InteractiveFly: GeneBrief

expanded : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - expanded Synonyms - Cytological map position - 21C2--3 Function - signaling Keywords - imaginal discs, proliferation, tissue polarity, tumor suppressor, Fat signaling pathway

Symbol - ex FlyBase ID: FBgn0004583 Genetic map position - 2-0.1 Classification - Band 4.1 protein with three potential SH3-binding sites Cellular location - cytoplasmic

BIOLOGICAL OVERVIEW

The first expanded (ex) mutation to be identified (Stern and Bridges, 1926) causes wide wings. Waddington (1940) characterized the ex phenotype in greater detail and concluded that the wing defect is probably due to effects on cell division. An allelic series of ex mutations has been characterized that causes varying degrees of hyperplastic overgrowth of discs. expanded has now been cloned and characterized. It encodes a protein with a canonical N-terminal 4.1 homology domain, with three potential SH3-binding sites (Boedigheimer, 1993). The SH3-binding sites may serve as docking sites for SH3-containing proteins, such as Discs large, Drosophila Abl oncogene or Drosophila Src (Boedigheimer, 1993). The 4.1 homology domain mediates interactions with cell membrane-bound proteins. It is tempting to speculate that the localization of Ex to adherens junctions (Boedigheimer, 1997), where a large number of signaling and structural proteins are also localized, may allow it to exert effects on multiple processes involved in disc development, including effects on cell proliferation, cell fate determination, and tissue polarity (Blaumueller, 2000).

Merlin, another member of the Protein 4.1 superfamily, also plays a role in the the regulation of cell proliferation; proteins in this family were previously thought to function primarily to link transmembrane proteins to underlying cortical actin. Loss of Merlin function in Drosophila results in hyperplasia of the affected tissue without significant disruptions in differentiation. Similar phenotypes have been observed for mutations in expanded. Because of the phenotypic and structural similarities between Merlin and expanded, it was asked whether Merlin and Expanded function together to regulate cell proliferation. Recessive loss of function of either Merlin or expanded can dominantly enhance the phenotypes associated with mutations in the other gene. Consistent with this genetic interaction, Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region (CNTR) of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones reveals that these proteins function to regulate differentiation in addition to proliferation in Drosophila. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for an understanding of the functions of other Protein 4.1 superfamily members (McCartney, 2000).

expanded plays a role in patterning of the eye, mainly at the level of planar polarity. Mutant ex^e1 clones exhibit penetrant phenotypes that are characterized by ommatidial chirality inversions, misrotations, and minor defects in photoreceptor differentiation. In order to determine whether these defects arise during early stages of development, an antibody against the Spalt protein, a marker for the R3 and R4 photoreceptor precursors (on which ommatidial rotation and chirality depend) was used to analyze rotation defects within mutant tissue in pupal imaginal discs. The random orientation of many of the R3/R4 photoreceptor precursor pairs within the clone at this early stage indicates that defects in planar polarity are likely to be a primary consequence of loss of ex function. ex does not affect the initiation of differentiation or the progression of the morphogenetic furrow (differentiation is evident in both imaginal disc and adult tissue), but ex does play a role in orchestrating the fine details of the ensuing cell fate specification and planar polarization events (Blaumueller, 2000).

Given the planar polarity phenotype of ex loss-of-function mutants, a test was made of the ability of ex alleles to genetically interact with gain-of-function genotypes generated by overexpression of components of the frizzled (fz) planar polarity pathway. The gain-of-function frizzled and disheveled (dsh) phenotypes (sev-fz, sev-dsh), and also sev-rho^AV14 and sev-rac^V12, have been successfully used in previous studies both to identify new components of the Fz/Dsh planar polarity pathway, and to genetically position known components with respect to others. This assay, dominant genetic modification of sev-fz, sev-dsh, sev-rho^AV14 and sev-rac^V12, was used to analyze in more detail the role of Ex in this process. These experiments did not reveal significant genetic interactions between ex and most of these genotypes. However, sev-dsh is dominantly enhanced by ex^e1, with an increase of ommatidia that are unscorable with respect to polarity because they lack one or more photoreceptors. This phenotypic modification could be achieved by several mechanisms, and is suggestive of complex cross-talk, either between multiple signaling pathways (dsh itself plays a role in multiple signaling pathways, including those represented by wingless, frizzled, and possibly Notch), or between signaling pathways and the mechanical processes required to carry out the instructions provided by the signaling pathways (Blaumueller, 2000).

Overexpression of Ex in the wing results in a tissue reduction phenotype, consistent with a growth inhibitory role (Boedigheimer, 1997). In order to test whether this role of Ex is conserved across disc types, adult eyes in which Ex was overexpressed using the UAS-GAL4 system were analyzed. Early expression was expected to affect cells anterior to the morphogenetic furrow (cells undergoing non-synchronized divisions prior to fate determination), whereas late expression would affect cells posterior to the morphogenetic furrow (cells dividing synchronously in the midst of differentiating cells). Externally visible adult phenotypes result from Ex expression under the control of a variety of early and late drivers including eyeless (ey), scabrous (sca), sevenless (sev), and glass multimer reporter (GMR). Early Ex overexpression throughout the developing eye disc under the ey driver (starting in embryonic development) results in a phenotype characterized by dramatic effects on the overall size and shape of the eye, as well as a mild roughening of the surface. Externally, eyes often bulge and are reduced in the ventral region. Retinal sections through these eyes reveal mild patterning defects that primarily affect the positioning of the equator, the border between the two chiral forms of ommatidia present in the dorsal and ventral halves of the eye. This defect is likely to be a secondary consequence of distortions of the eye disc (Blaumueller, 2000).

Overexpression of Ex under the remaining drivers, which are late-expressing, results in a distinct phenotype characterized by roughening and blistering externally, and patterning defects in sections. In the case of 32B (expression posterior to the morphogenetic furrow), external roughening of the eye reflects ommatidial misrotations and a loss of pigment cells (identifiable by yellow pigment granules). Similar effects are produced using the sca driver (expression in a small subset of cells posterior to the morphogenetic furrow). Overexpression of Ex under the control of either sev (transient expression in a subset of photoreceptor and all cone cell precursors), or GMR (expression in all cells within and posterior to the morphogenetic furrow throughout the remainder of development) results in severe effects on eye development. In both the latter cases, adult eyes are blistered and reduced in size. Sectioning through blistered regions reveal a mass of highly disorganized ommatidia, whereas the more mildly affected posterior regions of sev-ex eyes exhibit a phenotype similar to that seen in 32B-ex and sca-ex eyes. The most prominent features of this phenotype are a loss of pigment cells and defects in planar polarity. Occasionally, a photoreceptor cell is also missing within an ommatidium. The results are consistent with Ex overexpression causing an overall reduction in cell number. The small eye phenotype obtained by expressing Ex early under the ey driver can be explained by a reduction in cell number early in eye development. Likewise, the phenotypes generated by overexpression under the late drivers suggest that cell loss occurs, but at a point in time at which such effects impact tissue patterning as well as tissue size (Blaumueller, 2000).

An examination of eye imaginal discs in which Ex expression is driven early (ey-ex) reveals striking effects on disc size and shape. Discs were double-stained for markers of morphological differentiation (anti-Notch) and neuronal development (anti-ELAV). These stainings reveal that the eye imaginal discs are consistently small relative to the antennal portions of the same disc complex. Unlike wild-type discs, ey-ex discs are not flat. This effect could account for the misshapen adult eyes. In contrast to the dramatic effects on tissue size, those on cell fate specification and differentiation are mild, ommatidial preclusters looking fairly well-ordered. Together with the adult phenotype, these results indicate that the defects are caused mainly by early effects of Ex expression on cell number anterior to the furrow, prior to the time at which patterning events take place (Blaumueller, 2000).

An examination of discs in which Ex expression is driven posterior to the morphogenetic furrow reveals early defects in ommatidial polarity as well as effects on ommatidial precluster density and on cell number. The positions of specific subsets of photoreceptors were monitored using the svp-beta-galactosidase reporter (svp-beta-gal; expressed specifically in the R3/R4 photoreceptor precursors beginning just posterior to the furrow, and also in the R1/R6 precursors slightly further posterior in the disc), making it possible to analyze ommatidial polarity from the earliest point of its establishment. In wild-type discs, the R3/R4 and R1/R6 precursor pairs are aligned in precise diagonal rows. In the case of 32B-ex discs, this pattern is also fairly regular. However, several misrotated clusters are present, even in the anterior-most rows in which the R3/R4 pair can be identified. In the cases of sev-ex and GMR-ex discs, rotation defects are also prominent in anterior rows. Polarity could not be assessed in posterior regions of these discs because individual R3/R4 and R1/R6 pairs could not be distinguished in the dense mass of svp-beta-gal positive cells. In addition to its effects on planar polarity, Ex overexpression posterior to the furrow can lead to the loss of cells in which expression is driven. In sev-ex discs, one of the R3/R4 precursor pair is occasionally missing, consistent with the loss of photoreceptors in sections through adult tissue. Furthermore, the number of accessory cells that normally surround the photoreceptors appears to be greatly reduced. The adult phenotypic analysis suggests that both cone and pigment cells are affected: eye blistering is characteristically a consequence of defects in the cone cells that secrete the lens material, and pigment cells are clearly missing in retinal sections. Also, the svp-beta-gal stainings reveal that ommatidial preclusters are packed more densely than in wild-type discs. To test whether cone cells (in which sev also drives expression) are affected from early stages, sev-ex pupal discs were stained for the nuclear cone cell marker Cut. Whereas arrays of four Cut positive cells are present per precluster in wild-type discs, fewer are present in preclusters of sev-ex discs, and the ommatidial array is markedly disturbed. Hence, cone cell loss contributes to the phenotype seen in sev-ex flies, as does photoreceptor loss. These results are consistent with cell loss at, or after, the time of cell fate specification (Blaumueller, 2000).

