative growth. Moreover, cbt heterozygous mutant disks fail to proliferate and do not regenerate, and it is known that during regeneration, the JNK pathway triggers yki translocation to the nucleus to promote the proliferative response. Altogether, these data support a model for Cbt acting as a modulator of yki activity in the transcriptional regulatory mechanisms that control tissue growth (Ruiz-Romero, 2015).

Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila

In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). This paper reports studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, dramatic increase was observed in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

Wing and haltere are the dorsal appendages of second and third thoracic segments, respectively, of adult Drosophila. They are homologous structures, although differ greatly in their morphology. The homeotic gene Ultrabithorax (Ubx), which is required and sufficient to confer haltere fate to epithelial cells, is known to regulate many wing patterning genes to specify haltere, but the mechanism is still poorly understood (Singh, 2015).

There are a number of differences between wing and haltere at the cellular and organ levels. Wing is a large, flat and thin structure, while haltere is a small globular structure, although both are made up of 2-layered sheet of epithelial cells. Space between the two layers of cells in haltere is filled with haemocytes. Cuticle area of each wing cell is 8 fold more than a haltere cell. Haltere has smaller and fewer cells than the wing. Trichomes of wing cells are long and thin, while haltere trichomes are short and stout in morphology. The ratio of anterior to posterior compartment size in the haltere (~2.5:1) is much different from that in the wing (~1.2:1). Haltere also lacks wing-type vein and sensory bristles. Haltere cells are more cuboidal compared to flatter wing cells (Roch, 2000). Thus, cell number, size and shape all add to the differences in the size and shape of the two organs (Singh, 2015).

However, cells of the third instar larval wing and haltere discs are similar in size and shape. The difference between cell size and shape becomes evident at late pupal stages. Wing cells become much larger, compared to haltere cells. At pupal stages, they also exhibit differences in the organization of actin cytoskeleton elements viz. F-actin levels are much higher in haltere cells compared to wing cells (Singh, 2015).

In the context of final shape of wings and halteres, one needs to understand the mechanism by which Ubx influences cell size, shape and arrangement. It is possible that Ubx regulates overall shape of the haltere by regulating either cell size and/or shape. The current understanding of mechanisms by which wing and haltere differ at cellular, tissue and organ level is ambiguous (Sanchez-Herrero, 2013). For example, while removal of Ubx from the entire haltere, or at least from one entire compartment, leads to haltere to wing transformation with increased growth of Ubx minus tissues, mitotic clones of Ubx (using the null allele Ubx^6.28) show similar sized twin spot in small clones. Only when very large clones of Ubx^6.28Ubx^6.28 are generated, one can see increased growth compared to their twin spots. This suggests that unless a certain threshold level of growth factors is de-repressed, the haltere does not show any overgrowth phenotype (Singh, 2015).

There have been several efforts to identify functional and molecular mechanisms by which Ubx regulates genes/pathways to provide haltere its distinct morphology. Various approaches have been used to identify targets of Ubx that are expected to differentially express between wing and haltere, e.g., loss-of-function genetics, deficiency screens, enhancer-trap screening and genome wide approaches such as microarray analysis and chromatin immunoprecipitation (ChIP). Targets include genes involved in diverse cellular functions like components of the cuticle and extracellular matrix, genes involved in cell specification, cell proliferation, cell survival, cell adhesion, or cell differentiation, structural components of actin and microtubule filaments, and accessory proteins controlling filament dynamics (reviewed in Sanchez-Herrero, 2013; Singh, 2015).

Decapentaplegic (Dpp), Wingless (Wg), and Epidermal growth factor receptor (EGFR) are some of the major growth and pattern regulating pathways that are repressed by Ubx in the haltere. However, over-expression of Dpp, Wg, Vestigial (Vg) or Vein (Vn) provides only marginal growth advantage to haltere compared to the wildtype. In this context, additional growth regulating pathways amongst the targets of Ubx were examined. Genome wide studies have identified many components of Fat/Hippo and Insulin-insulin like/dAkt signalling (IIS/dAkt) pathways as potential targets of Ubx. The Fat/Hippo pathway is a crucial determinant of organ size in both Drosophila and mammals. It regulates cell proliferation, cell death, and cell fate decisions and coordinates these events to specify organ size. In contrast, the IIS/dAkt pathway is known to regulate cell size (Singh, 2015).

Recent studies have revealed that the Fat/Hippo pathway networks with other signalling pathways. For example, during wing development, Fat/Hippo pathway activities are dependent on Four-jointed (Fj) and Dachous (Ds) gradients, which are influenced by Dpp, Notch, Wg and Vg. Glypicans, which play a prominent role in morphogen signalling, are regulated by Fat/Hippo signalling (Baena-Lopez, 2008). EGFR activates Yorkie (Yki; effector of Fat/Hippo pathway) through its EGFR-RAS-MAPK signalling by promoting the phosphorylation of Ajuba family protein WTIP (Reddy, 2013). However, EGFR negatively regulates events downstream of Yki. The Fat/Hippo pathway is also known to inhibit EGFR signalling, which makes the interaction between the two pathways very complex and context-dependent. IIS/dAkt pathway is also known to activate Yki signalling and vice-versa. Thus, Fat/Hippo pathway may specify organ size by regulating both cell number (directly) and cell size (via regulating IIS/dAkt pathway) (Singh, 2015).

This study reports studies on the functional implication of regulation of Fat/Hippo and IIS/dAkt pathways by Ubx in specifying haltere development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded (ex), induced considerable increase in haltere size, mainly due to increase in cell number. Although activation of dAkt alone did not affect the organ size or the cell size, activation of Yki or down regulation of ex in the background of over-expressed dAkt caused dramatic increase in haltere size, much severe than Yki or ex alone. In this background, increase was observed in both cell size and cell number. The resulted haltere appeared flatter than wildtype haltere and the morphology of trichomes and their spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

The findings suggest that, downstream of Ubx, the Fat/Hippo pathway is critical for haltere specification. It is required for Ubx-mediated specification of organ size, sensory bristle repression, trichome morphology and arrangement. The Fat/Hippo pathway cooperates with the IIS/dAkt pathway, which is also a target of Ubx, in specifying cell size and compartment size in developing haltere. The fact that over-expression of Yki or downregulation of ex show haltere-to-wing transformations at the levels of organ size and shape, and trichome morphology and arrangement, suggest that regulation of the Fat/Hippo pathway by Ubx is central to the modification of wing identity to that of the haltere (Singh, 2015).

The observations made in this study pose new questions and suggest various interesting possibilities to study the Fat/Hippo pathway with a new perspective.

(1) It was observed that while Yki is nuclear in haltere discs, it appears to be non-functional. Yki is a transcriptional co-activator protein, which requires other DNA-binding partners for its activity. In this context, understanding the precise relationship between Yki and Ubx may provide an insight into mechanism of haltere specification (Singh, 2015).

(2) The Fat/Hippo pathway (along with the IIS/dAkt pathway) may be involved in the specification of cell size, trichome morphology and their arrangement, all of which are important parameters in determining organ morphology. Recent studies indicate that the Fat/Hippo pathway regulates cellular architecture and the mechanical properties of cells in response to the environment. It would be interesting to study the role of the Fat/Hippo pathway in regulating the cytoskeleton of epithelial cells during development. Haltere cells at pupal stages exhibit higher levels of F-actin than wing cells. One possible mechanism that is currently being investigated is lowering of F-actin levels in transformed haltere cells due to over-expression of Yki or down regulation of ex. This may cause flattening of cells during morphogenesis leading to larger organ size (Singh, 2015).

(3) Reversing cell size and number was sufficient to induce homeotic transformations at the level of haltere morphology. This suggests the importance of negative regulation of genetic mechanisms that determine cell size and number, in specifying an organ size and shape. As a corollary, Ubx-mediated regulation of Fat/Hippo and IIS/dAkt pathways provides an opportunity to study cooperative repression of cell number and cell size during organ specification (Singh, 2015).

(4) Certain genetic backgrounds investigated in this study showed severe effect on cell proliferation in haltere discs than in wing discs. This could be due to the fact that, the wing disc has already attained a specific size by the third instar larval stage (the developmental stage examined in this study), which is controlled by several pathways. Any change to this size may need more drastic alteration to the controlling mechanisms. As Ubx specifies haltere by modulating various wing-patterning events, there may still exist a certain degree of plasticity in mechanisms that determine the size of the haltere. However, in absolute terms, the haltere is also resistant to changes in growth control due to regulation by Ubx at multiple levels. Thus, differential development of wing and haltere provides a very good assay system to study not only growth control, but also to dissect out function of important growth regulators (tumour suppressor pathways) such as the Fat/Hippo pathway using various genome-wide approaches (Singh, 2015).

Zyxin antagonizes the FERM protein Expanded to couple F-actin and Yorkie-dependent organ growth

Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. A zyx mutant was generated in Drosophila using TALEN endonucleases, and this was used to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, this study shows that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). These results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity (Gaspar, 2015).

The control of tissue size represents a major unsolved question in developmental biology. The conserved Hippo (Hpo) signaling pathway is thought to sense mechanical and nutritional cues to restrict tissue growth. Activation of the Ste20-like kinase Hpo (MST1/2 in mammals) and subsequent phosphorylation of the downstream Ndr-like kinase Warts (Wts-LATS1/2 in mammals) inhibits the transcriptional co-activator Yorkie (Yki-YAP/TAZ in mammals), via phosphorylation at S168. This prevents the interaction of Yki with transcription factor partners, such as Scalloped (Sd-TEAD1-4 in mammals), thereby inhibiting expression of pro-growth and survival genes (Gaspar, 2015).

The known upstream stimuli for Hpo signaling involve a number of regulatory proteins, many of which are associated with the actin cytoskeleton. In particular, the Drosophila proteins Expanded (Ex) and Merlin (Mer), which belong to the FERM (Four point one, Ezrin, Radixin, Moesin) domain family, and the protocadherins Fat (Ft) and Dachsous (Ds), were identified as tumor suppressors that prevent expression of Yki target genes. Whether Ex/Mer and Ft/Ds signaling represent entirely distinct branches of Hpo signaling remains unclear. For instance, Ft depletion leads to a reduction in apical Ex localization. However, Ft and Ex have been implicated in distinct functions: Ft/Ds are involved in the control of planar cell polarity (PCP), while Ex has strong effects on eye differentiation. The proposed mechanisms of Ft and Ex function are also distinct. In particular, Ex promotes cytoplasmic sequestration of Yki through direct binding and by promoting Hpo-Wts kinase activity, while Ft antagonizes the growth-promoting function of the atypical myosin Dachs (D), which, in turn, destabilizes Wts (Gaspar, 2015).

Several reports have highlighted the contribution of the actin cytoskeleton to Hpo signaling. The actin Capping Protein αβ heterodimer (CP), which prevents addition of actin monomers to F-actin barbed ends, antagonizes Yki activity, and thereby restricts tissue growth. Accordingly, in mammals, CapZ and other factors that restrict F-actin levels, have growth-restrictive effects via the control of YAP/TAZ subcellular localization, particularly in response to mechanical cues. Interestingly, YAP and TAZ respond to mechanical cues dependent on actomyosin networks and formin-dependent actin polymerization. Recently, the actin-associated LIM (Lin11, Isl-1, and Mec-3) domain protein Zyxin (Zyx) has been shown to mediate the effects of Ft-Ds signaling on Yki target genes, by promoting Wts destabilization via its interaction with D (Rauskolb, 2011). Importantly, Zyx provides a link to the actin polymerization machinery, since it directly interacts with the actin-binding proteins Enabled (Ena)/VASP via conserved F/LPPPP motifs, and promotes Ena function in barbed-end F-actin polymerization (Gaspar, 2015 and references therein).

The analysis of Drosophila zyx has been limited by the absence of a mutant. This study generated a zyx mutation and describe its effects on growth and Hpo signaling. Zyx is shown to strongly antagonize Ex function in growth control, eye differentiation and F-actin accumulation, while being largely dispensable for Ft-mediated tissue growth. Finally, this work suggests that Zyx's growth-promoting function requires its ability to bind the actin polymerization factor Ena (Gaspar, 2015).

Zyx was previously shown to promote Wts degradation in a mechanism based on a Zyx/Dachs interaction (Rauskolb, 2011). However, this study reports that zyx and dachs (d) have additive effects on tissue growth. In addition, zyx loss has a modest effect on ft growth phenotypes, which, in contrast, are highly sensitive to d mutations, highlighting the possibility of additional functions for Zyx in tissue growth (Gaspar, 2015).

Characterization of the zyx mutant shows that Zyx acts in the Ex branch of the Hpo pathway to control tissue growth. This is in contrast to a previous study using RNAi knockdown of zyx and ex, which concluded that zyx expression had only minor effects on the Ex branch (Rauskolb, 2011). The current results indicate that zyx loss can significantly reverse the lethality and growth defects of ex mutant animals. This antagonistic function of Ex and Zyx is not confined to growth regulation but extends to tissue differentiation. This study shows that Zyx restricts eye differentiation antagonistically to Ex and in parallel to Dachs but independently of Ft. Consistent with these observations, simultaneous loss of ex and ft leads to additive, and therefore apparently independent effects on eye differentiation. Therefore, it is proposed that Zyx is a key modulator of Ex function (Gaspar, 2015).

In growth control, Zyx function may be partially independent of Hpo-Wts signaling, as zyx is partially required for the overgrowth of hpo and wts mutant eye and wing but has no major effect on wts overexpression in the wing or Yki phosphorylation by Wts. Ex has been reported to sequester Yki in the cytoplasm through a direct interaction. However, since ex mutant overgrowth is suppressed by zyx loss, it is unlikely that Zyx directly antagonizes Ex protein. Instead, it is suggested that the interplay between Zyx and Ex in growth control is mediated through their antagonistic effects on F-actin (Gaspar, 2015).

This work links F-actin barbed-end polymerization with Zyx/Ex in the control of Yki activity and tissue growth. The Zyx domain encompassing the conserved LPPPP motif, which binds Ena, is required for Zyx to promote growth and to antagonize Ex function. Moreover, Zyx and Ena synergize to promote tissue growth. This supports the idea that Zyx promotes tissue growth via its interaction with Ena. Conversely, CP antagonizes Zyx-induced tissue growth and functions together with Ex in preventing F-actin polymerization. Therefore, an attractive possibility is that antagonistic effects on Yki activity between the activators Zyx/Ena on one hand and the inhibitors Ex and CP on the other hand is played out indirectly through their effects on F-actin polymerization. Consistent with this hypothesis, Zyx antagonizes the effect of Ex on apical F-actin accumulation (Gaspar, 2015).

Recent data suggest that the actin cytoskeleton acts in parallel to the core kinase cascade to control YAP/TAZ activity, with CapZ being proposed as one of the 'gatekeepers' restricting its nuclear translocation. Yki/YAP/TAZ may respond to the relative activities of Ena and CP, either by being sensitive to the presence of polymerizing actin barbed ends, or because Ena produces a specialized set of cortical actin filaments necessary for Yki/YAP/TAZ activation. The study of the mechanism(s) coupling F-actin and Yki/YAP/TAZ should resolve these issues. This study has shown that Zyx cortical localization is relevant for its function in promoting tissue growth. Since Zyx has been shown to rapidly relocalize to strained or severed actin filaments in cultured mammalian cells and Drosophila follicular epithelial cells, it is possible that Zyx may also link mechanical forces to growth control (Gaspar, 2015).