The results of the experiments described above, coupled with previous loss-of-function and overexpression studies in the wing, suggest that ex regulates the growth of imaginal discs by inhibiting cell proliferation. The elimination of an inhibitor of proliferation in loss-of-function mutants could explain the overgrowth seen in these animals. Conversely, overexpression of such an inhibitor could account for the overall loss of tissue throughout the disc when expressed at early stages, and for the loss of late-born cells derived from the second mitotic wave when expressed at late stages. Moreover, phenotypes similar to those described for Ex overexpression posterior to the morphogenetic furrow have been obtained by inhibiting the second mitotic wave by overexpressing the cyclin-dependent kinase inhibitor p21 under the control of the GMR driver. The hypothesis that Ex overexpression negatively regulates proliferation was tested by staining for either the S-phase marker bromodeoxyuridine (BrdU), or the mitosis marker phospho-histone H3 in discs expressing Ex under the drivers discussed above. None exhibit a detectable change in the staining for either marker when compared to wild-type. Hence, it appears that Ex overexpression does not interfere with cell proliferation (Blaumueller, 2000).

This observation raises the question of whether Ex exerts its effects at the level of cell death. Discs overexpressing Ex were therefore tested for the incorporation of the cell death marker, Acridine Orange. Wild-type eye imaginal discs exhibit low levels of apoptosis both anterior and posterior to the morphogenetic furrow. In contrast, discs from both sev-ex and GMR-ex larvae exhibit dramatic increases in Acridine Orange incorporation. Moreover, the regions of incorporation overlap with the expression patterns of each driver. Whereas high levels of Acridine Orange incorporation are limited to a relatively narrow band just behind the morphogenetic furrow in sev-ex discs, in GMR-ex discs the staining pattern begins and ends further to the posterior. In the case of ey-ex, the incorporation of Acridine Orange in third instar larval discs is also increased, although this is more variable. Interestingly, in several ey-ex discs with high levels of Acridine Orange incorporation, the marker is regionalized to the ventral half, potentially accounting for the frequent loss of this region of the adult eye. These data demonstrate that Ex overexpression causes tissue reduction by inducing massive cell death. In order to determine whether this is also the case in other tissues, the effects of Ex overexpression on the wing and leg were tested. Misexpression of Ex using the optomotor blind (omb) driver, which is expressed throughout much of the region that gives rise to the wing pouch and also in a sector of the leg disc, results in discs incorporating high levels of Acridine Orange in a pattern overlapping that of omb-driven Ex overexpression. These flies have severely reduced wings with patterning defects including loss of vein material and disruption of the margin. Legs appears to be fragile and twisted. Similarly, overexpression using the MS1096 driver results in small, unpatterned wings and misshapen legs. The patterning defects generated by expression under both of these drivers probably reflect the extensive overlap of their expression domains with those of patterning elements for these tissues (Blaumueller, 2000).

The viral caspase inhibitor p35 is a known suppressor of apoptosis. Coexpression of this protein with Ex results in a dramatic rescue of the sev-ex phenotype. Externally, the eye is much smoother than in sev-ex and in sections most of the cell loss is rescued, although occasional photoreceptor and pigment cell loss is evident. Defects in planar polarity are still prominent. Taken together, the data presented here demonstrate that the tissue reduction phenotypes produced by Ex overexpression in all disc types are due to caspase-dependent, and therefore apoptotic, cell death (Blaumueller, 2000).

The most obvious consequence of loss of ex function in all imaginal discs is the deregulation of growth. Homozygous mutant ex clones have a significant growth advantage over their wild-type counterparts, and eye discs from larvae homozygous null for the gene are vastly overgrown. Hence, the apparent reduction of adult eye tissue originally observed in ex mutants may be a secondary consequence of a failure in disc eversion and morphogenesis of overgrown tissue. The overgrowth phenotypes in the eye are consistent with those seen in the wing (Boedigheimer, 1993; Boedigheimer, 1997). Like the loss-of-function phenotypes, those obtained with Ex over-expression are internally consistent. Regardless of disc type, Ex overexpression leads to tissue reduction that is a consequence of apoptosis in the region of overexpression, and its precise effects on size and patterning of the disc are dependent on its timing relative to decisions being made in the tissue (Blaumueller, 2000).

The finding that ex plays a common role in different disc types simplifies an understanding of the way in which this gene functions. However, the unexpected observation that the overexpression phenotype is a consequence of the induction of cell death, rather than of a repression of proliferation, complicates the issue. The most obvious model consistent with the overexpression data by itself would be that Ex normally acts as an inducer of apoptosis, and that loss of the protein leads to insufficient cell death in the disc, with consequent tissue overgrowth. However, apoptosis normally occurs at very low levels in larval imaginal discs and a lack of apoptosis on its own is not likely to cause the massive overgrowth that is evident in mutants as early as the third instar. This idea is supported by the fact that overexpression of the viral caspase inhibitor, p35 (which blocks apoptotic cell death in the eye), produces eyes that are only slightly larger than normal. If ex is required for controlling tissue growth, but is also capable of inducing apoptosis, how are these roles related to one another? One possibility is that Ex normally takes part only in regulating growth, but when expressed at high levels, interferes with growth and ultimately leads to apoptosis as a secondary effect. The fact that proliferation is not inhibited when Ex is overexpressed suggests that this is not the case. However, one cannot rule out the possibility of proliferation arrest at a late point within the cell cycle, which would not be detectable by BrdU or phospho-histone H3 labeling and might itself cause the cells to die. An alternative explanation is that Ex might play independent direct roles in both negatively regulating proliferation and positively regulating cell death. Its role as a negative regulator of growth might more easily be unmasked by loss-of-function mutations than by overexpression; Ex might be necessary for preventing overgrowth, but insufficient for inhibiting normal proliferation when overexpressed. Conversely, a role as a positive regulator of cell death might easily be detected when it is enhanced by overexpression, but not in the loss-of-function situation, where it could be masked by the overgrowth phenotype (Blaumueller, 2000).

Ex appears to play a direct role in certain aspects of tissue patterning. Both loss-of-function and overexpression mutants exhibit some patterning defects that cannot be explained as secondary effects. For example, in the case of Ex overexpression in sev-ex, rotation defects are present early in disc development, prior to the death of the cells in which it is expressed. Hence, this defect in planar polarity must be a direct consequence of Ex overexpression. The fact that the planar polarity defects exhibited by ex loss-of-function and overexpression mutants resemble one another, as well as being similar to those of previously characterized planar polarity genes, further supports the notion that Ex plays a direct role in this process. However, due to the lack of informative genetic interactions with components of the Fz pathway, the role of ex in planar polarity establishment remains unclear. It is possible that the observed phenotypic interaction between ex and sev-dsh reflects cross-talk between multiple signaling pathways (dsh itself has been implicated in several signaling pathways, or between signaling pathways and the mechanical processes involved in generating ommatidial polarity. However, regardless of whether ex plays a role in promoting cell signaling or in initiating the morphogenetic movements required for establishing planar polarity, its overall importance in organizing planar polarity is an exciting finding (Blaumueller, 2000). The possibility that Ex plays a role in the mechanics of planar polarity is particularly intriguing, since nothing is yet known about how the signaling events controlling planar polarity are translated into the physical movements that take place to establish this phenomenon in the eye. Hence, ex could provide a unique entry point en route to finding a solution for this problem. With regard to the potential connection of signaling and mechanical events by Ex, it is noted that the loss of ex does not lead to an obvious perturbation of the cellular architecture (as ascertained by staining of the eye disc for the junctional markers Notch (adherens junctions) and Coracle (septate junctions), suggesting that ex is not a global regulator of junctional structure. However, when overexpressed, Ex is mislocalized: in addition to being concentrated in apical regions of the membrane, the protein is also diffusely distributed throughout the cytoplasm. Hence, defects in differentiation, a process that relies heavily on cell-cell contacts, may arise as a consequence of Ex sequestering important signaling or structural components away from the appropriate junction, or perhaps even by allowing these molecules to act at inappropriate times and places. Certainly, this mislocalization could also account for other effects of Ex overexpression, for example, the induction of apoptosis (Blaumueller, 2000).

Spatial regulation of expanded transcription in the Drosophila wing imaginal disc

Growth and patterning are coordinated during development to define organ size and shape. The growth, proliferation and differentiation of Drosophila wings are regulated by several conserved signaling pathways. This study shows that the Salvador-Warts-Hippo (SWH) and Notch pathways converge on an enhancer in the expanded (ex) gene, which also responds to levels of the bHLH transcription factor Daughterless (Da). Separate cis-regulatory elements respond to Salvador-Warts-Hippo (SWH) and Notch pathways, to bHLH proteins, and to unidentified factors that repress ex transcription in the wing pouch and in the proneural region at the anterior wing margin. Senseless, a zinc-finger transcription factor acting in proneural regions, had a negative impact on ex transcription in the proneural region, but the transcriptional repressor Hairy had no effect. This study suggests that a complex pattern of ex transcription results from integration of a uniform SWH signal with multiple other inputs, rather than from a pattern of SWH signaling (Wang, 2018).

The development, differentiation and growth of cells and tissues require precisely regulated patterns of gene expression, depending on the time and spatial location of its activation, and its crosstalk with other signaling pathways. This study used the Drosophila wing disc as a model system to investigate how ex transcription is regulated. Ex is important as a negative growth regulator that acts through the SWH pathway. It restricts wing size in normal development, so that mutants have larger, 'expanded' wings, and can also be induced to block growth of cells with developmental perturbations, including those with emc mutations that over-express Da. In normal development, ex-LacZ is expressed most highly in the hinge region that surrounds the wing pouch, and expression decreases in a gradient until none is detected at the wing margin. Unlike expression patterns of ex-LacZ, a relatively ubiquitous distribution of Ex protein is seen in the wing imaginal discs. The discrepancy between ex reporter and Ex protein might be due to the post-translational control of Ex protein stability. Alternatively, Ex protein might be strongly influenced by expression patterns in earlier developmental stages. Regardless, because ex is itself a transcriptional target of the SWH pathway, acting through Sd and Yki, ex-LacZ is extensively used as a transcriptional readout of SWH signaling activity, if not of Ex protein distribution. This transcription pattern of ex could be interpreted to indicate a proximal-to-distal gradient of Sd/Yki activity, which would be consistent with certain models of wing growth regulation that propose that SWH activity represses growth in central regions of the disc whereas Yki activity is higher in proximal regions. Another mechanism predicted to repress growth in central regions of the wing disc is activation of Notch signaling there. Notch signaling induces expression of Vg, a protein that binds Sd in competition with Yki and is therefore predicted to reduce Yki activity (and ex transcription) in central regions of the wing pouch. Since this study has identified an ex enhancer whose activity reflects the ex-LacZ reporter in the endogenous gene, this enhancer can be explored to understand how these and other signals are integrated at the ex locus (Wang, 2018).

A deletion analysis (see Analysis of Intron3 enhancer)outlines several major features of ex regulation. First, the core of the enhancer, centered around the 'C' element and probably including contributions from the flanking 'B' and 'D' elements, is active throughout the wing disc. All this activity depends on both Sd and Yki, suggesting that Yki is active throughout the wing disc. This is consistent with the previous finding that yki is required for growth throughout the wing disc. ChIP-seq analysis revealed that Yki binding peaks over Ex^Intron3. Although Sd binding has not been mapped yet in Drosophila, it is likely that Sd binding strongly correlates with Yki, as seen for the mammalian YAP1 and TEAD1 proteins. Expression of the Sd/Yki-dependent BC and CD elements provided no evidence of a proximal-distal gradient of Sd/Yki activity. Instead, the overall reduction of ex expression in the central, wing pouch region of the wing disc requires both the B and D regions together, suggesting that two other inputs are both required to achieve this silencing. It does not seem that either of these silencing inputs corresponds to Notch signaling, because although it was confirmed that Notch is required to silence ex enhancer activity in the wing pouch, it is not required to silence the BCD element (Wang, 2018).

In the wing margin proneural region (high SWH activity; low Yki activity), ex is negatively regulated by inputs acting through elements A and F. The E-box site #2 (E2) is required for expression in the wing margin proneural cells while Da acts through E1 and E3 to regulate ex transcription. Sd/Yki regulates ex transcription through element BCD in wing pouch and hinge. Notch acting through element E and other inputs acting through elements B and C repress ex transcription in wing pouch (Wang, 2018).

An unexpected aspect of ex transcription regulation is independent regulation of ex enhancer activity in the proneural cells of the anterior wing margin. Activity here was encoded by the BC and CD elements, and depended on an E-box within C (E-box #2). Surprisingly, given the normal role of Notch signaling in lateral inhibition to prevent neural differentiation, the wing margin activity depended positively on N signaling. Anterior wing margin activity was dependent on Da, the obligate heterodimer partner of all proneural genes, indicating that it is possibly encoded by proneural genes of the AS-C. This regulation was unexpected because there was no evidence for ex-LacZ or Ex^intron3-GFP reporter activity at the anterior wing margin. This is because such expression was silenced by either one of two flanking elements, A and F, acting redundantly. The hypothesis was tested that Sens, which is expressed in the anterior wing margin proneural region and can act as a transcriptional repressor, was responsible for blocking the activity. Although Sens was sufficient to inhibit Ex^intron3-GFP activity at some ectopic locations, it was not required for Ex^intron3-GFP repression at any location, including at the anterior wing margin (Wang, 2018).

The ex enhancer was originally identified through its role in mediating ex transcription in response to elevated Da activity. The Da response was mapped to E-boxes #1 and #3 within elements B and D, respectively. Since Da can bind to DNA as a homodimer, and as no other Class I or Class II bHLH gene is known to be widely expressed in the wing pouch, it is possible that Da homodimers activate this site. The E1 site within element B matches the consensus CACCTG sequence that is preferred by Da-Da homodimers and by homologous E-protein homodimers in mammals. Endogenous Da protein levels are normally elevated at the anterior wing margin, because Emc levels are reduced there, so it is interesting that Ex^intron3 is activated at the anterior wing margin through distinct E-boxes that differ in sequence from E2 site. One possibility is that it is heterodimers of Da with proneural proteins encoded by AS-C that activates these E-boxes at the anterior wing margin, since it was previously shown that ectopically-expressed AS-C proteins can activate the intact enhancer. The consensus E-box site for Sc/Da has been described as GCAGC/GTG and the 5' flanking base G is essential for the binding specificity of Sc. The E2 site matches both the core sequence and the 5'4 flanking base. In part this could explain why emc loss is detrimental for proliferating progenitor cells in the rest of the wing disc and leads to their ex-dependent loss during growth, in contrast to the proneural cells that normally tolerate elevated Da levels, if Da proteins contribute to distinct dimers in these two situations (Wang, 2018).

This analysis shows that multiple regulatory inputs are integrated by the Ex^intron3 enhancer. Although this study confirms previous conclusions that Sd/Yki and Notch signaling regulate ex transcription, these studies indicate that the full enhancer, and hence the widely-used ex-LacZ line, are not straightforward reporters of Yki activity. Instead the smallest sequence that responds to Sd and Yki is active almost uniformly throughout the wing disc, suggesting that this may be the pattern of Yki activity. This may be a useful reporter for SWH activity in future studies. The final ex-LacZ pattern is strongly influenced by additional sequences that silence transcriptional activity in the wing pouch and at the anterior wing margin, in the latter case apparently preventing ex transcription in response to proneural gene activity while preserving sensitivity to ectopic Da expression (Wang, 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).

REGULATION

Salvador-Warts-Hippo pathway in a developmental checkpoint monitoring Helix-Loop-Helix proteins

The E proteins and Id proteins are, respectively, the positive and negative heterodimer partners for the basic-helix-loop-helix protein family and as such contribute to a remarkably large number of cell-fate decisions. E proteins and Id proteins also function to inhibit or promote cell proliferation and cancer. Using a genetic modifier screen in Drosophila, this study shows that the Id protein Extramacrochaetae enables growth by suppressing activation of the Salvador-Warts-Hippo (SWH) pathway of tumor suppressors, activation that requires transcriptional activation of the expanded gene by the E protein Daughterless. Daughterless protein bound to an intronic enhancer in the expanded gene, both activated the SWH pathway independently of the transmembrane protein Crumbs and bypassed the negative feedback regulation that targets the same expanded enhancer. Thus, the Salvador-Warts-Hippo pathway has a cell-autonomous function to prevent inappropriate differentiation due to transcription factor imbalance and monitors the intrinsic developmental status of progenitor cells, distinct from any responses to cell-cell interactions (Wang, 2015).

This study describes a process that prevents certain misspecified cells from differentiating into malformed organs. This process creates a requirement for the emc gene in imaginal disc cell growth, since emc loss results in high Da levels that trigger the pathway through transcriptional activation of the ex gene, an upstream regulator of the SWH tumor suppressor pathway. If ex or the downstream SWH genes are mutated, then cells with high Da levels not only survive and grow but also produce numerous ectopic neuronal structures. This surveillance function for SWH signaling does not require cell-cell signaling and is distinct from potential roles for SWH in limiting organ growth or preventing tumorigenesis. It may represent an adaptive function for SWH pathway hyperactivity (Wang, 2015).

The heterodimer partners of Da and Emc include proneural bHLH proteins that define proneural regions and neural progenitor cells and that are highly regulated in space and time. Da, by contrast, is expressed ubiquitously and controlled by emc. Inadequate emc expression permits higher levels of da expression and Da/bHLH heterodimers, leading to ectopic neural differentiation. Mammalian Id genes are similar feedback regulators of mammalian E-proteins. This study has shown that even if emc expression or its regulation is defective, abnormal neurogenesis is still restrained by SWH signaling that restricts the proliferation and survival of cells with abnormal Da expression. High Da levels directly activate transcription of the ex gene, thereby activating the SWH pathway of tumor suppressors in a cell-autonomous fashion. Because ex is a feedback inhibitor of SWH signaling that is transcriptionally activated by Yki, ex activation by high Da has the added effect of bypassing feedback control of SWH signaling, which likely contributes to the efficiency of removal of cells with high Da. Indeed, when ex is removed, cells with high Da are not removed but produce dramatic neural hyperplasia, in which ectopic bristles almost cover a clone in the thoracic epidermis. All these neurogenic defects would be maladaptive in nature, where the pattern of sensory bristles is highly selected (Wang, 2015).

These findings suggest that the Da/Emc balance is permissive for normal growth, and no evidence was found for regulation that determines normal organ size or growth rate. By contrast, Da/Emc imbalance outside the normal range in mutant cells triggers the SWH pathway to block growth and remove cells that will otherwise perturb developmental patterning. SWH activation in abnormal development might be analogous to the p53 tumor suppressor, which is inactive in most normal cells, but activated by DNA damage and other stresses. Interestingly a recent study reported that emc hypomorphic cells, which are less severely affected that emc null cells and can survive in imaginal discs, nevertheless exhibit a growth deficit caused by repression of the cell cycle gene string/cdc25, and that string/cdc25 is repressed directly by abnormally high Da. Thus there may be multiple, Da-dependent pathways that converge to select against progenitor cells with incorrect cell-fate specification (Wang, 2015).