Finally, it is also interesting to note the possible redundancy in growth control between Zyx and other Ena-interacting proteins. Like Zyx, Pico/Lamellipodin contains an EVH1-interacting L/FPPPP motif, and its interaction with Ena promotes tissue growth in Drosophila. Since Ena localization is not strictly dependent on Zyx, it is tempting to speculate that Ena recruitment by multiple membrane-associated proteins, such as Zyx and Pico, is a common denominator in the regulation of growth by the actin cytoskeleton (Gaspar, 2015).

C-terminal-mediated homodimerization of Expanded is critical for its ability to promote Hippo signalling in Drosophila

Hippo signalling plays key role in tissue growth and homeostasis, and its dysregulation is implicated in various human diseases. Expanded (Ex) is an important upstream activator of Hippo signalling; however, how Ex activates Hippo signalling is still poorly understood. This study demonstrate that Ex forms a homodimer via C-terminal interaction, and that the ExC2 region (912-1164 aa) is sufficient and essential for Ex dimerization. Functional analysis shows that ExC2 is required for Ex to promote the phosphorylation and inactivation of Yki in Drosophila cells. Further in vivo analysis shows that ExC2 is important for Ex to control Drosophila tissue growth. This study thus, uncovers a novel mechanism whereby Ex homodimerization mediates its full activation to promote Hippo signalling in growth control (Wang, 2022).

Crosstalk between mitochondrial fusion and the Hippo pathway in controlling cell proliferation during Drosophila development

Cell proliferation and tissue growth depend on the coordinated regulation of multiple signaling molecules and pathways during animal development. Previous studies have linked mitochondrial function and the Hippo signaling pathway in growth control. However, the underlying molecular mechanisms are not fully understood. This study identifies a Drosophila mitochondrial inner membrane protein ChChd3 as a novel regulator for tissue growth during larval development. Loss of ChChd3 leads to tissue undergrowth and cell proliferation defects. ChChd3 is required for mitochondrial fusion and removal of ChChd3 increases mitochondrial fragmentation. ChChd3 is another mitochondrial target of the Hippo pathway, although it is only partially required for Hippo pathway mediated overgrowth. Interestingly, lacking of ChChd3 leads to inactivation of Hippo activity under normal development, which is also dependent on the transcriptional co-activator Yorkie (Yki). Furthermore, loss of ChChd3 induces oxidative stress and activates the JNK pathway. In addition, depletion of other mitochondrial fusion components, Opa1 or Marf, inactivates the Hippo pathway as well. Taken together, the study proposes that there is a crosstalk between mitochondrial fusion and the Hippo pathway which is essential in controlling cell proliferation and tissue homeostasis in Drosophila (Deng, 2016).

The Hippo pathway targets Rae1 to regulate mitosis and organ size and to feed back to regulate upstream components Merlin, Hippo, and Warts

Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. This study identifies Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts Cyclin B levels and organ size while Rae1 over-expression increases Cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of Cyclin B and organ size; reducing Rae1 blocks Cyclin B accumulation and suppresses overgrowth caused by Hippo pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue 'synthetic lethality' phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, it is proposed that Rae1 also acts in a feedback circuit to regulate pathway homeostasis (Jahanshahi, 2016).

The Strip-Hippo pathway regulates synaptic terminal formation by modulating actin organization at the Drosophila neuromuscular synapses

Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. This study demonstrates that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. This study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization (Sakuma, 2016).

Since the Hippo (Hpo) pathway was discovered as the key regulator to ensure the appropriate final tissue size by coordinating cell proliferation and cell death, large-scale genetics studies have identified numerous regulators of the Hpo pathway. While most pathway components identified thus far are positive regulators of Hpo, some negative regulators were recently reported. One such negative regulator is the STRIPAK (STRiatin-Interacting Phosphatase And Kinase) complex, which is evolutionarily conserved and regulates various cellular processes including cell cycle control and cell polarity. The core component of the STRIPAK complex is the striatin family of proteins: striatins serve as B‴ subunits (one of the subfamily of regulatory B subunits) of the protein phosphatase 2A (PP2A) complex. Beyond this, the A and C subunits of PP2A, Mob3, Mst3, Mst4, Ysk1, Ccm3, Strip1, and Strip2 form the core mammalian STRIPAK complex. It has been previously reported that Strip, the Drosophila homolog of mammalian Strip1 and 2, is involved in early endosome formation, which is essential for axon elongation. Building on these findings, it was hypothesized that the Strip-Hpo pathway may also be involved in neuronal synaptic development (Sakuma, 2016).

The Drosophila larval neuromuscular junction (NMJ) is an ideal model for studying synaptic development because of its identifiable, stereotyped morphology, accessibility, broad complement of available reagents, and suitability for a wide range of experimental approaches. Furthermore, the Drosophila NMJ, like vertebrate central synapses, is glutamatergic, suggesting that the molecular mechanisms that regulate synaptic development in Drosophila NMJ might be applicable to vertebrates. Motor neuron axons are genetically hardwired to target specific muscles by the end of the embryonic stage. There, axonal growth cones subsequently differentiate into presynaptic termini, called boutons, each of which contains multiple active zones). During the larval stage, muscle size increases nearly 100-fold and boutons are continuously and proportionately added to maintain constant innervation strength. Various molecules can negatively or positively regulate the growth of synaptic termini. Amongst the many factors, elements of the actin cytoskeleton are key effectors of morphological change, functioning downstream of several cell surface receptors and signaling pathways. Of the two types of actin filaments (branched and linear), the activity of Arp2/3 complex, responsible for nucleation of branched F-actin, the first step of actin polymerization, should be strictly regulated. Arp2/3 hyperactivation results in synaptic terminal overgrowth characterized by excess small boutons emanating from the main branch that are termed satellite boutons (Sakuma, 2016).

This study shows that Strip negatively regulates the synapse terminal development through tuning the activity of the core Hpo kinase cassette. Loss or reduction of strip function in motor neurons increased the number of satellite boutons, which could be suppressed by reducing the genetic dosage of hpo. Similarly, activation of the core Hpo kinase cassette also increased satellite boutons. In this context, the presumptive downstream target of the core Hpo kinase cassette is Enabled (Ena), a regulator of F-actin assembly and elongation that was reported to antagonize the activity of Arp2/3. The canonical downstream transcriptional co-activator, Yorkie (Yki), appears dispensable for Hpo-mediated synaptic terminal development. It is proposed that the evolutionarily conserved Strip-Hpo pathway regulates local actin organization by modulating Ena activity during synaptic development (Sakuma, 2016).

This study has identified Strip and components of the Hpo pathway as regulators of synaptic morphology. In addition to the intensely investigated function of Hpo in growth control in mitotic cells, a few postmitotic roles of the Hpo pathway have recently been uncovered, such as dendrite tiling in Drosophila sensory neurons and cell fate specification of photoreceptor cells in Drosophila retina. This study now finds an additional postmitotic role for Hpo in synaptic terminal development. The results indicate that Strip and the core Hpo kinase cassette regulate satellite bouton formation by regulating the activity of Ena, an actin regulator that is involved in the initiation, extension, and maintenance of linear actin filaments at the barbed end. Although it cannot be excluded that there might be other targets of Yki in motor neurons than diap1 or bantam whose transcriptional activations were not observed in this study, Yki, a well-known downstream target of the core Hpo kinase cassette, was dispensable for proper synaptic morphology. Ena phosphorylation causes its inactivation; therefore, it was reasoned that Strip can act as a positive regulator of Ena by inactivating the Hpo pathway. A model is proposed for the regulation of satellite bouton formation by Strip and Hpo pathway components (see The Strip-Hpo pathway regulates satellite bouton formation with Ena, a regulator of F-actin organization). As the presynaptic localization of endogenous Strip was punctate and non-uniform, it is expected that Strip localization could be critical for regulating the phosphorylation status of Hpo, Wts, and Ena, which locally alters actin organization and eventually specifies the position of satellite bouton formation that could be a marker for new bouton outgrowth. When Strip is present, the core Hpo kinase cassette is inactivated, which in turn locally increases the expression of the active (unphosphorylated) form of Ena. However, the core Hpo kinase cassette can be activated in the absence of Strip, which subsequently phosphorylates and inactivates Ena. Ena prevents Arp2/3-induced branching, suggesting that Ena inactivation activates Arp2/3 and results in satellite bouton formation, similar to Rac activation. It is reported that Arp2/3 is involved in bouton formation and axon terminal branching downstream of WAVE/SCAR complex in NMJ. Indeed, the cureent findings support this hypothesis. F-actin organization was altered by strip knockdown in motor neurons. When the GFP-moe reporter was expressed in motor neuron, the GFP fused to the C-terminal actin-binding domain of Moesin, which is widely used as an F-actin reporter. The intensity of actin puncta became higher and puncta were unevenly distributed when strip was knocked down. This data suggests that Strip function is important for the proper organization of F-actin (Sakuma, 2016).

There are many indications that Strip and other STRIPAK components (Mst3, Mst4, and Ccm3) regulate the actin network. For example, Strip1, Strip2, Mst3, and Mst4 regulate the actomyosin contractions which regulate cell migration in cancer cells. In addition to regulating the actin network, STRIPAK has been suggested to function in microtubule organization. Mutants of Drosophila Mob4, a member of the core STRIPAK complex and homolog of mammalian Mob3, show abnormal microtubule morphology at NMJs and muscles. Furthermore, it has been reported that Strip forms a complex with Glued, the homolog of mammalian p150^Glued, a component of the dynactin complex required for dynein motor-mediated retrograde transport along microtubules. Strip also affects microtubule stability. As previously mentioned, microtubules are also key effectors of synaptic development downstream of several receptors and signaling pathways. Taken together, the STRIPAK complex can act as a regulatory hub for multiple cellular signals including Hpo pathway-mediated actin organization, endocytic pathway-dependent BMP signals, and microtubule stability for proper synaptic development (Sakuma, 2016).

The Hpo pathway has been reported to act as a sensor of the local cellular microenvironment, such as mechanical cues, apico-basal polarity and actin architecture to balance cell proliferation and cell death. Although synaptic morphogenesis is a postmitotic process, it is very plastic and depends on a dynamically changing extracellular environment, as exemplified by the nearly 100-fold expansion of muscle size during larval development. Thus, this study demonstrates an intriguing function for the Strip-Hippo pathway in the homeostatic control of neuronal synaptic morphology and function (Sakuma, 2016).

Localized JNK signaling regulates organ size during development

A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. This study shows that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control (Willsey, 2016).

Two independently generated antibodies that recognize the phosphorylated, active form of JNK (pJNK) specifically label a stripe in the pouch of developing wildtype third instar wing discs. Importantly, localized pJNK staining is not detected in hemizygous JNKK mutant discs, in clones of JNKK mutant cells within the stripe, following over-expression of the JNK phosphatase puckered (puc), or following RNAi-mediated knockdown of bsk using two independent, functionally validated RNAi lines (Willsey, 2016).

The stripe of localized pJNK staining appeared to be adjacent to the anterior-posterior (A/P) compartment boundary, a location known to play a key role in organizing wing growth, and a site of active Hedgehog (Hh) signaling. Indeed, pJNK co-localizes with the Hh target gene patched (ptc). Expression of the JNK phosphatase puc in these cells specifically abrogated pJNK staining, as did RNAi-mediated knockdown of bsk. Together, these data indicate that the detected pJNK signal reflects endogenous JNK signaling activity in the ptc domain, a region of great importance to growth control. Indeed, while at earlier developmental stages pJNK staining is detected in all wing pouch cells, the presence of a localized stripe of pJNK correlates with the time when the majority of wing disc growth occurs (1000 cells/disc at mid-L3 stage to 50,000 cells/disc at 20 hr after pupation, so it is hypothesized that localized pJNK plays a role in regulating growth (Willsey, 2016).

Inhibition of JNK signaling in the posterior compartment previously led to the conclusion that JNK does not play a role in wing development. The discovery of an anterior stripe of JNK activity spurred a reexamination of the issue. Since bsk null mutant animals are embryonic lethal, JNK signaling was conditionally inhibited in three independent ways in the developing wing disc. JNK inhibition was achieved by RNAi-mediated knockdown of bsk (bsk^RNAi#1or2), by expression of JNK phosphatase (puc), or by expression of a dominant negative bsk (bsk^DN). These lines have been independently validated as JNK inhibitors. Inhibition of JNK in all wing blade cells (rotund-Gal4, rn-Gal4) or specifically in ptc-expressing cells (ptc-Gal4) resulted in smaller adult wings in all cases, up to 40% reduced in the strongest cases. Importantly, expression of a control transgene (UAS-GFP) did not affect wing size. This contribution of JNK signaling to size control is likely an underestimate, as the embryonic lethality of bsk mutations necessitates conditional, hypomorphic analysis. Nevertheless, hypomorphic hep^r75/Y animals, while pupal lethal, also have smaller wing discs, as do animals with reduced JNK signaling due to bsk^DN expression. Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (rn-Gal4, UAS-bsk^DN), total animal body size is not altered (Willsey, 2016).

To test whether elevation of this signal can increase organ size, eiger (egr), a potent JNK activator, was expressed within the ptc domain (ptc-Gal4, UAS-egr). Despite induction of cell death as previously reporte and late larval lethality, a dramatic increase was observed in wing disc size without apparent duplications or changes in the shape of the disc. While changes in organ size could be due to changing developmental time, wing discs with elevated JNK signaling were already larger than controls assayed at the same time point. Similarly, inhibition of JNK did not shorten developmental time. Thus, changes in organ size by modulating JNK activity do not directly result from altering developmental time. Finally, the observed increase in organ size is not due to induction of apoptosis, as expression of the pro-apoptotic gene hid does not increase organ size. In contrast, it causes a decrease in wing size. Furthermore, co-expression of diap1 or p35 did not significantly affect the growth effect of egr expression, while the effect was dependent on Bsk activity (Willsey, 2016).

In stark contrast to known developmental morphogens, no obvious defects were observed in wing venation pattern following JNK inhibition, suggesting that localized pJNK may control growth in a pattern formation-independent manner. Indeed, even a slight reduction in Dpp signaling results in dramatic wing vein patterning defects. Second, inhibiting Dpp signaling causes a reduction in wing size along the A-P axis, while JNK inhibition causes a global reduction. Furthermore, ectopic Dpp expression increases growth in the form of duplicated structures, while increased JNK signaling results in a global increase in size. Molecularly, it was confirmed that reducing Dpp signaling abolishes pSMAD staining, while quantitative data shows that inhibiting JNK signaling does not. Furthermore, it was also found that Dpp is not upstream of pJNK, as reduction in Dpp signaling does not affect pJNK. Together, the molecular data are consistent with the phenotypic results indicating that pJNK and Dpp are separate programs in regulating growth. Consistent with these findings it has been suggested that Dpp does not play a primary role in later larval wing growth control (Akiyama, 2015). Finally, it was found that inhibition of JNK does not affect EGFR signaling (pERK) and that inhibition of EGFR does not affect the establishment of pJNK (Willsey, 2016).

A difference in size could be due to changes in cell size and/or number. Wings with reduced size due to JNK inhibition were examined and no changes in cell size or apoptosis were found, suggesting that pJNK controls organ size by regulating cell number. Consistently, the cell death inhibitor p35 was unable to rescue the decreased size following JNK inhibition. Indeed, inhibition of JNK signaling resulted in a decrease in proliferation, while elevation of JNK signaling in the ptc domain resulted in an increase in cell proliferation in the enlarged wing disc. Importantly, this increased proliferation is not restricted to the ptc domain, consistent with previous reports that JNK can promote proliferation non-autonomously (Willsey, 2016).

To determine the mechanism by which pJNK controls organ size, canonical JNK signaling through its target Jun was considered. Interestingly, RNAi-mediated knockdown of jun in ptc cells does not change wing size, consistent with previous analysis of jun mutant clones in the wing disc. Furthermore, in agreement with this, a reporter of canonical JNK signaling downstream of jun (puc-lacZ) is not expressed in the pJNK stripe. Finally, knockdown of fos (kayak, kay) alone or with jun^RNAi did not affect wing size. Together, these data indicate that canonical JNK signaling through jun does not function in the pJNK stripe to regulate wing size (Willsey, 2016).

In search of such a non-canonical mechanism of JNK-mediated size control, the Hippo pathway was considered. JNK signaling regulates the Hippo pathway to induce autonomous and non-autonomous proliferation during tumorigenesis and regeneration via activation of the transcriptional regulator Yorkie (Yki). Recently it has been shown that JNK activates Yki via direct phosphorylation of Jub. To test whether this link between JNK and Jub could account for the role of localized pJNK in organ size control during development, RNAi-mediated knockdown of jub was performed in the ptc stripe, and adults with smaller wings were observed. Indeed, the effect of JNK loss on wing size can be partially suppressed in a heterozygous lats mutant background and increasing downstream yki expression in all wing cells or just within the ptc domain can rescue wing size following JNK inhibition. These results suggest that pJNK controls Yki activity autonomously within the ptc stripe, leading to a global change in cell proliferation. This hypothesis predicts that the Yki activity level within the ptc stripe influences overall wing size. Consistently, inhibition of JNK in the ptc stripe translates to homogeneous changes in anterior and posterior wing growth. Similarly, overexpression or inhibition of Yki signaling in the ptc stripe also results in a global change in wing size (Willsey, 2016).

It is important to note that the yki expression line used is wild-type Yki, which is still affected by JNK signaling. For this reason, the epistasis experiment was also performed with activated Yki, which is independent of JNK signaling. Expression of this activated Yki in the ptc stripe caused very large tumors and lethality. Importantly, inhibiting JNK in this context did not affect the formation of these tumors or the lethality. Furthermore, inhibiting both JNK and Yki together does not enhance the phenotype of Yki inhibition alone, further supporting the idea that Yki is epistatic to JNK, instead of acting in parallel processes (Willsey, 2016).

Mutants of the Yki downstream target four-jointed (fj) have small wings with normal patterning, and fj is known to propagate Hippo signaling and affect proliferation non-autonomously. Although RNAi-mediated knockdown of fj in ptc cells does not cause an obvious change in wing size, it is sufficient to block the Yki-induced effect on increasing wing size . However, overexpression of fj also reduces wing size, which makes it not possible to test for a simple epistatic relationship. Overall, these data are consistent with the notion that localized pJNK regulates wing size not by Jun-dependent canonical JNK signaling, but rather by Jun-independent non-canonical JNK signaling involving the Hippo pathway (Willsey, 2016).

While morphogens direct both patterning and growth of developing organs, a link between patterning molecules and growth control pathways has not been established. pJNK staining is coincident with ptc expression, suggesting it could be established by Hh signaling. During development, posterior Hh protein travels across the A/P boundary, leading to activation of the transcription factor Cubitus interruptus (Ci) in the stripe of anterior cells. To test whether localized activation of JNK is a consequence of Hh signaling through Ci, RNAi-mediated knockdown of ci was performed, and it was found that the pJNK stripe is eliminated. Consistently, adult wing size is globally reduced. In contrast, no change was observed in pJNK stripe staining following RNAi-mediated knockdown of dpp or EGFR. Expression of non-processable Ci leads to increased Hh signaling. Expression of this active Ci in ptc cells leads to an increase in pJNK signal and larger, well-patterned adult wings. The modest size increase shown for ptc>Ci^ACT is likely due to the fact that higher expression of this transgene (at 25 ° C) leads to such large wings that pupae cannot emerge from their cases. For measuring wing size, this experiment was performed at a lower temperature so that the animals were still viable. Furthermore, inhibition of JNK in wings expressing active Ci blocks Ci's effects, and resulting wings are similar in size to JNK inhibition alone . Together, these data indicate that Hh signaling through Ci is responsible for establishing the pJNK stripe (Willsey, 2016).

To determine the mechanism by which Ci activates the JNK pathway, transcriptional profiles of posterior and ptc domain cells isolated by FACS from third instar wing discs were compared. Of the total 12,676 unique genes represented on the microarray, 50.4% (6,397) are expressed in ptc domain cells, posterior cells, or both. Hh pathway genes known to be differentially expressed were identified. It was next asked whether any JNK pathway genes are differentially expressed, and and it was found that dTRAF1 expression is more than five-fold increased in ptc cells, while other JNK pathway members are not differentially expressed (Willsey, 2016).

dTRAF1 is expressed along the A/P boundary and ectopic expression of dTRAF1 activates JNK signaling. Thus, positive regulation of dTRAF1 expression by Ci could establish a stripe of pJNK that regulates wing size. Indeed, Ci binding motifs were identified in the dTRAF1 gene, and a previous large-scale ChIP study confirms a Ci binding site within the dTRAF1 gene. Consistently, a reduction in Ci led to a 29% reduction in dTRAF1 expression in wing discs. Given that the reduction of dTRAF1 expression in the ptc stripe is buffered by Hh-independent dTRAF1 expression elsewhere in the disc, this 29% reduction is significant. Furthermore, inhibition of dTRAF1 by RNAi knockdown abolished pJNK staining. Finally, these animals have smaller wings without obvious pattern defects. Conversely, overexpression of dTRAF1 causes embryonic lethality, making it not possible to attempt to rescue a dTRAF1 overexpression wing phenotype by knockdown of bsk. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent. Finally, inhibition of dTRAF1 modulates the phenotype of activated Ci signaling. Together, these data reveal that the pJNK stripe in the developing wing is established by Hh signaling through Ci-mediated induction of dTRAF1 expression (Willsey, 2016).

Finally, localized centers of pJNK activity were detected during the development of other imaginal discs including the eye/antenna and leg. Inhibition of localized JNK signaling during development caused a decrease in adult antenna size and leg size. Conversely, increasing JNK signaling during development resulted in pupal lethality; nevertheless, overall sizes of antenna and leg discs were increased. Together, these data indicate that localized JNK signaling regulates size in other organs in addition to the wing, suggesting a more universal effect of JNK on size control (Willsey, 2016).

Intrinsic mechanisms of organ size control have long been proposed and sought after. This study reveals that in developing Drosophila tissues, localized, organ-specific centers of JNK signaling contribute to organ size in an activity level-dependent manner. Such a size control mechanism is qualitatively distinct from developmental morphogen mechanisms, which affect both patterning and growth. Aptly, this mechanism is still integrated in the overall framework of developmental regulation, as it is established in the wing by the Hh pathway. These data indicate that localized JNK signaling is activated by Ci-mediated induction of dTRAF1 expression. Furthermore,it is not canonical Jun-dependent JNK signaling, but rather non-canonical JNK signaling that regulates size, possibly through Jub-dependent regulation of Yki signaling, as described for regeneration. As the human dTRAF1 homolog, TRAF4, and Hippo components are amplified in numerous cancers, these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis. More importantly, these findings offer a new strategy for potential cancer therapies, as reactivating Jun in Hh-driven tumors could lead tumor cells towards an apoptotic fate (Willsey, 2016).

Hippo signaling promotes JNK-dependent cell migration

Overwhelming studies show that dysregulation of the Hippo pathway is positively correlated with cell proliferation, growth, and tumorigenesis. Paradoxically, the detailed molecular roles of the Hippo pathway in cell invasion remain debatable. Using a Drosophila invasion model in wing epithelium, this study shows that activated Hippo signaling promotes cell invasion and epithelial-mesenchymal transition through JNK, as inhibition of JNK signaling dramatically blocks Hippo pathway activation-induced matrix metalloproteinase 1 expression and cell invasion. Furthermore, bantam-Rox8 modules act as essential components downstream of Yorkie in mediating JNK-dependent cell invasion. Finally, YAP (Yes-associated protein) expression negatively regulates TIA1 (Rox8 ortholog) expression and cell invasion in human cancer cells. Together, these findings provide molecular insights into Hippo pathway-mediated cell invasion and also raise a noteworthy concern in therapeutic interventions of Hippo-related cancers, as simply inhibiting Yorkie or YAP activity might paradoxically accelerate cell invasion and metastasis (Ma, 2017).

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 fat^sum, 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).

Homeostatic response to blocking cell division in Drosophila imaginal discs: Role of the Fat/Dachsous (Ft/Ds) pathway

One major problem in developmental biology is the identification of the mechanisms that control the final size of tissues and organs. This issue was identified in the imaginal discs of Drosophila by analysing the response to blocking cell division in large domains in the wing and leg discs. The affected domains may be zones of restricted lineage like compartments, or zones of open lineage that may integrate cells from the surrounding territory. The results reveal the existence of a powerful homeostatic mechanism that can compensate for gross differences in growth rates and builds structures of normal size. This mechanism functions at the level of whole discs, inducing additional cell proliferation to generate the cells that populate the cell division-arrested territory and generating an active recruitment process to integrate those cells. The activation of this response mechanism is mediated by alterations in the normal activity of PCP genes of the Fat/Ds system: in discs mutant for dachs, ds or four jointed the response mechanism is not activated (Montes, 2017).

Opposite feedbacks in the Hippo pathway for growth control and neural fate

Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncogene, YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase, LATS/Warts (Wts), and growth regulator, Melted, generates mutually exclusive photoreceptor subtypes. This study show that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network-including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module-that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution (Jukam, 2013).

A fundamental strategy in animal development is to re-purpose the same signaling pathways for a diversity of functions. This study identified a tissue-specific transcription factor network that enables the otherwise homeostatic Hippo growth control pathway to act as a bistable switch for terminal cell fate. This alteration in network level properties -- such as positive vs. negative feedback -- within biochemically conserved pathways is an efficient means to re-use a signaling network in contexts as distinct as proliferation and terminal differentiation (Jukam, 2013).

How is the R8-specific Hippo regulatory circuit achieved? The two interlinked positive feedback loops (one with wts, one with melt) provide the R8 Hippo pathway with multiple points of potential regulation. Context-specific expression of wts and melt is defined by overlapping expression of four transcription factors: Otd, Tj, Pph13, and Sens. Otd and Pph13 are expressed in all photoreceptors and generate a permissive context that endows the initially equipotent R8s with the competence to become either subtype: Otd promotes melt/Rh5 whereas Pph13 promotes wts/Rh6 expression. This competence is further restricted by expression of Tj in R7 and R8, and Sens in R8s, which ensures that melt and wts cross-regulation is restricted to R8s. Importantly, it is the status of Yki activity and resulting feedback that assures the outcome of p vs. y fate: in pR8s, Yki functions with Otd and Tj to promote melt and Rh5; in yR8s, wts inhibits Yki, preventing melt and Rh5 expressionand allowing 'default' wts and Rh6 expression by Pph13 and Sens. Each of these four transcription factors regulates a partially overlapping subset of R8 subtype fate genes, and together, the network cooperates at multiple regulatory nodes to provide the specific context for repurposing the Hippo pathway (Jukam, 2013).

While other instances of pathways with both positive and negative feedback exist, these are conceptually different from R8 Hippo regulation. For example, in Sprouty (hSpry) regulation of Ras/MAPK-mediated EGFR signaling, EGFR induces hSpry2 expression but hSpry2 inhibits EGFR function (negative feedback); however, hSpry2 also promotes EGFR activity by preventing Cbl-dependent EGFR inhibition (positive feedback). hSpry2 positive feedback is likely coupled to its negative feedback to fine-tune the length and amplitude of receptor activation. In contrast, the opposite Hippo pathway feedbacks occur in vastly different cell types (mitotic epithelial cells vs. post-mitotic neurons), and both forms of feedback cannot co-exist in R8 since Yki's repression of wts expression (positive feedback) would make Yki up-regulation of Hippo regulators (negative feedback) inconsequential (Jukam, 2013).

Gaining positive feedback or losing negative feedback within Hippo signaling could permit oncogenesis. Indeed, the Yki ortholog, YAP, is an oncogene and is amplified in multiple tumors, and LATS1/2 (Wts) down-regulation is associated with non-small cell lung carcinomas, soft tissue sarcoma, metastatic prostate cancers, retinoblastoma, and acute lymphoblastic leukemia. Otx and MAF factors are also oncogenic in a number of tissues. Thus, understanding the regulatory networks identified here in other contexts will be crucial for deciphering how normal signaling pathways can go awry (Jukam, 2013).

The current findings also reveal that a Crx/Otd-Nrl/Tj feedforward module plays a conserved role in post-mitotic photoreceptor fate specification in both flies and mammals. Both induce one photoreceptor fate at the expense of another, and both regulate opsins with a feedforward loop wherein Crx/Otd activates Nrl/Tj expression and Crx-Nrl or Otd-Tj synergistically activate downstream targets. Given such deep evolutionary conservation, this module may be critical for generating photoreceptor diversity in other complex visual systems (Jukam, 2013).

This work has two main implications. First, although positive feedback is well documented in other switch-like, irreversible cell fate decisions such as in Xenopus oocyte maturation or cell cycle entry, this work suggests that positive feedback could have a broad role in terminal neuronal differentiation, which often requires permanent fate decisions to maintain a neuron's functional identity. Second, the changes in network topology in R8 photoreceptors allows a finely tuned growth control pathway to be used as a switch in a permanent binary cell fate decision. Context-specific regulation allows the feedback architecture to change in an otherwise conserved signaling module. This may be a general mechanism to endow signaling networks with new systems properties and diversify cell fates in development and evolution (Jukam, 2013).

A Drosophila tumor suppressor gene prevents tonic TNF signaling through receptor N-glycosylation

Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. This study reports that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. Ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, which results in increased binding to the continually circulating TNF. These results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues (de Vreede, 2018).

Dual role of a C-terminally truncated isoform of large tumor suppressor kinase 1 in the regulation of Hippo signaling and tissue growth

The considerable amount of experimental evidence has defined the Hippo pathway as a tumor suppressive pathway and increased expression and/or activity of its oncogenic effectors is frequently observed in cancer. However, clinical studies have failed to attribute cancer development and progression to mutations in the pathway. In explaining this conundrum, this study investigated the expression and functions of a C-terminally truncated isoform of large tumor suppressor kinase 1 (LATS1) called short LATS1 (sLATS1) in human cell lines and Drosophila. Intriguingly, through overexpression of sLATS1, it was demonstrated that sLATS1 either activates or suppresses the activity of Yes-associated protein (YAP; see Drosophila Yorkie), one of the effectors of the Hippo pathway, in a cell type-specific manner. The activation is mediated through inhibition of full-length LATS1, whereas suppression of YAP is accomplished through sLATS1-YAP interaction. In HEK293T cells, the former mechanism may affect the cellular response more dominantly, whereas in U2OS cells and developing tissues in Drosophila, the latter mechanism may be solely carried out. Finally, to find the clinical relevance of this molecule, the expression of sLATS1 was examined in breast cancer patients. The transcriptome analysis showed that the ratio of sLATS1 to LATS1 was increased in tumor tissues comparing to their adjacent normal tissues (Matsui, 2018).

Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis

To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. This study shows that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells (Bohere, 2018).

The results show that Shavenbaby is expressed and required for the maintenance of adult renal stem cells in flies, supporting the conclusion that the OvoL/Svb family of transcription factors plays a key and evolutionarily-conserved role in the behavior of progenitors/stem cells (Bohere, 2018).

The role of Svb in adult stem cell maintenance in flies requires both a fine control of its expression and of its transcriptional activity. Svb expression in RNSCs involves at least two separable enhancers, driving similar expression patterns. Svb was one of the first cases to reveal the functional importance of apparently redundant (or shadow) enhancers in the phenotypic robustness of regulatory networks across tissues and development stages. The data suggest that a similar cis-regulatory architecture is also underlying the control of adult stem cells (Bohere, 2018).

RNSCs maintenance further requires a proper post-translational maturation of the Svb protein, in response to Pri smORF peptides. During both embryonic and post-embryonic development, the main role of Pri peptides is to provide a temporal control of Svb activity, conveying systemic steroid signaling. It is therefore possible that Pri smORF peptides also connect genetic networks to hormonal control for the regulation of adult stem cells. Recent work has shown that various smORF peptides contribute to the regulation of developmental pathways, muscle formation and physiology, etc., and the current findings extend their influence to the control of adult stem cells. It has been proposed that the emerging field of smORF peptides may open innovative therapeutic strategies, for example peptidomimetic drugs, which might also be of interest for regenerative medicine (Bohere, 2018).

The current results establish that a main function of Svb in adult stem cells is mediated by a functional interplay with the Hippo pathway, well established for its roles in the control of adult stem cells. The results indicate that Svb behaves as a nuclear effector of Hippo, relying on a direct interaction with Yorkie in order to protect stem cells from apoptosis, at least in part through the regulation of DIAP1 expression. Analysis of genome-wide binding events further suggests that the Svb/Yki interaction is involved in the control of a broader set of Hippo-regulated genes, including during development. Since both Pri and Ubr3 are also essential for the survival of adult stem cells, it is interesting to note that Ubr3 protects the DIAP1 protein from degradation, and direct binding of Ubr3 on the activated form of DIAP1 is elicited in the presence of Pri peptides. Therefore, in addition to the control of DIAP1 expression (via Svb), Ubr3 and Pri could also stabilize the DIAP1 protein to protect stem cells from apoptosis. Although initially restricted to TEAD transcription factors, the number of Yorkie (YAP/TAZ) nuclear partners is rapidly growing. Interestingly, recent work has shown a direct interaction of YAP/TAZ with the pro-EMT factors Snail/Slug, in the control of stem cell renewal and differentiation. As previously reported for intestinal stem cells, this study shows that pro-EMT regulators are also required for preventing premature differentiation of renal stem cells. While pro-EMT and OvoL factors are often viewed as antagonistic factors, in vivo studies in Drosophila stem cells show that they both contribute to their maintenance, Svb/Yki preventing their apoptosis and EMT factors their differentiation. Since many studies have implicated the Hippo pathway, pro-EMT and OvoL/Svb factors in various tumors, new insights into their functional interactions in adult stem cells may provide additional knowledge directly relevant to understand their connections in human cancers (Bohere, 2018).

Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination

The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. Previous work has identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and has shown that Fat's regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. This study characterizes a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, Dlish was found to binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. It was further shown that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex (Wang, 2019).

The intracellular domain (ICD) of the giant Drosophila protocadherin Fat reduces cell proliferation in imaginal disc tissues by regulating the Hippo pathway, an effect potentiated by heterophilic binding between Fat and the protocadherin Dachsous (Ds). The Fat ICD increases the activity of NDR family kinase Warts, the Drosophila homolog of vertebrate LATS and the final effector kinase in the Hippo pathway, and decreases the activity of the Warts target Yorkie (Yki), the Drosophila homolog of the vertebrate YAP and TAZ transcriptional coactivators. Active Warts phosphorylates and inhibits Yki by increasing the binding between Yki and its cytoplasmic tethers. In the absence of Fat, Warts activity is reduced, increasing the proportion of Yki that enters the nucleus with its TEAD-family cofactor Scalloped (Sd), driving transcription and overgrowth of imaginal disc epithelia (Wang, 2019).

While Fat is one of the best-studied transmembrane regulators of the Hippo pathway, the biochemical pathways that link Fat's ICD to changes in Warts and Yki activity have not been fully elucidated. The portions of Fat's ICD that suppress growth lack obvious catalytic or protein-binding motifs and, until recently, binding partners. However, recent work indicates that Fat's ICD binds to the cytoplasmic SH3-domain protein Dlish (also known as Vamana), reducing Dlish levels and activity, and thereby regulating a Dlish-binding partner, the atypical type XX myosin Dachs (Zhang, 2016; Misra, 2016) (see Model of inputs from the Fat, Ds, and Crumbs ICDs into the Hippo growth control pathway). Fat, Dlish, Dachs, and Warts are all concentrated at the subapical cell cortex of disc epithelial cells near their adherens junctions, although some subapical Warts is also concentrated at nonjunction sites, and for both Dlish and Dachs this depends on the formation of a Dlish-Dachs complex. When Fat is lost, the subapical levels of Dlish and Dachs greatly increase, an effect specific to the Fat branch of the Hippo pathway. The increased Dachs binds and inhibits Warts by altering its conformation and reducing its levels, thereby inducing Yki-mediated overgrowth (Wang, 2019).

Previous evidence suggested that the increased Dlish of fat mutants stimulates growth only by increasing subapical Dachs, rather than through any direct effect on Warts or Yki. Unlike Dachs, Dlish does not bind Warts. And while Dlish is necessary and sufficient for the localization and activity of wild-type Dachs, the overgrowth induced by a membrane-targeted Dachs construct is not reduced by the loss of Dlish; concentrating Dachs at the membrane is sufficient to bypass Dlish. Instead, Dlish provides a physical link between Dachs and the Fat ICD, along with the DHHC palmitoyltransferase Approximated (App), which can bind Fat and Dachs and palmitoylate Dlish. Loss of either Dlish or App disrupts Fat's ability to regulate Dachs levels and Yki activity. In the simplest view, the Fat ICD binds and inhibits Dlish and App, reducing their ability to regulate Dachs; when Fat is lost, App and palmitoylated Dlish are free to bind Dachs and concentrate it near the subapical cell membrane where it inhibits Warts. Dachs is also needed to concentrate the Dlish/Dachs complex in the cortex, likely by binding to the actin cytoskeleton or other scaffolds; thus, the reduction of Dachs by the Fat-tethered ubiquitin ligase Fbxl7 provides a parallel route for regulating Dlish and Dachs activity. The Ds ICD also binds and recruits Dachs and Dlish, but it is uncertain how strongly the endogenous Ds ICD affects the Hippo pathway (Wang, 2019).

While Dachs plays a major role in growth control through its ability to inhibit Warts, this study shows that this is not the sole mediator of Dlish activity. Rather, Dlish also regulates a parallel pathway mediated by the growth-inhibiting FERM domain protein Expanded (Ex). Ex regulates the Hippo pathway at multiple levels that are distinct from the Dachs-mediated alterations in Warts conformation. Ex binds Hippo, Warts, and the pathway modulators Merlin and Kibra and stimulates the phosphorylation and activity of Warts; Ex can also bypass Warts by binding and inhibiting Yki (Wang, 2019).

Indeed, Fat was originally linked to the Hippo pathway, not through Dachs, but through Ex: loss of Fat decreases Ex levels in the subapical cell cortex. The Ex decrease is particularly striking as it is at odds with the increased Yki-driven ex transcription caused by loss of Fat, indicating that the effect is posttranscriptional; the effect is also specific for the Fat branch of the Hippo pathway, as increasing Yki activity through other branches increases both ex transcription and Ex protein levels as part of a negative feedback loop (Wang, 2019).

This study shows that mimicking the effects of Fat loss by increasing Dlish levels has a growth-inducing activity that is independent of the Dachs myosin, and thus Dachs-mediated inhibition of Warts.Two of the three SH3 domains of Dlish bind directly to multiple sites in Ex, Dlish decreases Ex protein levels in wing imaginal discs independently of ex transcription, and that without Dlish the loss of Fat no longer reduces Ex protein levels. Previous studies showed that Ex levels are reduced by ubiquitination mediated by the Ex-binding F-box E3 ubiquitin ligase Slimb and the Skp-Cullin-F-box (SCF) complex, a process stimulated by the Ex-binding transmembrane protein Crumbs. This study will confirm and extend previous finding that Dlish binds to Slimb and shows that Dlish stimulates the Slimb-dependent ubiquitination of Ex in vitro (Wang, 2019).

Expanded directly binds conserved regions of Fat to restrain growth via the Hippo pathway

The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. This study showed that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. Direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, this study found Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth (Fulford, 2023).

Recruitment of Jub by alpha-catenin promotes Yki activity and Drosophila wing growth

The Hippo signaling network controls organ growth through YAP family transcription factors, including the Drosophila Yorkie protein. YAP activity is responsive to both biochemical and biomechanical cues, with one key input being tension within the F-actin cytoskeleton. Several potential mechanisms for biomechanical regulation of YAP proteins have been described, including tension-dependent recruitment of Ajuba family proteins, which inhibit kinases that inactivate YAP proteins, to adherens junctions (AJ). This study investigated the mechanism by which the Drosophila Ajuba family protein, Ajuba LIM protein (Jub) is recruited to adherens junctions, and the contribution of this recruitment to the regulation of Yorkie. Alpha-catenin is identifed as the mechanotransducer responsible for tension-dependent recruitment of Jub by identifying a region of α-catenin that associates with Jub, and by identifying a region, which when deleted, allows constitutive, tension-independent recruitment of Jub. Increased Jub recruitment to alpha-catenin is associated with increased Yorkie activity and wing growth, even in the absence of increased cytoskeletal tension. These observations establish alpha-catenin as a multi-functional mechanotransducer and confirm Jub recruitment to alpha-catenin as a key contributor to biomechanical regulation of Hippo signaling (Alegot, 2019).

To evaluate the role of α-catenin in the recruitment of Jub to AJ, Jub localization was examined in wing discs isolated from animals expressing truncated forms of α-catenin. This was achieved by using RNAi to knock-down expression of endogenous α-catenin and expressing RNAi-insensitive Venus- or V5-tagged α-catenin transgenes under UAS-Gal4 control. Transgenes were expressed in posterior cells using an en-Gal4 driver, so that anterior cells could serve as a control for Jub localization. Full-length α-catenin expressed under UAS-Gal4 control rescued both the lethality associated with knockdown of α-catenin, and Jub localization. α-catenin has similarity to Vinculin in N-terminal, middle, and C-terminal regions termed VH1, VH2, and VH3. An initial series of constructs deleted regions of α-catenin centered around these Vinculin homology regions, as well as separate deletions of either the N-terminal or C- terminal half of VH2. Constructs lacking either the N- or C-terminus of α-catenin (ΔVH1 or ΔVH3) could not rescue the lethality associated with α-catenin knockdown. Thus, to enable visualization of the consequences of these deletions on Jub, conditional knockdown and expression of α-catenin was achieved using a temperature sensitive-allele of Gal80 (Gal80ts) that represses Gal4-driven expression at 18°C but not at 29°C. After a 30 hour shift to higher temperature, Jub was mostly lost from apical cell junctions. However, E-cadherin was also mostly lost, indicating that these regions of α-catenin are required to maintain AJ. This is consistent with the roles of F-actin and α-catenin in stabilizing AJ, as the VH1 region is required for association with β-catenin and thus localization to AJ, and the VH3 region is required for association with F-actin. The ΔVH1 and ΔVH3 constructs also did not themselves localize to junctions. The loss of Jub from cell junctions under these conditions is consistent with studies indicating its association with AJ, but does not provide specific information about its interactions with α-catenin (Alegot, 2019).

Conversely, deletions within the central region of α-catenin increased Jub recruitment to AJ. Deletion of the entire VH2 region increased Jub recruitment but was also associated with abnormal cell morphology and E-cadherin localization. However, two smaller deletions comprising the N- and C-terminal halves of the VH2 region (ΔVH2-N and ΔVH2-C) rescued knock-down of α-catenin, resulting in cells that appear to have normal localization of E-cadherin and α-catenin. The ΔVH2-C deletion also appears to have normal Jub localization. In contrast, the ΔVH2-N deletion is associated with increased Jub localization at AJ. Moreover, in cells with the ΔVH2-N deletion, Jub is distributed relatively uniformly around cell junctions, whereas in cells with wild-type α-catenin, Jub localization to junctions is often punctate. The ΔVH2-N deletion was not only associated with increased Jub recruitment when expressed in place of endogenous α-catenin; it could also promote Jub recruitment even when expressed in the presence of endogenous α-catenin. Thus, expression of this isoform dominantly increases Jub recruitment to AJ (Alegot, 2019).

To further investigate the influence of α-catenin on Jub, smaller deletion constructs were created. To aid in the design of these additional constructs, Phyre2 was used to predict the structure of Drosophila α-catenin, based on its sequence similarity to mammalian α-catenin proteins with experimentally-determined structures. Mammalian α-catenin consists largely of a series of α-helical bundles: two N-terminal 4-helix bundles (N1 and N2) that share one long α-helix; three central 4-helix bundles (M1, M2, and M3), and a C-terminal 5-helix bundle. These structural features are also predicted for Drosophila α-catenin (Alegot, 2019).

The ΔVH2-N deletion that increases Jub recruitment to AJ deletes both the M1 and M2 helical bundles. Thus, separate deletions were created of either the M1 or M2 bundles. Deletion of the M2 bundle (ΔM2) slightly increased Jub localization to AJ, but a much stronger increase in Jub binding was detected when the M1 bundle was deleted (ΔM1a or ΔM1b). Recruitment of Jub to AJ is normally promoted by cytoskeletal tension. To examine the possibility that M1 deletions increase Jub recruitment by increasing tension, levels were examined of junctional myosin (using a GFP-tagged myosin light chain) that correlate with junctional tension, but no difference was observed. F-actin levels were similarly unaffected. Staining was carried out specifically for the phosphorylated (activated) form of myosin regulatory light chain (pMLC) in discs expressing Jub:GFP. This revealed similar levels of pMLC in control cells and cells expressing α-catenin ΔM1b, whereas expression of rok RNAi or an activated form of Rok visibly decreased or increased, respectively, junctional levels of both pMLC and Jub. Since Jub associates with α-catenin, the observation that deletion of the M1 bundle increases Jub localization to AJ without increasing tension suggests that deletion of this region alters the structure of α-catenin in a way that makes a Jub-binding region more accessible. The M1 bundle might thus act as an 'inhibitory' region that prevents Jub binding, with this inhibition normally alleviated in wild-type α-catenin by a conformational change induced by high tension. Alternatively, deletion of the M1 bundle might destabilize a non-Jub binding conformation of α-catenin and thereby indirectly increase accessibility of a Jub-binding region (Alegot, 2019).