Mammalian E-proteins and Id-proteins are well-established tumor suppressors and proto-. In normal development, E proteins and Id-proteins regulate the coordination of differentiation with cell-cycle arrest and the expansion of mammary epithelial cells in response to pregnancy and lactation. At least in part, these growth controls relate to the transcriptional activation of cyclin-dependent kinase inhibitor genes by E-proteins, such that E-proteins are required for cellular senescence, counteracted by Id-proteins. The senescence mechanisms may not be conserved between mammalian and Drosophila cells, but other pathways of tumor suppression by mammalian E-proteins exist, and in certain contexts, E-proteins can be tumor promoting and Id-proteins tumor suppressive (Wang, 2015).

The distinctive phenotype of SWH pathway mutations is dramatically enhanced growth and organ size. The normal biological functions of the pathway are still debated. Reduced SWH activity is implicated in wound healing and regenerative growth. Mice mutant for Mst1, Mst2, Lats1, or Lats2 are tumor prone, suggesting that tumor growth could mimic wound healing or regeneration. Epigenetic silencing of these genes has been reported in human cancer, where other SWH components are mutated, such as NF2 in neurofibromatosis. Yap is amplified in cancers of the liver, colon, lung, and ovary (Wang, 2015).

Clearly, SWH activity is normally maintained between a low threshold necessary to prevent hyperplasia and a high threshold that blocks growth and kills cells. Reduced SWH activity is associated with regenerative responses. In principle, increased SWH might be hyperactivated to eliminate potential tumors, perhaps because of imbalanced expression of E-proteins and Id-proteins; tumor cells might evolve to evade such a checkpoint. Microarray data from E2A-deficiency mice that exhibit high incidence of T cell leukemia suggest that FRMD6, a mammalian homolog of ex, is an E2A target, which would be consistent with this hypothesis (Wang, 2015).

This work shows directly that in Drosophila hyperactivation of the SWH tumor suppressor pathway can select against cells that express certain developmental errors, which may be adaptive for development. It will be interesting to discover whether SWH signaling can be hyperactivated to remove other kinds of dysfunctional cells besides those expressing inappropriate bHLH protein levels, whether in development or in cancer (Wang, 2015).

Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila

The conserved Hippo tumor suppressor pathway is a key kinase cascade that controls tissue growth by regulating the nuclear import and activity of the transcription co-activator Yorkie. This study reports that the actin-Capping Protein αβ heterodimer, which regulates actin polymerization, also functions to suppress inappropriate tissue growth by inhibiting Yorkie activity. Loss of Capping Protein activity results in abnormal accumulation of apical F-actin, reduced Hippo pathway activity and the ectopic expression of several Yorkie target genes that promote cell survival and proliferation. Reduction of two other actin-regulatory proteins, Cofilin and the cyclase-associated protein Capulet, cause abnormal F-actin accumulation, but only the loss of Capulet, like that of Capping Protein, induces ectopic Yorkie activity. Interestingly, F-actin also accumulates abnormally when Hippo pathway activity is reduced or abolished, independently of Yorkie activity, whereas overexpression of the Hippo pathway component expanded can partially reverse the abnormal accumulation of F-actin in cells depleted for Capping Protein. Taken together, these findings indicate a novel interplay between Hippo pathway activity and actin filament dynamics that is essential for normal growth control (Fernández, 2011).

The Hippo pathway has emerged as a crucial regulator of tissue size in both Drosophila and mammals. In Drosophila, the Hpo pathway comprises a kinase cascade in which the Hpo kinase binds the WW domain adaptor protein Salvador (Sav) to phosphorylate and activate the Warts (Wts) kinase. Wts, in turn, facilitated by Mats, phosphorylates and prevents nuclear translocation of the transcriptional co-activator Yorkie (Yki). This leads to transcriptional downregulation of target genes that positively regulate cell growth, survival and proliferation, including the Drosophila inhibitor of apoptosis protein 1 (Diap1; thread - FlyBase), expanded (ex), Merlin (Mer) and wingless (wg) in the inner distal ring, within the proximal wing imaginal disc. The upstream components Ex, Hpo and Wts are also thought to regulate Yki through a phosphorylation-independent mechanism, by directly binding to Yki, sequestering it in the cytosol and thereby abrogating its nuclear activity (Fernández, 2011).

Multiple upstream inputs are known to regulate the core Hpo kinase cassette at various levels. Thus, the atypical cadherin Fat was identified as an upstream component of the Hpo pathway and was proposed to transduce signals from the atypical cadherin Dachsous (Ds) and Four-jointed (Fj), a Golgi-resident kinase that phosphorylates Fat and Ds. Moreover, the two Ezrin-Radixin-Moesin (ERM) family members, Ex and Mer have been reported to lie upstream of the Hpo pathway. Mer and Ex can heterodimerize and are believed to exert their growth suppression activity by activating the Hpo kinase. However, how the different inputs that feed into the core kinase cassette are coordinated to regulate Yki activity is unknown (Fernández, 2011).

ERM proteins form a structural linkage between transmembrane components and actin filaments (F-actin). For instance, mammalian Mer binds numerous cytoskeletal factors, including actin, and appears to act as an inhibitor of actin polymerization. Interestingly, the Merlin-actin cytoskeleton association is required for growth suppression and inhibition of epidermal growth factor (EGFR) signaling. Moreover, F-actin depolymerization promotes activation of the Hpo orthologs MST1 and MST2 in mouse fibroblasts (Densham, 2009). These observations suggest a role for F-actin dynamics in modulating Hpo pathway activity (Fernández, 2011).

Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Among these, the Capping Protein (CP) heterodimer, composed of α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer to restrict the accessibility of the filament barbed end, inhibiting addition or loss of actin monomers (Cooper, 2008). In Drosophila, mutations in either cpa or cpb, lead to accumulation of F-actin within the cell and give rise to identical developmental phenotypes that are tissue specific. In the wing blade (BL), the most distal domain of the imaginal disc, cpa and cpb prevent cell extrusion and death, but they are not required for this function in the most proximal domain, the prospective body wall and the hinge wing imaginal disc (Janody, 2006). The Cofilin homolog Twinstar (Tsr) and the Cyclase-associated protein Capulet (Capt) also restrict actin polymerization: Tsr severs filaments and enhances dissociation of actin monomers from the pointed end, whereas Capt sequesters actin monomers, preventing their incorporation into filaments (Fernández, 2011).

This study investigated the relationship between the control of the actin cytoskeleton and Hpo pathway activity. Actin-binding proteins CP and Capt, but not Tsr, were shown to enhance Hpo signaling activity. Moreover, a new relationship was uncovered between the Hpo pathway and the machinery that regulates F-actin, and it was revealed that Hpo signaling activity, like CP, limits F-actin accumulation at apical sites independently of Yki. Finally, it is proposed that regulation of an apical F-actin network by Hpo signaling activity and CP sustains Hpo pathway activity, thereby limiting Yki nuclear import and the activation of proliferation and survival genes (Fernández, 2011).

This report shows an interdependency between Hpo signaling activity and F-actin dynamics in which CP and Hpo pathway activities inhibit F-actin accumulation, and the reduction in F-actin in turn sustains Hpo pathway activity, preventing Yki nuclear translocation and upregulation of proliferation and survival genes (Fernández, 2011).

It is suggested that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki. ERM proteins can form a structural linkage between transmembrane components and the actin cytoskeleton. Mammalian Mer appears to act as an inhibitor of actin polymerization. Moreover, the Mer-actin cytoskeleton association has a crucial role for growth suppression and inhibition of EGFR signaling. In Drosophila, Mer and Ex are structurally related and appear to have partially redundant functions but vary in their requirement depending on the tissue or developmental stage. In imaginal discs, loss of ex shows stronger phenotypes when compared with those of Mer. Ex might also have a stronger requirement on F-actin dynamics, as loss of ex, but not that of Mer, triggered F-actin accumulation. Surprisingly, loss of hpo, sav, mats or wts also triggered apical F-actin accumulation. Ex is likely to affect F-actin through activation of the Hpo kinase cassette because in most ex mutant clones, overexpressing hpo suppressed F-actin accumulation. Some clones seemed to contain increased F-actin. However, these clones also constricted apically, suggesting that the effect on F-actin levels results from a reduction of the apical cell surface and that in the absence of ex, differential activity of overexpressed hpo triggers cell shape changes. Together, these observations argue that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki (Fernández, 2011).

Loss of Hpo pathway activity or CP triggerw apical F-actin accumulation. Ex localizes to the sub-apical region of epithelial cells, and colocalizes with an HA-tagged form of Cpa, Ex, Hpo, Sav and Wts all interact with each other through WW and PPXY motifs (Oh, 2009; Reddy, 2008). Therefore, a pool of Hpo, Sav and Wts, localized at apical sites, could directly regulate an actin-regulatory protein. Hpo pathway activity might act downstream of CP on F-actin. In agreement with this, ex overexpression significantly suppresses F-actin accumulation in cells with reduced CP levels. The role of Hpo signaling activity might be to inhibit an actin-nucleating factor, which adds new actin monomers to filament barbed ends free of the capping activity of CP. However, it cannot be excluded that ex overexpression enhances the activity of residual Cpa in cells knocked down using RNAi, nor that Hpo pathway activity acts in parallel to CP on F-actin. Interestingly, although endogenous Ex is upregulated in cells lacking CP, mutant cells still accumulated F-actin. wts mutant clones also upregulated Ex, which, when overexpressed, suppresses growth of wts mutant clones. Therefore, the increased levels of endogenous Ex in cells lacking either CP or wts appears to be insufficient to fully suppress the effects of loss of CP or wts on F-actin and growth, respectively (Fernández, 2011).