While both Jub and Vinculin exhibit tension-dependent association with α-catenin, the results suggest that their interactions are distinct. A region initially identified as inhibitory for Vinculin binding, corresponds to the M3 helical bundle and the linker between VH2 and VH3. Moreover, the M1 helical bundle includes the Vinculin binding interface. To directly compare the influence of α-catenin deletions on Jub and Vinculin binding, advantage was taken of a Drosophila genomic GFP-tagged Vinculin (Vinc:GFP). Both of the M1 deletions substantially reduced, but did not eliminate, Vinculin localization to AJ, which fits with observations that Vinculin interacts with sequences in the M1 bundle, but implies that additional mechanisms also contribute to Vinculin localization. Deletion of the M2 bundle increased Vinculin localization to AJ, consistent with observations that M2 participates in interactions that stabilize a conformation of α-catenin that is 'closed' with respect to Vinculin binding (Alegot, 2019).

If M1 deletions mimic the influence of cytoskeletal tension on the ability of α-catenin to bind to Jub, the increased recruitment of Jub in M1 deletion isoforms could occur even in the absence of tension. To test this, Jub localization was examined in flies expressing α-catenin constructs and with cytoskeletal tension decreased by RNAi of Rho-associated protein kinase (Rok), a promoter of myosin activity. In the presence of full-length α-catenin, rok RNAi decreases Jub recruitment to junctions. Conversely, in the presence of deletions that include the M1 bundle, Jub recruitment to junctions remains elevated. Quantitation of Jub at junctions, normalized to E-cadherin, revealed a decrease in Jub recruitment when tension is lowered even in cells expressing M1 deletions, but Jub recruitment nonetheless remains above that in control cells. Thus, these deletions recruit Jub even under low-tension conditions, which implicates α-catenin as the key mechanotransducer responsible for tension-dependent recruitment of Jub (Alegot, 2019).

Cytoskeletal tension promotes Yki activity, and Yki activity is suppressed by knockdown of Jub. However, other mechanisms by which tension might increase Yki activity have also been suggested, such as an influence on spectrins. The observation that deletions of the M1 bundle lead to increased Jub at AJ without increasing myosin identifies a condition under which it was possible to determine whether recruitment of Jub to junctions is sufficient to increase Yki activity, and thus distinguish the contribution of Jub from other potential influences of cytoskeletal tension (Alegot, 2019).

Yki activity was evaluated by examining ex-lacZ expression, which is a reporter for the Yki target gene expanded (ex). Expression of α-catenin with deletion of the M1 bundle increased ex-lacZ, whereas expression of full length α-catenin did not. Increased Yki activity is associated with increased accumulation of Yki in the nucleus, and nuclear levels of Yki were slightly increased in wing disc cells expressing M1 region deletions. To further examine the influence of the M1 deletion, adult wing size was examined, as wing growth is promoted by Yki activity. Expression of M1 deletion isoforms throughout the developing wing under nub-Gal4 control increased wing size as compared to wings expressing wild-type α-catenin. The increased Yki activity and wing growth in M1 deletion mutants are Jub-dependent, because they are reversed by RNAi of jub. As mutation or knock-down of Vinculin has only minor phenotypic consequences in Drosophila, the effects of M1 deletion on Yki activity and wing growth cannot be attributed to loss of Vinculin. Altogether, these observations establish that increased Jub recruitment to junctions can be sufficient to elevate Yki activity. This strongly supports the conclusion that Jub recruitment to AJ is a key component of the biomechanical response linking cytoskeletal tension to Yki activity (Alegot, 2019).

However, analysis of the larger ΔVH2N deletion suggests that the role of α-catenin in promoting Yki activity is more complex. Although expression of ΔVH2N α-catenin increased Jub recruitment at AJ, it did not detectably increase ex-lacZ expression or wing size. Similarly, even though deletion of the M2 helical bundle modestly increased Jub recruitment to AJ, it did not detectably increase ex-lacZ expression or wing size. Thus, it is inferred that additional features of α-catenin may contribute to Yki regulation (Alegot, 2019).

Jub requires α-catenin to localize to junctions. To map regions of α-catenin that mediate association with Jub, co-immunoprecipitation experiments were performed in Drosophila S2 cells expressing transfected constructs. Initially, co- immunoprecipitation of Jub with α-catenin full length, VH1, VH2, and VH3 region constructs was compared. Significant association of Jub and a VH1 region construct was detected. Conversely, association of Jub with full length, VH2, or VH3 constructs was weaker, and close to the non- specific background. The association with the VH1 region of α-catenin was not as strong as binding of Jub to Warts, which was included as a positive control. Association of Jub with the VH1 region is consistent with reports that the mammalian Ajuba protein can associate with an N- terminal fragment of α-catenin, corresponding roughly to VH1. The stronger association of Jub with a VH1 fragment as compared to full length α-catenin is consistent with normal binding depending upon junctional tension, as S2 cells are not epithelial, so it would be expected that the transfected α-catenin would be in a low-tension conformation. Moreover, co-immunoprecipitation experiments comparing M1 bundle deletion constructs to full length α-catenin revealed that M1 deletions significantly increased association of Jub with α-catenin (Alegot, 2019).

To further refine the Jub-binding region, the predicted Drosophila α-catenin structure was used to design constructs corresponding to the N1 or N2 helical bundles. No significant co-immunoprecipitation with the N1 bundle was detected, whereas co- immunoprecipitation with N2 was comparable to that for the VH1 region construct. Thus, it is inferred that the primary site of association of Jub with α-catenin is within N2. While the simplest model would be that Jub directly binds to this region of α-catenin, it remains possible that their association is mediated through other proteins. The N2 bundle is near the M1 bundle, but they do not directly contact each other in the predicted structure. Thus, the effect of M1 deletion on Jub binding may be an indirect consequence of a conformational change in α-catenin when this region is deleted, or reduced stability of a more closed conformation, rather than the M1 bundle directly obscuring a binding site within N2. Attempts were also mad to examine the consequences of loss of Jub recruitment to junctions by expressing α-catenin with N2 deleted in vivo. However, this protein failed to rescue the viability of wing disc cells expressing α-catenin RNAi, so this could not be examined. Moreover, when expressed in wild-type cells, the ΔN2 construct failed to localize to adherens junctions (Alegot, 2019).

These results establish key features of α-catenin mechanotransduction and tension- dependent regulation of Yki activity. They implicate α-catenin as a multi-functional mechanotransducer that associates with both Vinculin and Jub upon a tension-induced conformational change in α-catenin, but through distinct sites. The complete structure of the open conformation of α-catenin has not been determined, but it is inferred that it makes Jub- associating regions accessible. M1 deletions also make the Jub-associating region more accessible, but it's not clear whether they do so in a manner similar to, or distinct from, the effect of tension. The observation that M1 deletions increase Yki activity also supports the crucial role of Jub recruitment to AJ in promoting Yki activity. This does not exclude the possibility that of other consequences of cytoskeletal tension contribute to Yki regulation, but clearly implicates recruitment of Jub as a key factor (Alegot, 2019).

Opposing transcriptional and post-transcriptional roles for Scalloped in binary Hippo-dependent neural fate decisions

The Hippo tumor suppressor pathway plays many fundamental cell biological roles during animal development. Two central players in controlling Hippo-dependent gene expression are the TEAD transcription factor Scalloped (Sd) and its transcriptional co-activator Yorkie (Yki). Hippo signaling phosphorylates Yki, thereby blocking Yki-dependent transcriptional control. In post-mitotic Drosophila photoreceptors, a bistable negative feedback loop forms between the Hippo-dependent kinase Warts/Lats and Yki to lock in green vs blue-sensitive neuronal subtype choices, respectively. Previous experiments indicate that sd and yki mutants phenocopy each other's functions, both being required for promoting the expression of the blue photoreceptor fate determinant melted and the blue-sensitive opsin Rh5. This study demonstrates that Sd ensures the robustness of this neuronal fate decision via multiple antagonistic gene regulatory roles. In Hippo-positive (green) photoreceptors, Sd directly represses both melt and Rh5 gene expression through defined TEAD binding sites, a mechanism that is antagonized by Yki in Hippo-negative (blue) cells. Additionally, in blue photoreceptors, Sd is required to promote the translation of the Rh5 protein through a 3'UTR-dependent and microRNA-mediated process. Together, these studies reveal that Sd can drive context-dependent cell fate decisions through opposing transcriptional and post-transcriptional mechanisms (Xie, 2019).

Ensuring that the correct complement of genes remains on or off in any given cell type is an essential feature of multicellular organisms. This is particularly critical in the peripheral nervous system, where exclusive sensory receptor expression is necessary for selective and specific activation of a given sensory neuron. Such exclusion is well-established in the visual system of most animals, where individual photoreceptors (PRs) express a single opsin photopigment and repress the expression of others to prevent sensory overlap. The gene regulatory mechanisms underlying this mutual exclusion, however, are still under investigation (Xie, 2019).

The Drosophila eye has long served as a powerful model to understand the functions and architecture of gene regulatory networks underlying PR subtype cell fate specification. Each of the approximately 750 individual eye units (ommatidia) in the Drosophila compound eye contains 8 PRs. Based on the specific opsin that is expressed in the R8 photoreceptor, two major ommatidial subtypes, pale (p) and yellow (y), are present in the adult eye. Pale ommatidia are primarily defined based on the expression of the blue-sensitive opsin, Rhodopsin 5 (Rh5), while yellow ommatidia express the green-sensitive opsin, Rh6. These ommatidial subtypes are randomly distributed through the eye in a 30:70 blue:green ratio, and are established and maintained through a bistable negative feedback loop between two signaling molecules: the pleckstrin homology-containing protein Melted (Melt) and the Hippo signaling kinase Warts (Wts, aka Lats) (Xie, 2019).

Wts is a core component of the Hippo kinase complex that phosphorylates and inactivates the transcriptional co-activator Yorkie (Yki). Hippo signaling is best understood in the context of growth regulation, where Wts and Yki function in a homeostatic feedback loop: Wts blocks Yki function and Yki initiates its own inactivation by promoting Hippo pathway gene expression. In contrast, in post-mitotic PR fate decisions, Yki promotes the expression of the wts repressor, melt, generating a double-negative 'on/off' feedback loop between wts and Yki that ensures two stably maintained fate choices. In green PRs, Hippo signaling promotes the expression of green fate determinants (wts and Rh6), and prevents the expression of Yki-dependent blue fate determinants (melt and Rh5). In blue PRs, Yki promotes melt, thereby repressing wts and inhibiting Hippo signaling, further promoting Yki-dependent activation of blue fate effectors and suppression of green fate effectors. Thus, Wts-positive (Yki-inactive) cells adopt the default green/wts/Rh6 fate, while Wts-negative (Yki-active) cells acquire the blue/melt/Rh5 fate (Xie, 2019).

Yki, a YES-associated protein (YAP), is a transcriptional co-activator that does not bind DNA itself, but instead requires a DNA-binding partner. The primary binding partners for Yki/Yap factors are members of the TEAD family of transcription factors. In Drosophila, the single TEAD family member is encoded by Scalloped (Sd). Sd/TEAD and Yki/YAP can physically interact and together activate TEAD-site-containing reporter expression in vitro. Furthermore, in ectopic yki conditions, sd/TEAD is essential for yki/YAP to induce tissue overgrowth and activate target gene expression. However, in vivo, sd mutants do not phenocopy yki growth phenotypes and sd mutants do not show changes in yki target gene expression. These data suggest that Sd and Yki use distinct mechanisms to control tissue size. Studies aimed at addressing this conundrum have shown that in developing wing, eye, and follicle cells, Sd functions as a transcriptional repressor under 'Hippo-on' conditions to inhibit cell growth, and that in 'Hippo-off' cells, Yki antagonizes Sd repression to promote growth regulatory genes. This suggests that Sd and Yki can play opposite roles during growth (Xie, 2019).

In post-mitotic PRs, it has been previously shown that sd mutants phenocopy yki's knockdown phenotype in PR subtype fate specification: both sd and yki are necessary to promote blue PR fate and inhibit green PR fate. Combined, these findings suggest that sd and yki function together in this cell fate specification event. This study investigated the molecular basis underlying this interaction. Sd was found to play roles at both the transcriptional and post-transcriptional level to ensure blue vs green PR subtype fate decisions. At the transcriptional level, Sd directly represses blue fate effector gene expression in Hippo (Wts)-positive green PRs, and Yki antagonizes this repression in Hippo (Wts)-negative blue PRs. This is consistent with previously reported antagonism between Sd and Yki. In addition to this function, it was found that Sd promotes blue fate through a post-transcriptional, microRNA (miRNA)-dependent process in Wts-negative blue PRs, revealing a cooperative interaction with Yki in promoting blue PR fate. Together, these new findings elucidate a multi-tiered regulatory network involving the Drosophila TEAD transcription factor that functions at both the transcriptional and post-transcriptional level to precisely specify neuronal subtype fate (Xie, 2019).

The mutually exclusive expression of sensory receptor genes in sense organs is essential to prevent sensory input overlap in the mature organism. This study shows that, in the fly retina, the TEAD factor Sd achieves this in blue and green PRs using two different mechanisms: direct transcriptional repression of the blue fate determinant melt and blue Rh5 opsin genes in green photoreceptors, and relief of post-transcriptional control of the Rh5 mRNA in blue photoreceptors. In addition, Yki, a major Sd cofactor, antagonizes Hippo-specific and Sd-dependent repression of melt and Rh5 to promote blue PR fate. Thus, Sd and Yki play multiple roles to ensure a robust bistable cell fate decision in post-mitotic sensory neurons (Xie, 2019).

The antagonistic relationship between Sd repression and Yki de-repression is similar to the model previously proposed in cell cycle control. Nevertheless, the mechanisms by which Sd represses gene expression in green PRs remains unknown. In cell growth, for instance, repression is mediated in part through Tgi, a Tondu domain containing protein, which Yki competes with to alleviate repression. However, no significant change was detected in Rh5 protein or reporter expression with knockdown of Tgi in PRs, suggesting the existence of another Sd co-repressor in this system. Indeed, a zinc finger protein Nerfin-1 was recently identified as a Tgi-independent Sd co-repressor that participates in Hippo-dependent cell growth and competition during Drosophila eye development (Guo, 2019). Preliminary studies showed that knockdown of nerfin-1 led to an expansion of Rh5-expressing blue PRs at the expense of green PRs, comparable to the expanded expression of Sd site mutants in the melt and Rh5 reporters. Therefore, Nerfin-1 is very likely to be at least one Sd co-repressor during blue- and green PR fate specification in the Drosophila eye. Combined, these findings suggest Sd repression activity is a general mechanism in controlling the output of the Hippo pathway (Xie, 2019).