The data indicate that CP inhibits Yki nuclear accumulation, activation of Yki target genes, and consequently overgrowth of the proximal wing epithelium. Interestingly, Yki was also found to accumulate in nuclei of wild-type cells adjacent to the clone border. Consistent with a non-autonomous effect of CP loss on Hpo pathway activity, ex-lacZ and diap1-lacZ were upregulated in wild-type cells adjacent to CP mutant clones. However, Ex levels were reduced in wild-type neighboring cells. Cells expressing different amounts of ds and fj also upregulate ex-lacZ, but show reduced levels of Ex. Therefore, loss of CP might affect Fj or Ds levels, creating a sharp boundary of their expression. However, in contrast to clones overexpressing ds or mutant for fj, cell lacking CP also upregulated Ex and Mer inside the mutant clones, indicating that CP also acts cell-autonomously to promote Hpo signaling activity. CP might facilitate Yki phosphorylation by the Hpo kinase cassette as cpa-depleted tissues contain decreased phospho-Yki levels. But, the possibility cannot be excluded that CP also favors the direct binding of non-phosphorylated Yki to Ex, Hpo or Wts (Oh, 2009). Further analysis will be required to elucidate the mechanisms by which CP restricts Yki activity cell autonomously and in wild-type neighboring cells (Fernández, 2011).

The results argue for a constitutive role of CP in Hpo pathway activity, since Yki target genes are upregulated in the whole wing and eye imaginal discs. However, loss of CP did not fully recapitulate the phenotype for core components of the hpo pathway. Despite that, on average, cpb mutant clones located in the proximal wing disc domain were 25% larger than wild-type twin spots; 60% of mutant clones failed to grow. Moreover, in the distal wing epithelium, reducing CP levels induces mislocalization of the adherens junction components Armadillo and DE-Cadherin, extrude and death. Furthermore, in Drosophila, CP also prevents retinal degeneration (Delalle, 2005; Johnson, 2008). This indicates that although loss of CP can, under certain conditions, result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue. Therefore, in addition to promoting Hpo pathway activity, CP has additional developmental functions in epithelia. However, the possibility cannot be excluded that, like most upstream inputs that feed into the Hpo pathway, CP has a tissue-specific requirement in Hpo pathway activity (Fernández, 2011).

CP, Capt and Tsr all restrict F-actin assembly directly. CP and Capt control F-actin formation near the apical surface and inhibit ectopic expression of Yki target genes, whereas Tsr acts around the entire cell cortex and has no effect on Yki target genes. This argues that Hpo signaling activity is not affected by the excess of F-actin per se but provides significant support to the view that stabilization of an apical F-actin network by CP, Capt and Hpo signaling activity feeds back on the Hpo pathway to sustain its activity (Fernández, 2011).

These findings do not lead to an understanding of where F-actin accumulation intersects Hpo signaling activity because both Hpo signaling activity and F-actin dynamics feedback to each other. For instance, hpo or ex overexpression suppressed growth of CP-depleted cells. But, overexpressed ex and possibly hpo also suppress F-actin accumulation of Cpa-depleted cells. The control of F-actin by Hpo signaling activity and CP might constitute a parallel input, which sustains Hpo pathway activity. Alternatively, F-actin could act upstream of the core kinase cascade, which in turn feeds back to F-actin, to maintain its activity. The identification of additional actin cytoskeletal components that either promote Hpo pathway activity or act downstream of Hpo pathway activity on F-actin would help to discriminate between these possibilities (Fernández, 2011).

How F-actin influences Hpo signaling activity remains to be determined. The apical F-actin network, which regulates the formation and movement of endocytic vesicles from the plasma membrane, might promote the recycling or degradation of Hpo pathway components. Increased F-actin at apical sites would, therefore, affect protein turnover. Alternatively, apical F-actin might act as a scaffold to tether Hpo pathway components apically. In support of this, Ex, Hpo, Sav, Wts and Yki could all interact between each other through WW and PPXY motifs at apical sites (Oh, 2009; Reddy, 2008). Moreover, expression of a membrane-targeted form of Mats enhances Hpo signaling (Ho, 2010). Although Ex and Mer are properly localized in CP mutant cells, other members of the pathway might be mislocalized in the presence of excess F-actin. Interestingly, in mouse fibroblasts, the Hpo orthologs MST1 and MST2 colocalize with F-actin structures and are activated upon F-actin depolymerization (Densham, 2009), suggesting that by tethering Hpo pathway components, F-actin dynamics modulates their activity. Finally, the F-actin network might act as a mechanical transducer. Most of the mechanosensitive responses require tethering to force-bearing actin filaments. Tissue surface tension has been proposed to be a stimulus for a feedback mechanism that could regulate tissue growth. The tension exerted by neighboring cells might be sensed at the cell membrane by the actin cytoskeleton and translated to the regulation of cell proliferation through the Hpo signaling pathway (Fernández, 2011).

Drosophila PI4KIIIα is required in follicle cells for oocyte polarization and Hippo signaling

In a genetic screen mutations were isolated in CG10260, which encodes a phosphatidylinositol 4-kinase (PI4KIIIα). PI4KIIIα was found to be is required for Hippo signaling in Drosophila ovarian follicle cells. PI4KIIIα mutations in the posterior follicle cells lead to oocyte polarization defects similar to those caused by mutations in the Hippo signaling pathway. PI4KIIIα mutations also cause misexpression of well-established Hippo signaling targets. The Merlin-Expanded-Kibra complex is required at the apical membrane for Hippo activity. In PI4KIIIα mutant follicle cells, Merlin fails to localize to the apical domain. This analysis of PI4KIIIα mutants provides a new link in Hippo signal transduction from the cell membrane to its core kinase cascade (Yan, 2011).

DV asymmetry of the Drosophila oocyte is established during mid-oogenesis through a repolarization process initiated in the posterior follicle cells PFCs. In response to an unknown signal from the PFCs the oocyte nucleus migrates from the posterior end to the dorsal-anterior corner of the oocyte. As a consequence, the Gurken (Grk) protein no longer accumulates at the posterior cortex of the oocyte, but is now found in the dorsal-anterior membrane overlying the oocyte nucleus where it activates EGFR to initiate DV patterning. In a genetic screen directed at FC components affecting this repolarization process, a complementation group with six lethal mutant alleles was isolated, and initially named after a representative allele, GS27. When the PFCs were mutant for the GS27 gene product, the oocyte nucleus frequently remained at the posterior end of the oocyte. This phenotype was confirmed by the abnormal posterior localization of Grk in late egg chambers (Yan, 2011).

The lethality of the GS27 complementation group was mapped through duplication and deficiency mapping to the X-chromosomal region 3A4-3A8, which contains 16 genes. Sequencing of candidate genes showed that four alleles of the GS27 complementation group contained mutations that lead to premature stop codons in the coding region of CG10260, a predicted phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinases (PI4Ks) catalyze the generation of PIP₄. Phosphoinositides, including PIP₄, are important phospholipids in the cell membrane that participate in numerous signaling events. Four classes of PI4Ks have been identified in mammalian cells that localize to different cellular compartments and are likely to perform non-redundant functions. Three PI4K genes have been annotated in the fly genome: four wheel drive (fwd; PI4KIIIβ), CG2929 (PI4KIIα) and CG10260 (PI4KIIIα) (Yan, 2011).

To investigate the oocyte polarization defects caused by PI4KIIIα mutations, the localization of well-established oocyte polarity markers was examined. The microtubule cytoskeleton is polarized in the oocyte. The microtubule plus-end marker Kinesin (Kin, or Khc) fused to β-gal (Kin-β-gal), which normally forms a crescent at the posterior of the oocyte after stage 8, was examined. When the PFCs were mutant for PI4KIIIα, Kin-β-gal either localized to the center of the oocyte or was diffuse in the oocyte. Staufen localizes to the posterior pole of wild-type oocytes after stage 8 and is required for the localization of maternal RNAs. In PFC clones mutant for PI4KIIIα, Staufen also frequently mislocalized to the center of the oocyte or became dispersed in the oocyte. Therefore, in combination with the mislocalization of the oocyte nucleus, these results demonstrate that PI4KIIIα is required in the PFCs for all aspects of the establishment of correct oocyte polarity (Yan, 2011).

Oocyte polarization relies on the integrity of four signaling pathways in the PFCs: Notch, JAK/STAT, EGFR and Hippo. To examine whether the polarization defect observed in PI4KIIIα mutants was caused by disruption of one of these signaling pathways, well-established downstream targets of each pathway were examined in PI4KIIIα mutants (Yan, 2011).

The EGFR signaling reporter kekkon-lacZ (kek-lacZ) is highly expressed in the PFCs at stage 7 and 8 as a result of EFGR activation by Grk. In PFCs mutant for PI4KIIIα, the kek-lacZ expression level was comparable to that of wild-type PFCs, indicating that EFGR signaling was unaffected. The JAK/STAT signaling reporter 10×STAT92E-GFP is normally turned on in the PFCs during stage 7 and 8 in response to JAK/STAT activation. Apparently normal levels of GFP were detected in the nuclei of PI4KIIIα mutant PFCs, suggesting that JAK/STAT signaling was also intact (Yan, 2011).

Notch signaling is required for FCs to exit the mitotic cell cycle at stage 6 and switch to an endocycle. PI4KIIIα mutant PFCs maintained a mitotic cell cycle after stage 6, as indicated by the sustained staining of the mitotic marker phosphorylated Histone H3 (PH3), which is only seen up to stage 6 in wild-type FCs. Consistent with a failure to exit the mitotic cycle, the PI4KIIIα mutant PFCs often lost their monolayered epithelial structure and had smaller nuclei than neighboring cells. The expression of two Notch signaling targets, Cut and Hindsight (Hnt; Pebbled) was examined. In wild-type FCs, Cut expression is downregulated whereas Hnt expression is upregulated upon Notch activation at stage 6. PI4KIIIα mutant PFCs frequently failed to downregulate Cut and upregulate Hnt expression. Interestingly, PI4KIIIα mutant cells on the lateral side of the egg chambers showed no defect in Notch signaling. These results suggest that PI4KIIIα mutations compromise Notch signaling in the PFCs only (Yan, 2011).