If the role of Sd in green PRs were solely to repress Rh5 transcription, then Rh5 mRNA levels might be expected to be elevated in sd mutants relative to controls. Instead, a ~50% reduction was observed. This observation could reflect two possibilities, which are not mutually exclusive. First, based on previous and unpublished findings that Otd cooperates with Yki to activate Rh5 in Hippo-negative blue PRs, it is expected that in sd mutants, where all R8s switch to Hippo-positive (and hence Yki-inactive) green PRs, Rh5 activation in green PRs would be reduced. Second, since the current studies suggest a new role for miRNAs in the post-transcriptional control of Rh5, it is possible that Rh5 mRNA stability is affected in sdmutants (Xie, 2019).

In terms of the post-transcriptional control of Rh5, it was demonstrated that the Rh5 3' UTR was required to prevent its co-expression with Rh6 in sd knockdown green PRs. In addition, the simultaneous knockdown of sd and miRNA processing machinery genes led to Rh5 protein de-repression (and co-expression with Rh6) in a substantial subset of green R8 cells. Together, these data suggest miRNA-dependent regulation of Rh5 depends on Sd, either directly or indirectly. It is posited that, as a transcription factor, Sd prevents the transcription of Rh5-directed miRNA genes. However, follow-up studies will be important for defining the complete repertoire of miRNA-dependent events involved in this Hippo-directed cell fate decision. For example, possible differences in an pRh5 reporter and endogenous Rh5 protein were reported in retinas mutant for the transcription factor PvuII-PstI homology 13 (pph13). While this disparity could be due to the rhabdomere defects observed in pph13 mutants, there is potential for a role for Pph13 in Rh5 post-transcriptional regulation. Finally, it is possible that the Rh5 3'UTR recruits other non-coding RNAs or proteins to regulate its expression (Xie, 2019).

Combined, the bimodal functions of Sd in Yki-vs Wts-positive cells form a feedforward regulatory module in post-mitotic PR fate decisions, robustly preventing sensory receptor overlap. Feedforward modules between transcription factors and miRNAs have been previously reported in neuronal differentiation and other biological processes. For example, the proto-transcription factor c-Myc can directly activate E2F1 transcription, but also limit E2F1 translation by activating miR-175p and miR-20a. In contrast to the c-Myc-miRNAs-E2F1 activation module, which fine-tunes a proliferative signal in dividing cells, however, the Sd-miRNA-Rh5 repression module ensures a robust ON-OFF switch in the terminal PR differentiation process. If similar mechanisms take place during Hippo-dependent cell growth remains to be determined (Xie, 2019).

Whether yki is also involved in Sd's post-transcriptional control in blue PRs remains unresolved, as yki itself is essential for blue PR fate, and hence, Rh5-expressing cells. Previous studies have demonstrated that Yki is important for the activation of at least one miRNA to promote cell growth (i.e. bantam). However, in the case of Rh5 regulation, the miRNA must be repressed in Yki-expressing cells, rather than activated. In this context, it is worth noting that the Yki ortholog YAP has been shown to mediate widespread miRNA suppression in tumor cells (Hippo-negative) by sequestering an RNA helicase p72/DDX-17, a regulatory component of microRNA-processing machinery. Comparably, the results suggest that the miRNA(s) is/are inactive in Yki-positive blue PRs in order to allow Rh5 protein expression. These findings raise the possibility that YAP/Yki- and TEAD/Sd-dependent regulation of miRNA biogenesis is a universal mechanism in control of the Hippo signaling pathway in tissue growth and neuronal cell fate decisions (Xie, 2019).

The role of lysine palmitoylation/myristoylation in the function of the TEAD transcription factors

The TEAD transcription factors are the most downstream elements of the Hippo pathway. Their transcriptional activity is modulated by different regulator proteins and by the palmitoylation/myristoylation of a specific cysteine residue. This report shows that a conserved lysine present in these transcription factors can also be acylated, probably following the intramolecular transfer of the acyl moiety from the cysteine. Using Scalloped (Sd), the Drosophila homolog of human TEAD, as a model, we designed a mutant protein (Glu352Gln(Sd)) that is predominantly acylated on the lysine (Lys350(Sd)). This protein binds in vitro to the three Sd regulators-Yki, Vg and Tgi-with a similar affinity as the wild type Sd, but it has a significantly higher thermal stability than Sd acylated on the cysteine. This mutant was also introduced in the endogenous locus of the sd gene in Drosophila using CRISPR/Cas9. Homozygous mutants reach adulthood, do not present obvious morphological defects and the mutant protein has both the same level of expression and localization as wild type Sd. This reveals that this mutant protein is both functional and able to control cell growth in a similar fashion as wild type Sd. Therefore, enhancing the lysine acylation of Sd has no detrimental effect on the Hippo pathway. However, we did observe a slight but significant increase of wing size in flies homozygous for the mutant protein suggesting that a higher acylation of the lysine affects the activity of the Hippo pathway. Altogether, these findings indicate that TEAD/Sd can be acylated either on a cysteine or on a lysine, and suggest that these two different forms may have similar properties in cells (Mesrouze, 2022).

Hippo reprograms the transcriptional response to Ras signaling

Hyperactivating mutations in Ras signaling are hallmarks of carcinomas. Ras signaling mediates cell fate decisions as well as proliferation during development. It is not known what dictates whether Ras signaling drives differentiation versus proliferation. This study shows that the Hippo pathway is critical for this decision. Loss of Hippo switches Ras activation from promoting cellular differentiation to aggressive cellular proliferation. Transcriptome analysis combined with genetic tests show that this excessive proliferation depends on the synergistic induction of Ras target genes. Using ChIP-nexus, Hippo signaling was found to keep Ras targets in check by directly regulating the expression of two key downstream transcription factors of Ras signaling: the ETS-domain transcription factor Pointed and the repressor Capicua. These results highlight how independent signaling pathways can impinge on each other at the level of transcription factors, thereby providing a safety mechanism to keep proliferation in check under normal developmental conditions (Pascual, 2017).

Mechanical strain regulates the Hippo pathway in Drosophila

Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole Drosophila homolog of which is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP and TAZ. This study shows that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin, and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts clones causes Yki nuclear localisation even in columnar follicle cells. No evidence was found for involvement of other pathways, such as Src42A kinase, in regulation of Yki. Finally, the results in follicle cells appear generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening (Fletcher, 2018).

Pits and CtBP Control Tissue Growth in Drosophila melanogaster with the Hippo Pathway Transcription Repressor, Tgi

The Hippo pathway is an evolutionary conserved signalling network that regulates organ size, cell fate control and tumorigenesis. The central Hippo signalling effector is the transcriptional co-activator Yorkie, which controls gene expression in partnership with different transcription factors, most notably Scalloped. When it is not activated by Yorkie, Scalloped can act as a repressor of transcription, at least in part due to its interaction with the corepressor protein Tgi. The mechanism by which Tgi represses transcription is incompletely understood and therefore this study sought to identify proteins that potentially operate together with it. Using an affinity purification and mass-spectrometry approach this study identified Pits and CtBP as Tgi-interacting proteins, both of which have been linked to transcriptional repression. Both Pits and CtBP were required for Tgi to suppress the growth of the Drosophila melanogaster eye and CtBP loss suppressed the undergrowth of yorkie mutant eye tissue. Furthermore, as reported previously for Tgi, overexpression of Pits repressed transcription of Hippo pathway target genes. These findings suggest that Tgi might operate together with Pits and CtBP to repress transcription of genes that normally promote tissue growth. The human orthologues of Tgi, CtBP and Pits (VGLL4, CTBP2 and IRF2BP2) have previously been shown to physically and functionally interact to control transcription, implying that the mechanism by which these proteins control transcriptional repression is conserved throughout evolution (Vissers, 2020).

A new perspective on the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and in this study on human VGLL3 show that these proteins interact with TEAD (Drosophila homolog: Scalloped) via a conserved amino acid motif called the TONDU domain. Surprisingly, these studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, it is proposed that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors (Mesrouze, 2020).

The Hippo pathway coactivator Yorkie can reprogram cell fates and create compartment-boundary-like interactions at clone margins

During development, tissue-specific patterns of gene expression are established by transcription factors and then stably maintained via epigenetic mechanisms. Cancer cells often express genes that are inappropriate for that tissue or developmental stage. This study shows that high activity levels of Yki, the Hippo pathway coactivator that causes overgrowth in Drosophila imaginal discs, can also disrupt cell fates by altering expression of selector genes like engrailed (en) and Ultrabithorax (Ubx). Posterior clones expressing activated Yki can down-regulate en and express an anterior selector gene, cubitus interruptus (ci). The microRNA bantam and the chromatin regulator Taranis both function downstream of Yki in promoting ci expression. The boundary between Yki-expressing posterior clones and surrounding wild-type cells acquires properties reminiscent of the anteroposterior compartment boundary; Hedgehog signaling pathway activation results in production of Dpp. Thus, at least in principle, heterotypic interactions between Yki-expressing cells and their neighbors could activate boundary-specific signaling mechanisms (Bairzin, 2020).

Human cancers are characterized by multiple genetic lesions, a subset of which are driver mutations that are thought to be responsible for their tumorous characteristics. It is estimated that most cancers have two to eight driver mutations. This makes it difficult to evaluate the contribution of each mutation to any particular characteristic of the tumor. This study has taken advantage of the ability of single-gene manipulations to cause overgrowth in Drosophila imaginal discs to assess the ability of three different oncogenes to destabilize established patterns of selector gene expression; yki, the Drosophila ortholog of Yap and Taz, is especially potent in doing so. The patterns of expression of En, Ci, and Ubx are established relatively early in embryogenesis and maintained stably in imaginal discs during the larval stages of development. These patterns of expression can be disrupted in clones expressing an activated form of Yki. Expression of a wild-type form of Yki is capable of disrupting these expression patterns in combination with other genetic manipulations such as overexpression of ban or tara. This latter scenario is more likely to apply to human cancers; increased Yap or Taz activity has been described in multiple human cancers, which often also have other genetic lesions (Bairzin, 2020).

These studies show that sd, ban, and tara make important contributions to the pathway by which Yki^CA destabilizes gene expression; reducing the expression of any of these in clones expressing Yki^CA greatly reduces ectopic Ci expression, and increasing expression of both genes can cause ectopic Ci expression. It is likely that other mechanisms function in parallel to destabilize selector gene expression since combined overexpression of ban and tara increased ectopic Ci expression but did not reduce En expression (Bairzin, 2020).

Changing selector gene expression within an overgrowing clone can create interactions at the clone margin that are reminiscent of compartment boundaries and result in the production of morphogens. A recent study showed that forced expression of En in lgl clones can elicit similar phenomena in anterior clones. In addition, Yki^CA clones are often extruded, consistent with previous observations that heterotypic interactions caused by overexpressing patterning genes also promotes extrusion. Previous work found that ci RNA levels were increased in wts mutant tissue, this study did not see ectopic Ci protein expression in wts mutant clones or wild-type Yki-overexpressing clones. This work shows therefore that sustained expression of very high Yki levels is necessary to destabilize expression of selector genes. However, even under these conditions, the effect on ectopic Ci expression is Sd dependent. Moreover, this study shows that even wild-type Yki can, in combination with increased expression of ban or tara, induce ectopic Ci expression. While these changes in gene expression are most obvious with above-physiological levels of Yki, they nevertheless reflect a previously unknown ability of this pathway to alter patterning gene expression and furthermore to change the growth characteristics of neighboring wild-type cells. Differences in selector gene expression between human cancers or precancerous lesions and their wild-type neighbors have received relatively little attention, and the current results call attention to tumor margins as sites where heterotypic interactions could create signaling centers that affect the behavior of tumor cells (Bairzin, 2020).

CORO7 functions as a scaffold protein for the core kinase complex assembly of the Hippo pathway

The Hippo pathway controls organ size and tissue homeostasis through the regulation of cell proliferation and apoptosis. However, the exact molecular mechanisms underpinning Hippo pathway regulation are not fully understood. This study identified a new component of the Hippo pathway: coronin 7 (CORO7), a coronin protein family member that is involved in organization of the actin cytoskeleton. pod1, the Drosophila ortholog of CORO7, genetically interacts with key Hippo pathway genes in Drosophila. In mammalian cells, CORO7 is required for the activation of the Hippo pathway in response to cell-cell contact, serum deprivation, and cytoskeleton damage. CORO7 forms a complex with the core components of the pathway and functions as a scaffold for the Hippo core kinase complex. Collectively, these results demonstrate that CORO7 is a key scaffold controlling the Hippo pathway via modulating protein-protein interactions (Park, 2020).

The Hippo pathway controls myofibril assembly and muscle fiber growth by regulating sarcomeric gene expression

Skeletal muscles are composed of gigantic cells called muscle fibers, packed with force-producing myofibrils. During development the size of individual muscle fibers must dramatically enlarge to match with skeletal growth. How muscle growth is coordinated with growth of the contractile apparatus is not understood. This study used the large Drosophila flight muscles to mechanistically decipher how muscle fiber growth is controlled. Regulated activity of core members of the Hippo pathway were found to be required to support flight muscle growth. Interestingly, Dlg5 and Slmap were identified as regulators of the STRIPAK phosphatase, which negatively regulates Hippo to enable post-mitotic muscle growth. Mechanistically, the Hippo pathway was shown to control timing and levels of sarcomeric gene expression during development and thus regulates the key components that physically mediate muscle growth. Since Dlg5, STRIPAK and the Hippo pathway are conserved a similar mechanism may contribute to muscle or cardiomyocyte growth in humans (Kaya-Copur, 2021).

Negative feedback couples Hippo pathway activation with Kibra degradation independent of Yorkie-mediated transcription

The Hippo (Hpo) pathway regulates tissue growth in many animals. Multiple upstream components promote Hpo pathway activity, but the organization of these different inputs, the degree of crosstalk between them, and whether they are regulated in a distinct manner is not well understood. Kibra (Kib) activates the Hpo pathway by recruiting the core Hpo kinase cassette to the apical cortex. This study shows that the Hpo pathway downregulates Drosophila Kib levels independently of Yorkie-mediated transcription. Hpo signaling complex formation promotes Kib degradation via SCF(Slimb)-mediated ubiquitination. This effect requires Merlin, Salvador, Hpo, and Warts, and this mechanism functions independently of other upstream Hpo pathway activators. Moreover, Kib degradation appears patterned by differences in mechanical tension across the wing. It is proposed that Kib degradation mediated by Hpo pathway components and regulated by cytoskeletal tension serves to control Kib-driven Hpo pathway activation and ensure optimally scaled and patterned tissue growth (Tokamov, 2021).

Misshapen Disruption Cooperates with Ras(V12) to Drive Tumorigenesis

Although RAS family genes play essential roles in tumorigenesis, effective treatments targeting RAS-related tumors are lacking, partly because of an incomplete understanding of the complex signaling crosstalk within RAS-related tumors. A large-scale genetic screen in Drosophila eye imaginal discs identified Misshapen (Msn) as a tumor suppressor that synergizes with oncogenic Ras (Ras(V12)) to induce c-Jun N-terminal kinase (JNK) activation and Hippo inactivation, then subsequently leads to tumor overgrowth and invasion. Moreover, ectopic Msn expression activates Hippo signaling pathway and suppresses Hippo signaling disruption-induced overgrowth. Importantly, it was further found that Msn acts downstream of protocadherin Fat (Ft) to regulate Hippo signaling. Finally, msn as a Yki/Sd target gene that regulates Hippo pathway in a negative feedback manner. Together, these findings identified Msn as a tumor suppressor and provide a novel insight into RAS-related tumorigenesis that may be relevant to human cancer biology (Kong, 2021).

Shaggy regulates tissue growth through Hippo pathway in Drosophila

The evolutionarily conserved Hippo pathway coordinates cell proliferation, differentiation and apoptosis to regulate organ growth and tumorigenesis. Hippo signaling activity is tightly controlled by various upstream signals including growth factors and cell polarity, but the full extent to which the pathway is regulated during development remains to be resolved. This study reports the identification of Shaggy, the homolog of mammalian Gsk3β, as a novel regulator of the Hippo pathway in Drosophila. These results show that Shaggy promotes the expression of Hippo target genes in a manner that is dependent on its kinase activity. Loss of Shaggy leads to Yorkie inhibition and downregulation of Hippo pathway target genes. Mechanistically, Shaggy acts upstream of the Hippo pathway and negatively regulates the abundance of the FERM domain containing adaptor protein Expanded. These results reveal that Shaggy is functionally required for Crumbs/Slmb-mediated downregulation of Expanded in vivo, providing a potential molecular link between cellular architecture and the Hippo signaling pathway (Wu, 2022).

Myotubularin functions through actomyosin to interact with the Hippo pathway

The Hippo pathway is an evolutionarily conserved developmental pathway that controls organ size by integrating diverse regulatory inputs, including actomyosin-mediated cytoskeletal tension. Despite established connections between the actomyosin cytoskeleton and the Hippo pathway, the upstream regulation of actomyosin in the Hippo pathway is less defined. This study identified the phosphoinositide-3-phosphatase Myotubularin (Mtm) as a novel upstream regulator of actomyosin that functions synergistically with the Hippo pathway during growth control. Mechanistically, Mtm regulates membrane phospholipid PI(3)P dynamics, which, in turn, modulates actomyosin activity through Rab11-mediated vesicular trafficking. PI(3)P dynamics were revealed to be a novel mode of upstream regulation of actomyosin and established Rab11-mediated vesicular trafficking as a functional link between membrane lipid dynamics and actomyosin activation in the context of growth control. This study also shows that MTMR2, the human counterpart of Drosophila Mtm, has conserved functions in regulating actomyosin activity and tissue growth, providing new insights into the molecular basis of MTMR2-related peripheral nerve myelination and human disorders (Hu, 2022).

Metabolic control of progenitor cell propagation during Drosophila tracheal remodeling

Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. In response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling (Li, 2022).

Transcriptomic analysis provides insight into the mechanism of IKKbeta-mediated suppression of HPV18E6-induced cellular abnormalities

High-risk Human Papillomaviruses (HPV) 16 and 18 are responsible for more than 70% of cervical cancers and majority of other HPV-associated cancers world-wide. Recently, a Drosophila model of HPV18E6 plus human E3 ubiquitin ligase (hUBE3A; see Drosophila Ubiquitin protein ligase E3A) was developed, and it was demonstrated that the E6-induced cellular abnormalities are conserved between humans and flies. Subsequently, it was demonstrated that reduced level and activity of IKKβ, a regulator of NF-κB, suppresses the cellular abnormalities induced by E6 oncoprotein and that the interaction of IKKβ and E6 is conserved in human cells. In this study, transcriptomic analysis was performed to identify differentially expressed genes that play a role in IKKβ-mediated suppression of E6-induced defects. Transcriptome analysis identified 215 genes whose expression were altered due to reduced levels of IKKβ. Out of these 215 genes 151 genes showed annotations. These analyses were followed by functional genetic interaction screen using RNAi, overexpression, and mutant fly strains for identified genes. The screen identified several genes including genes involved in Hippo and Toll pathways as well as junctional complexes whose downregulation or upregulation resulted in alterations of E6-induced defects. Subsequently, RT-PCR analysis was performed for validation of altered gene expression level for a few representative genes. These results indicate an involvement for Hippo and Toll pathways in IKKβ-mediated suppression of E6 + hUBE3A-induced cellular abnormalities. Therefore, this study enhances understanding of the mechanisms underlying HPV-induced cancer and can potentially lead to identification of novel drug targets for cancers associated with HPV (Collins, 2023).

Proteomic analysis reveals oxidative stress-induced activation of Hippo signaling in thiamethoxam-exposed Drosophila

Thiamethoxam (THIA) is a widely used neonicotinoid insecticide. However, the toxicity and defense mechanisms activated in THIA-exposed insects are unclear. This study used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics technology to identify changes in protein expression in THIA-exposed Drosophila. It was found that the antioxidant proteins Cyp6a23 and Dys were upregulated, whereas vir-1 was downregulated, which may have been detoxification in response to THIA exposure. Prx5 downregulation promoted the generation of reactive oxygen species. Furthermore, the accumulation of reactive oxygen species led to the induction of antioxidant defenses in THIA-exposed Drosophila, thereby enhancing the levels of oxidative stress markers (e.g., superoxide dismutase, glutathione S-transferase, and glutathione) and reducing catalase expression. Furthermore, the Hippo signaling transcription coactivator Yki was inactivated by THIA. These results suggesting that Hippo signaling may be necessary to promote insect survival in response to neonicotinoid insecticide toxicity (Li, 2023).

Dimerization and autophosphorylation of the MST family of kinases are controlled by the same set of residues

The Hippo pathway controls tissue growth and regulates stem cell fate through the activities of core kinase cassette that begins with the Sterile 20-like kinase MST1/2. Activation of MST1/2 relies on trans-autophosphorylation but the details of the mechanisms regulating that reaction are not fully elucidated. Proposals include dimerization as a first step and include multiple models for potential kinase-domain dimers. Efforts to verify and link these dimers to trans-autophosphorylation were unsuccessful. The link between dimerization and trans-autophosphorylation for MST2 and the entire family of MST kinases was explored. Crystal lattice contacts of structures of MST kinases was examined, and an ensemble of kinase-domain dimers compatible with trans-autophosphorylation was identified. These dimers share a common dimerization interface comprised of the activation loop and αG-helix while the arrangements of the kinase-domains within the dimer varied depending on their activation state. The dimerization interface was identfied, and its function was determined using MST2. Variants bearing alanine substitutions of the αG-helix prevented dimerization of the MST2 kinase domain both in solution and in cells. These substitutions also blocked autophosphorylation of full-length MST2 and its Drosophila homolog Hippo in cells. These variants retain the same secondary structure as wild-type and capacity to phosphorylate a protein substrate, indicating the loss of MST2 activation can be directly attributed to a loss of dimerization rather than loss of either fold or catalytic function. Together this data functionally links dimerization and autophosphorylation for MST2 and suggests this activation mechanism is conserved across both species and the entire MST family (Weingartner, 2023).

Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling

The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. This study demonstrates that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics. This study show sthat while the apical polarity network, in part via atypical protein kinase C (aPKC), tethers Kibra at the junctional cortex to silence its activity, medial actomyosin flows promote Kibra-mediated Hippo complex formation at the medial cortex, thereby activating the Hippo pathway. This study provides a mechanistic understanding of the relationship between the Hippo pathway, polarity, and actomyosin cytoskeleton, and it offers novel insights into how fundamental features of epithelial tissue architecture can serve as inputs into signaling cascades that control tissue growth, patterning, and morphogenesis (Tokamov, 2023).

Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Protein

The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional cofactors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIM domain-containing protein 1 (LIMD1) and Wilms tumor protein 1-interacting protein [WTIP]) can bind and inactivate large tumor suppressor kinases 1 and 2, (LATS1/2) two of the Hippo pathway key kinases. WTIP, furthermore, connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton. This study used garland cell nephrocytes of Drosophila to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced the Djub level. Both Djub knockdown and Hippo activation led to morphological changes in the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins. It is concluded that Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism (Gilhaus, 2023).

Coordinated growth of linked epithelia is mediated by the Hippo pathway

An epithelium in a living organism seldom develops in isolation. Rather, most epithelia are tethered to other epithelial or non-epithelial tissues, necessitating growth coordination between layers. This study investigated how two tethered epithelial layers of the Drosophila larval wing imaginal disc, the disc proper (DP) and the peripodial epithelium (PE), coordinate their growth. DP growth is driven by the morphogens Hedgehog (Hh) and Dpp, but regulation of PE growth is poorly understood. This study found that the PE adapts to changes in growth rates of the DP, but not vice versa, suggesting a 'leader and follower' mechanism. Moreover, PE growth can occur by cell shape changes, even when proliferation is inhibited. While Hh and Dpp pattern gene expression in both layers, growth of the DP is exquisitely sensitive to Dpp levels, while growth of the PE is not; the PE can achieve an appropriate size even when Dpp signaling is inhibited. Instead, both the growth of the PE and its accompanying cell shape changes require the activity of two components of the mechanosensitive Hippo pathway, the DNA-binding protein Scalloped (Sd) and its co-activator (Yki), which could allow the PE to sense and respond to forces generated by DP growth. Thus, an increased reliance on mechanically-dependent growth mediated by the Hippo pathway, at the expense of morphogen-dependent growth, enables the PE to evade layer-intrinsic growth control mechanisms and coordinate its growth with the DP. This provides a potential paradigm for growth coordination between different components of a developing organ (Friesen, 2023).

Long non-coding RNA CR46040 is essential for injury-stimulated regeneration of intestinal stem cells in Drosophila

Long non-coding RNAs (lncRNAs) play important regulatory roles in stem cells self-renewal, pluripotency maintenance and differentiation. Till now, there is very limited knowledge about how lncRNAs regulate intestinal stem cells (ISCs), and lncRNAs mediating ISCs regeneration in Drosophila have yet been characterized. This study identify a lncRNA, CR46040, that is essential for the injury-induced ISCs regeneration in Drosophila. Loss of CR46040 greatly impairs ISCs proliferation in response to tissue damage caused by dextran sulfate sodium (DSS) treatment. This study demonstrates that CR46040 is a genuine lncRNA that has two isoforms transcribed from the same transcription start site, and works in trans to regulate intestinal stem cells. Mechanistically, CR46040 knock-out flies are failed to fully activate JNK, JAK/STAT and HIPPO signaling pathways after tissue damage, which are required for ISCs proliferation after intestinal injury. Moreover, CR46040 knock-out flies are highly susceptible to DSS treatment and enteropathogenic bacteria Erwinia carotovora ssp. carotovora 15 (Ecc15) infection. These findings characterize, for the first time, a lncRNA that mediates damage-induced ISCs proliferation in Drosophila, and provide new insights into the functional links among the long non-coding RNAs, ISCs proliferation and tissue homeostasis (Xu, 2023).

Modulation of Hippo signaling by Mnat9 N-acetyltransferase for normal growth and tumorigenesis in Drosophila

Hippo signaling is a conserved mechanism for controlling organ growth. Increasing evidence suggests that Hippo signaling is modulated by various cellular factors for normal development and tumorigenesis. Hence, identification of these factors is pivotal for understanding the mechanism for the regulation of Hippo signaling. Drosophila Mnat9 is a putative N-acetyltransferase that is required for cell survival by affecting JNK signaling. This study shows that Mnat9 is involved in the negative regulation of Hippo signaling. RNAi knockdown of Mnat9 in the eye disc suppresses the rough eye phenotype of overexpressing Crumbs (Crb), an upstream factor of the Hippo pathway. Conversely, Mnat9 RNAi enhances the eye phenotype caused by overexpressing Expanded (Ex) or Warts (Wts) that acts downstream to Crb. Similar genetic interactions between Mnat9 and Hippo pathway genes are found in the wing. The reduced wing phenotype of Mnat9 RNAi is suppressed by overexpression of Yorkie (Yki), while it is suppressed by knockdown of Hippo upstream factors like Ex, Merlin, or Kibra. Mnat9 co-immunoprecipitates with Mer, implying their function in a protein complex. Furthermore, Mnat9 overexpression together with Hpo knockdown causes tumorous overgrowth in the abdomen. These data suggest that Mnat9 is required for organ growth and can induce tumorous growth by negatively regulating the Hippo signaling pathway (Mok, 2022).

Bip-Yorkie interaction determines oncogenic and tumor-suppressive roles of Ire1/Xbp1s activation

Unfolded protein response (UPR) is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. This study reports that the Ire1/Xbp1s pathway has surprisingly oncogenic and tumor-suppressive roles in a context-dependent manner. Activation of Ire1/Xbp1s up-regulates their downstream target Bip, which sequesters Yorkie (Yki), a Hippo pathway transducer, in the cytoplasm to restrict Yki transcriptional output. This regulation provides an endogenous defensive mechanism in organ size control, intestinal homeostasis, and regeneration. Unexpectedly, Xbp1 ablation promotes tumor overgrowth but suppresses invasiveness in a Drosophila cancer model. Mechanistically, hyperactivated Ire1/Xbp1s signaling in turn induces JNK-dependent developmental and oncogenic cell migration and epithelial-mesenchymal transition (EMT) via repression of Yki. In humans, a negative correlation between XBP1 and YAP (Yki ortholog) target gene expression specifically exists in triple-negative breast cancers (TNBCs), and those with high XBP1 or HSPA5 (Bip ortholog) expression have better clinical outcomes. In human TNBC cell lines and xenograft models, ectopic XBP1s or HSPA5 expression alleviates tumor growth but aggravates cell migration and invasion. These findings uncover a conserved crosstalk between the Ire1/Xbp1s and Hippo signaling pathways under physiological settings, as well as a crucial role of Bip-Yki interaction in tumorigenesis that is shared from Drosophila to humans (Yang, 2022).

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, this study investigated models of compensatory proliferation in Drosophila imaginal discs. Compensatory proliferation was found to be associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models (Crucianelli, 2023).

Drosophila USP22/nonstop polarizes the actin cytoskeleton during collective border cell migration

Polarization of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarization is directly controlled by the Hippo signaling complex, which resides at contacts between border cells in the cluster. This study identified, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for nonstop/USP22 in the expression of Hippo pathway components expanded and merlin. Loss of nonstop (not) function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarized actin protrusions, and tumbling of the border cell cluster. Nonstop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA's histone acetyltransferase module, which does not bind to Expanded or Merlin, is dispensable for migration. Taken together, these results uncover novel roles for SAGA-independent nonstop/USP22 in collective cell migration, that may help guide studies in other systems where USP22 is necessary for cell motility and invasion (Badmos, 2021).