The phenotypes described above are similar to those caused by mutations in Hippo pathway components. In particular, the observation that only PFCs appear affected is characteristic of mutations in the Hippo pathway, which are reported to affect Notch signaling only in this group of FCs. When the expression of a Hippo pathway target, ex, was checked using the enhancer trap line ex-lacZ, a much higher level of β-galin PI4KIIIα mutant FCs was detected than in wild-type cells. This upregulation was observed in all FCs, regardless of their position. Another Hippo pathway target, Diap1, monitored with the enhancer trap line diap1-lacZ, was mildly upregulated in the PI4KIIIα mutant FCs. These results indicate that the polarization defect in the PI4KIIIα mutants is likely to be caused by defective Hippo signaling (Yan, 2011).

Multiple lines of evidence suggest that the apical localization of the Expanded-Merlin-Kibra complex is crucial for Hippo signaling activity as it is proposed to function as a platform to bring the core Hippo components into close proximity and facilitate the phosphorylation reactions. In addition, it has been reported that Expanded directly interacts with Yki and functions to sequester Yki in the cytoplasm (Yan, 2011).

To investigate how mutations in PI4KIIIα lead to defective Hippo signaling, the apical localization of the Merlin-Expanded-Kibra complex was exmined. The complex is confined to the apical domain in wild-type FCs. In the PI4KIIIα mutant cells, a loss of apical Merlin staining was observed, whereas Expanded and Kibra were upregulated at the apical membrane. In addition to being Hippo pathway regulators, Expanded and Kibra are also targets of the Hippo signaling pathway. Mutations in Hippo pathway components lead to upregulation of Expanded and Kibra. In addition, it has been reported that the apical sorting of Merlin, Expanded and Kibra occur independently of each other. Therefore, the absence of Merlin from the apical membrane in PI4KIIIα mutant cells is the likely cause of the signaling defect, and the upregulation of Expanded and Kibra would be an expected secondary consequence of the disrupted Hippo signaling (Yan, 2011).

When PI4KIIIα mutant clones were examined in the imaginal eye discs of early second instar larvae, an absence of Merlin from the apical and junctional region was observed. However, no overgrowth phenotype typical of Hippo pathway mutations was observed. In fact, adults with mutant eye clones had smaller eyes than wild-type adults. Eye discs from late L2 larvae exhibited pyknotic nuclei staining in PI4KIIIα mutant clones, indicating death of the mutant cells (data not shown) (Yan, 2011).

Multiple classes of PI4Ks exist in eukaryotic cells that participate in producing various phosphoinositide species in distinct cellular compartments. Three PI4K genes have been annotated in the fly genome. When the intracellular distribution and level of PIP₂ was examined using a Ubi-PH-PLCδ-GFP reporter, a complete absence of PIP₂ from PI4KIIIα mutant FCs was observed in rare cases. In most cases, the PIP₂ reporter was specifically lost from the apical plasma membrane in the mutant cells. The yeast homolog of PI4KIIIα, Stt4p, localizes to patches on the plasma membrane where it is required for normal actin cytoskeleton organization. When the actin cytoskeleton of PI4KIIIα mutant FCs was examined by phalloidin staining, they exhibited abnormal actin-enriched spike structures on their apical domain that were positively marked by the microvillus marker Cad99C, suggesting that the spikes were malformed microvilli. As mutations in the Hippo pathway have been reported to lead to apical domain expansion, one possibility is that the malformed microvilli are caused by defective Hippo signaling. However, the morphology of the actin-enriched spikes in PI4KIIIα mutant cells is distinct from that caused by mutations in the Hippo pathway, suggesting that the loss of PI4KIIIα might also have a Hippo-independent effect on apical membrane structure (Yan, 2011).

How could PI4KIIIα mutations cause Merlin mislocalization? Expanded and Merlin are ERM (Ezrin, Radixin and Moesin)-related proteins, which are key linkers of the plasma membrane and cytoskeleton. Classical ERM proteins bind to PIP₂ in the membrane to switch from a closed to an open conformation for their activation. Significantly, in PI4KIIIα mutant cells, phosphorylated ERM proteins were absent from the apical microvilli region as indicated by a phospho-ERM-specific antibody. The malformed microvillus structure might therefore indicate a general failure of ERM protein activation in the PI4KIIIα mutant cells. For Merlin, the closed conformation is the active form, opposite to other ERM proteins. Nevertheless, Merlin undergoes a similar conformational switch to the other ERM proteins and contains an ERM PIP₂-binding site. Given these observations, it is possible that PIP₂ binding activates and/or stabilizes Merlin in the apical membrane, and a depletion of this lipid species due to the absence of PI4KIIIα might directly lead to the loss of Merlin (Yan, 2011).

In summary, this study has shown that PI4KIIIα is required in the FCs for Merlin localization and Hippo signaling. PI4KIIIα mutations in the PFCs lead to a Notch signaling defect and the subsequent failure of oocyte repolarization, which are precisely the phenotypes reported for Hippo mutations in the FCs. This effect is likely to be caused by a change in lipid composition in the membrane. How the abnormal actin structures are generated in the mutant cells, and whether they have a direct role in Merlin localization, remain to be investigated (Yan, 2011).

Protein Interactions

Removal by genetic mutation of either Merlin or Expanded does not appear to affect the subcellular localization of the other, indicating that physical interaction between these proteins is not required for proper subcellular localization in tissues. Studies of ERM proteins have shown that they exist in multiple conformations that appear to regulate their ability to interact with transmembrane and other interacting proteins. Thus, the formation of homotypic and heterotypic dimers or oligomers via interactions between the conserved NH₂-terminal region (CNTR) and C-terminal domains may function to maintain ERM proteins in either an active or inactive state. Recent studies of human Merlin indicate that it can form folded monomers, homotypic dimers and heterotypic dimers with ERM proteins. The CNTR of Expanded interacts directly with the C-terminal domains of both Expanded and Merlin, suggesting that Expanded can exist in a similar range of conformations. Based on these results, it is proposed that Merlin and Expanded form a heterodimeric complex that actively suppresses proliferation. According to this model, in the heterodimer, the N-terminal domain of Merlin would be free to interact with other proteins, and therefore would be in an activated state. Consistent with this view, removal of the 35 amino acid C-terminal region of Merlin results in a constitutively active form of the protein that contains all essential Merlin functions. In addition, both Merlin and Expanded must possess growth suppression functions that are independent of each other, because the observed Mer^-;ex^- double mutant phenotype is much more severe than either mutation alone (McCartney, 2000).

Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Drosophila Merlin and Expanded are shown to be components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes (Hamaratoglu, 2006).

The FERM-domain protein Expanded regulates Hippo pathway activity via direct interactions with the transcriptional activator Yorkie

The Hippo kinase pathway plays a central role in growth regulation and tumor suppression from flies to man. The Hippo/Mst kinase phosphorylates and activates the NDR family kinase Warts/Lats, which phosphorylates and inhibits the transcriptional activator Yorkie/YAP. Current models place the FERM-domain protein Expanded upstream of Hippo kinase in growth control. To understand how Expanded regulates Hippo pathway activity, affinity chromatography and mass spectrometry were used to identify Expanded-binding proteins. Surprisingly it was found that Yorkie is the major Expanded-binding protein in Drosophila S2 cells. Expanded binds Yorkie at endogenous levels via WW-domain-PPxY interactions, independently of Yorkie phosphorylation at S168, which is critical for 14-3-3 binding. Expanded relocalizes Yorkie from the nucleus, abrogating its nuclear activity, and it can regulate growth downstream of warts in vivo. These data lead to a new model whereby Expanded functions downstream of Warts, in concert with 14-3-3 proteins to sequester Yorkie in the cytoplasm, inhibiting growth activity of the Hippo pathway (Badouel, 2009).

Current models propose that ex and mer function together to restrict tissue growth upstream of hpo. Mer and Ex colocalize with cortical actin in the apical region of the cell. Both genetic and physical interactions have been observed between Mer and Ex: loss of one copy of mer dominantly enhanced wing overgrowth in ex mutants. In addition, fragments of Mer and Ex protein can interact physically with each other in far-western experiments or when overexpressed in cultured cells. Clones doubly mutant for mer and ex have more dramatic overgrowth than either single mutant, and mer,ex double mutants phenocopy hpo mutants (Badouel, 2009).

However, despite the widespread acceptance that ex and mer function upstream of Hpo, some data are difficult to reconcile with ex acting strictly upstream of hpo in activation of the pathway. Genetic analysis indicates that ex is downstream of dachs, which has been shown to be directly upstream of wts in growth control. Biochemical analysis of the effects of overexpressing Mer and Ex also suggested that ex may not act simply upstream of hpo. For example, overexpression of Mer in S2 cells leads to a shift in Wts mobility, whereas overexpression of Ex does not alter Wts mobility. In vivo analysis has also suggested that mer and ex may have different roles in controlling growth and apoptosis (Badouel, 2009).