This study reports that Drosophila USP22, encoded by not, is necessary for F-actin polarity and collective cell migration of invasive BCs. Collective BC migration requires actomyosin polymerization and contraction at the cortex around the cluster as it moves over the nurse cell substrate; F-actin is effectively excluded from the center of the cluster where polarity determinants acting via the Hippo complex block the activity of the F-actin regulator Enabled. Not has been reported to regulate the actin cytoskeleton directly by promoting the stability of Scar/WAVE. However, this study did not observe a reduction in Scar levels in not mutant clones, and scar loss of function did not disrupt F-actin polarity. Furthermore, no significant change was observed in the number of actin protrusions following not loss of function. This might be expected if Scar were a target in BCs. Interestingly, scar RNAi weakly suppressed not loss of function, suggesting that accumulation of branched actin, mediated by Scar at BC-BC junctions, may contribute to disrupted cell polarity and impaired migration. The data suggest that not regulates inside-out F-actin polarity by regulating the expression of Hippo signaling components ex and mer, that are direct Not targets, in a yki-independent manner. Reanalysis of ChIP-Seq data from embryos indicates that Not and Ada2b bind other core Hippo pathway components, so expression of multiple components may be affected by loss of SAGA components. However, ex and mer are targets for Not, but not Ada2b, which is largely dispensable for migration. Notably, this study found that overexpression of ex suppressed not¹-induced F-actin accumulation at inner BC junctions, consistent with partial restoration of Hippo function and inhibition of Enabled function. It was also observed that cpb overexpression rescued loss of not, again consistent with disruption of Enabled function due to competitive binding of Cpb to F-actin barbed ends and the inhibition of F-actin polymerization at inner BC junctions. Incomplete rescue of not¹ with overexpressed ex or cpb means that other parallel downstream targets that contribute to not function may exist. Interestingly, the data suggest that not is dispensable in polar cells for BC migration. It will be interesting to examine whether the requirement for not in Hippo pathway function is limited to situations where the Hippo complex acts in a yki-independent fashion. The nature of putative noncell autonomous signaling mediated by not controlling polar cell number remains to be elucidated, but altered signaling may be an indirect consequence of changes in polarity or via direct changes in the expression of affected signaling molecules (Badmos, 2021).

A striking effect of not loss of function in BCs is the redistribution of Crb from inner to outer BC junctions. When possible effects of this on other polarity determinants were examined, it was found that localization of aPKC to the inside apical junction between BCs was disrupted, consistent with studies showing that Crb, acting together with the Par complex and endocytic recycling machinery, is necessary for ensuring its correct distribution. Mislocalized aPKC generates protrusions at the side and back of BCs, just as were seen in not¹ clusters. Why is Crb mislocalized to the cortex of the BC complex? Complementation experiments suggest that this is partially accounted for by loss of expression of the FERM domain proteins Ex and Mer, which in follicle cells act together with Moe to recruit Crb to the apical surface. Moe stabilizes Crb at the apical membrane of epithelia by linking Crb to cortical actin. Although the physical interaction between Moe and Crb may be weak, Moe is an important regulator of dynamic Crb localization because it acts to antagonize interactions between Crb and aPKC at the marginal zone of the apical membrane domain while stabilizing interactions between Crb and the apical surface. Importantly, in BCs, Moe is cortically localized where it organizes a supercellular actin cytoskeleton network and promotes cortical stiffness. An attractive hypothesis, therefore, is that Moe, along with other proteins, is a sink for Crb at the cortex of the BC cluster following loss of Ex and Mer at inner BC junctions in not mutants. When ex was overexpressed, the normal pattern of Crb localization was partially restored in support of there being competitive binding. Interestingly, weak rescue of Crb localization was also observed following Cpb overexpression. This might be because Moe, or other proteins that tether Crb on the outer membrane, is only accessible in the absence of a strong supercellular F-actin cortex and that restoration of cortical F-actin in not¹ cpb⁺ cells displaces Crb. In WT BCs, Crb needs to be constantly moved from the outside membrane in a dynamin- and Rab5-dependent manner. Another possibility therefore, which is not mutually exclusive from the first, is that polarization of the F-actin cytoskeleton is important for correct trafficking of Crb in BCs as it is in follicle cells (Badmos, 2021).

The growth, specification, and migration of cells during tissue development requires precisely regulated patterns of gene expression that depend on numerous cues for temporal and spatial gene activation involving crosstalk with multiple signaling pathways. Strikingly, it has emerged that factors once considered to be ubiquitous regulators of transcription, including the SAGA chromatin-modifying complex, can have specific roles in discrete developmental processes. Although it has been suggested that SAGA is required for all transcribed genes in some contexts, numerous studies have shown that loss of SAGA components affects the expression of only a subset of genes and that different components modulate distinct and overlapping subsets. These differences in expression are likely to explain their different physiological roles; for instance, during female germline development in Drosophila, ada2B affects the expression of many genes and is required for oogenesis, whereas not affects relatively few and is dispensable. Genome-wide ChIP studies indicate that even though both DUB and HAT modules bind the same genes, many of the targets do not require the DUB module for expression, explaining the observed dependencies. These experiments also revealed nonoverlapping sites of chromatin occupancy for the DUB and HAT modules of SAGA in Drosophila, but the significance of differences in transcriptional targeting for cell function had not been established. Notably, in this respect, this study found that the requirement for not in BC migration is not matched by a requirement for HAT components, including ada2b or gcn5. Furthermore, Ada2b has not been found to bind the ex and mer promoters, providing a molecular explanation for not's SAGA-independent role. Importantly, these findings challenge the perceived view that transcriptional roles for not/USP22 are mediated solely by SAGA. This may have broader relevance to situations where USP22, but not other members of SAGA, is associated with human disease states, particularly where cell polarity is frequently disrupted, such as cancer. Current efforts are directed at identifying SAGA-independent factors that facilitate Not's chromatin binding and function (Badmos, 2021).

Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. This study uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, this study identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. This study found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength in this approach in predicting novel human obesity genes and signalling pathways and their site of action (Agrawal, 2021).

The ZO-1 protein Polychaetoid as an upstream regulator of the Hippo pathway in Drosophila

The generation of a diversity of photoreceptor (PR) subtypes with different spectral sensitivities is essential for color vision in animals. In the Drosophila eye, the Hippo pathway has been implicated in blue- and green-sensitive PR subtype fate specification. Specifically, Hippo pathway activation promotes green-sensitive PR fate at the expense of blue-sensitive PRs. In this study, using a sensitized triple heterozygote-based genetic screening approach, the identification of the single Drosophila zonula occludens-1 (ZO-1) protein Polychaetoid (Pyd) was identified as a new regulator of the Hippo pathway during the blue- and green-sensitive PR subtype binary fate choice. Pyd acts upstream of the core components and the upstream regulator Pez in the Hippo pathway. Furthermore, Pyd was found to repress the activity of Su(dx), a E3 ligase that negatively regulates Pez and can physically interact with Pyd, during PR subtype fate specification. Together, these results identify a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification (Sang, 2021).

Hippo signaling suppresses tumor cell metastasis via a Yki-Src42A positive feedback loop

Metastasis is an important cause of death from malignant tumors. It is of great significance to explore the molecular mechanism of metastasis for the development of anti-cancer drugs. This study found that the Hippo pathway hampers tumor cell metastasis in vivo. Silence of hpo or its downstream wts promotes tumor cell migration in a Yki-dependent manner. Furthermore, inhibition of the Hippo pathway promotes tumor cell migration through transcriptional activating src42A, a Drosophila homolog of the SRC oncogene. Yki activates src42A transcription through direct binding its intron region. Intriguingly, Src42A further increases Yki transcriptional activity to form a positive feedback loop. Finally, it was shown that SRC is also a target of YAP and important for YAP to promote the migration of human hepatocellular carcinoma cells. Together, these findings uncover a conserved Yki/YAP-Src42A/SRC positive feedback loop promoting tumor cell migration and provide SRC as a potential therapeutic target for YAP-driven metastatic tumors (Ding, 2021).

Prickle isoform participation in distinct polarization events in the Drosophila eye

Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pk^sple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. By characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, this study showed that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pk^sple and pk^pk can enforce robust R3/R4 fate decisions, but only pk^sple can correctly orient them along the dorsal-ventral axis. In contrast, pk^sple and pk^pk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. It is proposed that expression dynamics and competitive interactions determine isoform participation in these processes. It is proposed that the selective requirement for pk^sple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation (Cho 2022).

Fat body-derived Spz5 remotely facilitates tumor-suppressive cell competition through Toll-6-α-Spectrin axis-mediated Hippo activation

Tumor-suppressive cell competition is an evolutionarily conserved process that selectively removes precancerous cells to maintain tissue homeostasis. Using the polarity-deficiency-induced cell competition model in Drosophila, this study identify Toll-6, a Toll-like receptor family member, as a driver of tension-mediated cell competition through α-Spectrin (α-Spec)-Yorkie (Yki) cascade. Toll-6 aggregates along the boundary between wild-type and polarity-deficient clones, where Toll-6 physically interacts with the cytoskeleton network protein α-Spec to increase mechanical tension, resulting in actomyosin-dependent Hippo pathway activation and the elimination of scrib mutant cells. Furthermore, this study show that Spz5 secreted from fat body, the key innate organ in fly, facilitates the elimination of scrib clones by binding to Toll-6. These findings uncover mechanisms by which fat bodies remotely regulate tumor-suppressive cell competition of polarity-deficient tumors through inter-organ crosstalk and identified the Toll-6-α-Spec axis as an essential guardian that prevents tumorigenesis via tension-mediated cell elimination (Kong, 2022).

Epithelial cells possess intrinsic mechanisms to outcompete and eliminate early precancerous cells to maintain homeostasis. For instance, in a mouse model of esophageal carcinogenesis, the majority of newly developed tumor clones are eliminated through cell competition by adjacent normal epithelium. Similarly, surveillance mechanisms also exist in Drosophila epithelium to actively remove oncogenic clones composed of polarity-deficient cells. Genetic studies in flies have uncovered numerous mechanisms that regulate tumor-suppressive cell competition, including c-Jun-N-terminal kinase (JNK) signaling activation-mediated cell elimination, direct cell-cell interaction, secreted factors from epithelial cells, and inter-organ crosstalk between insulin-producing cells and precancerous cell-bearing discs (Kong, 2022).

Initially identified in Drosophila, the Hippo pathway is an evolutionarily conserved signaling cascade that plays crucial roles in various physiological and pathological contexts, ranging from tumor progression and embryogenesis to stem cell renewal and immune surveillance. Apart from its well-established roles in controlling cell proliferation and cell death, numerous studies have proved that the Hippo pathway also functions as a key mechanotransducer to sense mechanical changes in the microenvironment. Despite the identification of multiple essential mechanosensitive signaling molecules including RAP2, MAP4K, Agrin, and Spectrin, it remains poorly understood how mechanical stimuli are transmitted from plasma membrane localized receptors to activate Hippo signaling cascade-mediated cellular responses, especially in intact tissues. This study, through a genetic screen in Drosophila, uncovered a regulatory mechanism whereby mechanical tension drives tumor-suppressive cell competition though the Hippo pathway. The genetic and biochemistry data uncovered Toll-6 as an essential regulator of Hippo signaling and further identified α-Spec as an essential downstream component that regulates cell competition via tension-mediated actomyosin activation. Moreover, this study further demonstrated that inter-organ communication is critical for the removal of precancerous cells at a systemic level and discovered fat body (FB)-derived Spz5 as a crucial ligand (Kong, 2022).

This study demonstrated that polarity-deficient oncogenic clones are eliminated through tension-dependent cell competition and has identified Toll-6 as a key membrane receptor that physically interacts and acts through α-Spec to activate the Hippo pathway. It has long been recognized that both extrinsic cues such as ligands and intrinsic factors such as stiffness and cell-cell contact-mediated mechanical cues can determine cell fate and affect cell proliferation, yet relatively little is known about how the cytoskeleton system contributes to the elimination of precancerous cells during cell competition in vivo. The data show that both &alpha-Spec and Rho1, two essential cytoskeleton regulators, accumulate and facilitate the elimination of scrib clones. In addition, α-Spec as a crucial linker that bridges Toll-6 activation-induced tensional changes to cytosolic Hippo pathway activation. Interestingly, studies in the mammalian system showed that RhoA (human Rho1 ortholog) is responsible for mechanical force-induced cell extrusion. Thus, a similar tension-mediated cell-elimination mechanism might exist in the mammalian system to actively remove unfitted precancerous cells (Kong, 2022).

TLRs play critical roles in the innate immune response. The Drosophila genome encodes nine TLRs, of which only Toll (Tl/Toll-1) has a clear function in innate immunity. Interestingly, a paradoxical role of Tl in regulating cell competition has been reported. Activation of Tl in polarity-deficient clones suppresses the elimination of losers, whereas in the Myc-induced cell competition model, increased Tl activity accelerates the elimination of losers. Apart from Tl, Toll-2, Toll-3, Toll-7, Toll-8, and Toll-9 have been implicated in regulating cell competition in different contexts, while Toll-4 and Toll-5 have little effect. It is noteworthy that none of above studies has investigated the role of Toll-6 in cell competition. The current data not only reveal Toll-6 as a crucial regulator of tumor-suppressive cell competition but also show how the mechanical tension-mediated Hippo cascade is initiated from the cell membrane through the Toll-6-&alpha-Spec axis. Notably, this study found that Toll-6 was not required for Myc-induced cell competition. Given that TLRs are highly conserved in vertebrates and the elimination of scrib-depleted cells also exists in the mammalian system, further experiments are necessary to determine whether analogous mechanisms exist in mammals and humans to regulate mechanical tension-induced, Hippo pathway-mediated tumor-suppressive cell competition (Kong, 2022).

Inter-organ communication is essential for proper development and homeostasis maintenance of multicellular organisms under both physiological and pathological conditions. The tumor progression process is also shaped by the interactions between tumor and other organs, including the immune system. Recent studies in Drosophila have provided insightful understanding of the complex crosstalk between organs during tumorigenesis. The FB is the major immune organ of Drosophila, and it has been shown that intestinal tumor progression or abdominal tumor transplantation promotes the wasting behavior of FBs. The current findings that the transcription of spz5 is increased in the FB from scrib clone bearing larvae to facilitate tumor-suppressive cell competition may provide an in vivo mechanistic understanding of the inter-organ communications between FBs and remotely colonized precancerous clones. Together, the ism) around the boundary between losers and winners, which recruits α-Spec and provokes Hippo pathway-dependent elimination of scrib^-/- clones. Meanwhile, the presence of scrib^-/- loser cells in the eye disc will trigger a systemic effect on the distal organs, including FBs, which results in the transcription upregulation and secretion of Spz5, in turn forming a feedforward loop to reinforce the tumor-suppressive cell competition by binding to Toll-6 (Kong, 2022).

Although biochemical and genetic data demonstrate that Toll-6 physically interacts with α-Spec and that α-Spec is required for the elimination of scrib clones, these experiments were unable to explain the molecular mechanisms by which Toll-6 recruits α-Spec and initiates the downstream signaling transduction. Another limitation is that because the substantial analysis relies heavily on genetics to infer mechanism, enough rigorous biochemistry data was not included to prove how the binding of Toll-6 with α-Spec triggers Hippo signaling activation. Additionally, this study found that FB-derived Spz5 is essential for the elimination of scrib clones through inter-organ communications, but it is not understood completely how the spz5 mRNA level is upregulated systematically in the FBs of larvae that bear scrib mutant clones. Future work will be required to dissect the transcriptome changes of FBs upon precancerous clone induction in distal organs. Finally, this study showed that Spz5 acts through Toll-6 to regulate cell competition, and it is known that Spz5 can bind to other TLRs to regulate both cell death and survival through a three-tier mechanism (Foldi, 2017), suggesting that Spz5 can trigger intracellular signal transduction through ligand receptor binding. Nonetheless, a question that remains unsolved is why a signaling network that relays cell mechanical properties (Toll-6-α-Spec axis) should be regulated by a chemical ligand/receptor interaction; it would be interesting to further explore the underlying mechanisms (Kong, 2022).

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Zygotically transcribed genes

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