Using biochemical purification and mass-spectrometic analysis, Yki as a major Ex-binding protein in Drosophila S2 cells was identified. Binding of Yki to Ex is direct and is mediated by a WW domain-PPxY interaction. This interaction is independent of Wts-dependent phosphorylation at S168, a site previously shown to be essential for strong interactions of Yki with 14-3-3 proteins. Consistent with the biochemical analysis, it was shown that ex can act downstream of wts in the regulation of growth in eye imaginal discs and can repress the pupal lethality caused by excessive growth of wts clones. In addition, it was found that loss of hpo does not alter the ability of ex to regulate yki activity, as indicated by transcriptional assays in S2 cells, and that expression of Ex is sufficient to relocalize Yki to the cytoplasm. These data lead to a model in which Ex functions to repress Yki activity at least in part by keeping Yki out of the nucleus. Intriguingly, the Yki homolog, YAP, was first identified as a protein that binds Src family kinases at the cell membrane. Subsequent studies have focused on the role of Yki in the nucleus. Interestingly, immunohistochemical analysis reveals that a portion of Yki colocalizes with Ex at the cell membrane in Drosophila imaginal discs (Badouel, 2009).

Once Yki is phosphorylated by Wts, it can bind 14-3-3 proteins and can be transported out of the nucleus. However, since 14-3-3-bound Yki can also shuttle back into the nucleus, Ex binding Yki provides an anchor that can effectively dampen Yki activity. 14-3-3 shuttling activity results in an equilibrium of distribution of Yki between the nucleus and the cytoplasm. This equilibrium is biased in favor of Yki in the cytoplasm in the presence of Ex acting as an anchor. The presence of a tether of Yki in the cytoplasm was already suggested based on the distribution of YkiS168A between the cytoplasm and the nucleus, instead of predominantly in the nucleus (Badouel, 2009).

The strong nuclear localization of Yki is seen in Drosophila tissues only in cases of pathological stimulation of growth, such as in wts loss-of-function clones, which lead to massive overgrowth. The lack of detectable Yki nuclear localization during normal growth regulation suggests that Yki is an exceedingly potent growth regulator, and points to why there are many layers of regulation of Yki localization. The need for Ex to dampen Yki signaling in the nucleus is reflected by the increase of Cyclin E and Diap1 transcription in ex mutant clones (Badouel, 2009).

It is speculated that the regulation of the Hpo pathway by combined loss of Ex and Mer is so potent because one acts as the brake and the other controls the accelerator. Ex restricts Yki to the cytoplasm, thus blocking activity downstream, whereas Mer activates Hpo activity, thereby restricting Yki via inhibitory phosphorylation. Thus, loss of Ex on its own does not have a dramatic effect on cell proliferation and apoptosis, since the activity of the kinase cascade is regulated via Mer. Conversely, as long as Ex is present, excessive pathway activity induced by loss of Mer can be effectively modulated by the dampening activity of Ex (Badouel, 2009).

The data strongly suggest that Ex regulates Yki activity downstream of wts, by directly binding Yki and inhibiting Yki nuclear localization and transcriptional activity. The possibility cannot, however, be excluded that Ex also has additional upstream roles in regulating Hpo activity. Interestingly, overexpressed Ex does not induce apoptosis in a wts mutant background, although it can block growth, suggesting that ex is upstream of wts in apoptosis control, yet downstream of wts in growth control. Genetic dissection of this pathway is complicated by the well-documented feedback loops in the Hpo pathway: for example, Yki regulates the expression of both mer and ex. In addition, genetic evidence suggests that ex and mer function together to regulate endocytosis and growth factor signaling. Further biochemical dissection of Hpo pathway activity will be required to fully elucidate the diverse ways in which growth and apoptosis are controlled in response to various developmental and environmental signals (Badouel, 2009).

The binding of Ex to Yki is likely to be 14-3-3 independent, as mutation of S168, the predominant Wts phosphorylation site, impairs 14-3-3 binding, yet does not affect the ability of Ex to bind Yki. Thus, Ex binding to Yki could provide a pool of Yki that is nonphosphorylated and poised for release by upstream growth regulators. The binding of 14-3-3 to Yki can protect Yki from dephosphorylation. This provides a problem for the cell, since 14-3-3 must dissociate from Yki to allow it to become dephosphorylated, thus releasing a potent activator of proliferation and inhibitor of cell death, allowing it to re-enter the nucleus. The ability of Ex to bind both phosphophorylated and dephosphorylated Yki provides a mechanism by which to anchor dephosphorylated Yki in the cytoplasm (Badouel, 2009).

An appealing model is that the binding of Ex to Yki may be modulated, once in the cytoplasm, as an additional control point for the Hpo pathway. FERM-domain proteins frequently form inhibitory intramolecular associations, blocking the activity of the protein until the repression is relieved. Modifications of the Ex FERM domain or linker region could thus alter the ability of Ex to bind Yki in vivo. Ex localization at apical junctions is at least partially dependent upon the atypical cadherin Fat, which can regulate Hpo pathway activity. The recruitment of Ex complexes (directly or indirectly) to Fat may modify the ability of Ex to interact with Yki (Badouel, 2009).

The in vivo analysis indicates that the N-terminal FERM domain of Ex contains apical localization elements, whereas the C-terminal region contains junctional localization elements. Thus, each of these localization elements might be regulated independently and might impact on the ability of Ex to sequester Yki. Identification of which protein(s) Ex binds at junctions may illuminate the mechanisms by which Ex responds to external inputs to regulate Yki activity (Badouel, 2009).

All of the components of the Hpo pathway are well conserved in mammals and have been shown to have conserved functions in regulating growth. Loss of Hpo and Wts orthologs and overexpression of the Yki ortholog, YAP, have been implicated in a variety of human cancers. FERM6, the human ortholog of Ex, also regulates Hpo pathway activity in mammals. Future studies will determine if FERM6 directly binds YAP, and if disrupting YAP-FERM6 interactions is a tumor-promoting event (Badouel, 2009).

Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded

The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. This study identified Kibra as another upstream component of the Hippo signaling pathway. This study shows that Kibra functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function and implicate Kibra as a potential tumor suppressor with relevance to neurofibromatosis (Yu, 2010).

In multicellular organisms, cell growth, proliferation, and death must be coordinated in order to attain proper organ size during development and to maintain tissue homeostasis in adult life. Recent studies in Drosophila have led to the discovery of the Hippo signaling pathway as a key mechanism that controls organ size by impinging on cell growth, proliferation, and apoptosis. Central to the Hippo pathway is a kinase cascade comprised of four tumor suppressors, including the Ste20-like kinase Hippo (Hpo) and its regulatory protein Salvador (Sav), the NDR family kinase Warts (Wts) and its regulatory protein Mats. The Hpo-Sav complex phosphorylates and activates the Wts-Mats complex, which in turn phosphorylates and inactivates the oncoprotein Yki, which normally functions as a coactivator for the TEAD/TEF family transcription factor Scalloped (Sd). Recent studies have also implicated the atypical cadherin Fat (Ft) as well as the membrane-associated FERM-domain proteins Expanded (Ex) and Merlin (Mer) as upstream components of the Hippo pathway. How these proteins are biochemically linked to the Hippo kinase cascade remains largely unknown, although Ex can at least partially regulate the Hippo pathway by directly binding and sequestering Yki in the cytoplasm. Ft differs from Ex, Mer, and core components of the Hippo kinase cascade in that, besides tissue growth, Ft also regulates planar cell polarity (PCP), for which it interacts with another cadherin Dachsous (Ds). Most recently, it was shown that a gradient of Ds activity in imaginal discs can modulate Hippo-mediated growth regulation, thus potentially linking PCP to the Hippo kinase cascade, although the biochemical mechanism of this linkage remains to be determined (Yu, 2010).

The physiological function of the Hippo pathway is best understood in Drosophila imaginal discs, where inactivation of the Hippo pathway tumor suppressors, or overexpression of the Yki oncoprotein, results in tissue overgrowth characterized by excessive cell proliferation, diminished apoptosis, and increased transcription of Hippo pathway target genes such as the cell death inhibitor diap1 and the microRNA bantam, as well as ex and mer as part of a negative feedback regulatory loop. Recent studies further implicated the Hippo pathway as a conserved mechanism of organ size control and tissue homeostasis in mammals. Thus, the mammalian homologs of Hpo (Mst1/2), Sav (WW45), Wts (Lats1/2), and Yki (YAP) constitute an analogous kinase cascade, and transgenic overexpression of YAP or inactivation of Mst1/2 led to massive organomegaly and rapid progression to tumorigenesis. Furthermore, NF2, the mammalian homolog of mer, is a well-established tumor suppressor gene whose mutations lead to neurofibromatosis (Yu, 2010 and references therein).

Besides its prominent role in controlling imaginal disc growth, the Hippo pathway is required during Drosophila oogenesis for the proper maturation of posterior follicle cells (PFCs). In the absence of Hippo signaling, the PFCs fail to undergo a Notch-mediated mitotic cycle-endocycle switch and accumulate in extra layers of follicular epithelium. The PFC maturation defects, in turn, lead to a disruption of the anterior-posterior (AP) polarity of the underlying oocyte, which manifests itself as mislocalization of the oocyte nucleus and AP axis determinants such as the RNA-binding protein Staufen (Stau). Interestingly, the oocyte polarity defect is observed in mutants for components of the Hippo kinase cascade as well as ex and mer, but not ft, suggesting that the canonical Hippo pathway may integrate different signals in different developmental contexts (Yu, 2010).

This study identifies Kibra as an upstream component of the Hippo pathway. Loss of kibra leads to oogenesis defects, imaginal disc overgrowth, and aberrant gene expression characteristic of defective Hippo signaling. Kibra functions together with Mer and Ex in an apical protein complex, which, through direct binding to the Hpo-Sav complex, regulates the Hippo kinase cascade and thus Yki phosphorylation. These findings uncover an important missing link in the Hippo signaling pathway and shed light on the molecular mechanism of the Ex and Mer tumor suppressor proteins (Yu, 2010).

In a genetic screen for oocyte polarity mutants based on FRT/FLP-induced mitotic clones in follicle cells, four lethal P element insertion lines on chromosome 3R were identified that caused mislocalization of Stau-GFP and Stau to the center of the oocyte when the PFCs were made homozygous mutant for the P element insertions. This polarity defect was observed with variable penetrance depending on the specific P element line analyzed, likely due to their hypomorphic nature. These lethal lines (264/09, 1156/7, f06952, and EP3494) fail to complement each other and all carry a P element insertion near the 5' UTR or within the first intron of CG33967. CG33967 encodes a 1288 amino acid protein that shares 39% identity with KIBRA, a cytoplasmic protein named after its predominant expression in kidney and brain in humans. Both CG33967 and KIBRA contain two N-terminal WW domains and one C-terminal C2 domain. CG33967 is referred to as kibra to distinguish it from its human ortholog KIBRA (Yu, 2010).

This study identifies Kibra as a tumor suppressor and an essential component of the Hippo pathway. A model is proposed in which Kibra functions together with Mer and Ex in an apical protein complex to transduce growth-regulatory signals to the Hpo-Sav complex, which, through the canonical Hippo kinase cascade, controls Yki phosphorylation and target gene transcription. Of note, the findings do not exclude the possibility that Kibra, Ex, or Mer may interact with additional Hippo pathway components besides Hpo-Sav, especially given the recent report that Ex can directly bind Yki. How Kibra, Ex, and Mer function together to integrate upstream signals remains to be determined. One possibility is that these proteins function redundantly in receiving signals from the same upstream regulator(s). Alternatively, each protein may be regulated by distinct upstream regulator(s) (Yu, 2010).

A commonly used assay for Hippo signaling in Drosophila S2 cells involves examining mobility shifts of the Wts protein on SDS-PAGE. Given its large size and that not all protein phosphorylation causes discernable mobility shift on SDS-PAGE, this assay is less sensitive in detecting Wts phosphorylation than the phospho-specific antibody used in the present study. Indeed, overexpression of Ex in S2 cells has no effect on Wts mobility, yet this study demonstrates that Ex induces robust Wts phosphorylation at its hydrophobic motif. The fact that this hydrophobic motif is a well-established direct phosphorylation site by Hpo homologs in mammalian cells further suggests that Ex, as well as Mer and Kibra, regulates Wts through the canonical Hippo kinase cascade. Indeed, it was found that Ex-induced Wts phosphorylation is Hpo dependent. These results are not incompatible with recent report that Ex can also regulate the Hippo pathway in a kinase-independent manner. Using a well-established assay for Yki transcriptional activity, this study found that while Ex, Mer plus Kibra, or Hpo could all suppress the activity of a Yki-Gal4 fusion protein, only Ex was able to suppress the activity of a Yki-Gal4 fusion protein in which all the possible Wts-phosphorylation sites are mutated. These observations are consistent with the view that Ex can regulate the Hippo pathway through both Wts-dependent and -independent mechanisms (Yu, 2010).

A comparison of the loss-of-function phenotypes of mer, ex, and kibra in egg chambers and imaginal discs reveals tissue-specific differences in the relative contribution of each gene to Hippo pathway regulation. For example, loss of ex alone, but not mer or kibra, is sufficient to cause robust diap1 upregulation in imaginal discs, suggesting that ex has a more essential role in diap1 transcriptional regulation. However, the converse is true in the ovary, where loss of mer or kibra results in stronger oocyte polarity and Notch signaling defects than loss of ex. In fact, the severity of mer or kibra mutant phenotypes in oogenesis are comparable to those of core components of the Hippo pathway such as hpo and sav, even though the former display much milder overgrowth than the latter in imaginal discs. Perhaps the most extreme case of tissue-specific requirement is provided by the ft tumor suppressor gene, which is required for Hippo pathway regulation in the imaginal discs but dispensable in developing egg chambers. While the underlying molecular basis remains to be determined, such tissue-specific requirements suggest that the core Hippo kinase cascade may function as a signal integrator of multiple inputs in a dynamic and versatile manner, and that additional cell surface receptors besides Ft may signal to the Hippo pathway (Yu, 2010).

Considerable efforts have been directed at identifying the key signaling pathways regulated by the NF2/Merlin tumor suppressor protein. These investigations have led to the identification of a number of effector mechanisms downstream of NF2/Merlin, such as growth control pathways mediated by Ras, Rac, STAT, or PI3K, contact inhibition mediated by cell surface receptors or adherens junctions, and endocytosis/degradation of various membrane proteins. The recent identification of Mer as an upstream regulator of Hpo in Drosophila provides yet another plausible mechanism through which Mer functions as a tumor suppressor protein. The identification of Kibra as a regulator of the Hippo pathway further strengthens the case for a functional link between NF2/Mer and the Hippo pathway. The observation that NF2/Mer and KIBRA can synergistically stimulate Lats1/2 phosphorylation in mammalian cells not only supports an NF2/Mer-Hippo connection, but further implicates KIBRA as a potential tumor suppressor in humans with relevance to neurofibromatosis (Yu, 2010).

The identification of Kibra as an upstream regulator of the Hippo pathway has implications for understanding memory-related functions of the human KIBRA gene. Besides its well-established roles in growth control, the Hippo pathway is also required for differentiation and morphogenesis of certain postmitotic neurons in Drosophila. It is speculated that modulation of the Hippo pathway may influence the growth or differentiation of memory-related neuronal structures, a hypothesis that can be directly tested by genetic manipulation of Hippo signaling activity in animal models (Yu, 2010).

Kibra is a regulator of the Salvador/Warts/Hippo signaling network

The Salvador (Sav)/Warts (Wts)/Hippo (Hpo) (SWH) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core of the pathway consists of a MST and LATS family kinase cascade that ultimately phosphorylates and inactivates the YAP/Yorkie (Yki) transcription coactivator. The FERM domain proteins Merlin (Mer) and Expanded (Ex) represent one mode of upstream regulation controlling pathway activity. This study identified Kibra as a member of the SWH network. Kibra, which colocalizes and associates with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kibra/Mer association is conserved in human cells. Finally, Kibra complexes with Wts and kibra depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. It is suggested that Kibra is part of an apical scaffold that promotes SWH pathway activity (Genevet, 2010).

An in vivo screen was performed in the fly wing in order to identify genes implicated in growth control. Transgenic flies bearing RNA interference (RNAi) constructs generated by the Vienna Drosophila RNAi Centre (VDRC) were crossed to the hedgehog-GAL4 (hh-GAL4) driver, leading to target gene silencing in the posterior compartment of the wing. A collection was screened of 12,000 lines targeting genes conserved between Drosophila and mammals. Expressing an RNAi line directed against kibra induced overgrowth of the posterior wing compartment compared to control flies. This phenotype was also observed upon wts depletion. Driving the same kibra RNAi line in the eye also led to increased organ size, similarly to a wts RNAi line. Adult eye sections revealed that kibra knockdown retinas present an excess of interommatidial cells (IOCs). The IOCs, the last population of cells to differentiate in the eye primordium, give rise to the secondary and tertiary pigment cells that optically isolate the ommatidia in the compound eye from each other. Extra IOCs are produced during normal development but are then eliminated by apoptosis at the pupal stage to give rise to the adult lattice. The presence of extra IOCs is a hallmark of SWH network loss of function, which reduces retinal apoptosis, as seen in wts RNAi adult eye sections. Thus, depletion of kibra elicits a similar phenotype to SWH network mutants, suggesting a potential role for Kibra in Hpo signaling (Genevet, 2010).

To study kibra loss of function, the kibra^Δ32 allele loss of function allele was generated by imprecise excision of the EP747 transposon. This deletion allele, which removes the translation initiation site, is homozygous lethal and may be a null allele for kibra. kibra^Δ32 FLP/FRT mutant clones in 40 hr after-puparium-formation (APF) retinas present extra IOCs, similarly to what was observed in adult eyes with kibra knockdown. Duplication of bristles or missing bristles can also be observed. Apoptotic indexes were determined during the retinal apoptosis wave (28 hr APF) in pupal retinas containing kibra mutant clones stained with an anti-active Caspase-3 antibody. kibra mutant tissue presents a reduced apoptotic index compared with wild-type (WT) areas in the same retinas. Thus, extra IOCs persist in kibra mutant clones as a result of decreased developmental apoptosis (Genevet, 2010).

The proliferation rate of kibra mutant cells was assessed in imaginal discs, the larval precursors to the adult appendages. By using the FLP/FRT system under the control of the heat-shock promoter, kibra mutant cells and their WT sister clones were generated through single recombination events from heterozygous mother cells. After several rounds of divisions, the sizes of mutant clones (no GFP) and WT twin spots (two copies of GFP) were compared, allowing estimation of the relative proliferation rates of mutant versus WT cells. The total kibra clone area is 1.57-fold larger than the control twin spot area, compared to a ratio of 0.98 when both clones and twin spots are WT, indicating that kibra^Δ32 mutant cells grow 1.6 times faster than WT cells (Genevet, 2010).

In addition to cell cycle rates, the timing of cell cycle exit can readily be measured in the eye disc, where cell divisions follow a spatially determined pattern. During the third larval instar, the morphogenetic furrow, a wave of differentiation, sweeps the eye disc from posterior to anterior. Anterior to the furrow cells still proliferate asynchronously, while in the furrow cells synchronize in G1. Immediately posterior to the furrow, cells enter a final round of synchronous S phases, the second mitotic wave (SMW). Posterior to the SMW, most cells permanently exit the cell cycle. Thus, in WT discs, no S phases can be observed posterior to the SMW. As expected, hpo mutant cells fail to exit from the cell cycle in a timely manner and present ectopic EdU-positive staining posterior to the SMW. kibra mutant cells exhibit a less pronounced but similar phenotype. Thus, kibra mutant tissues have a proliferative advantage and an apoptosis defect, consistent with an involvement in the SWH network. The overgrowth defect appears more subtle than that of core pathway members such as wts and is more akin to upstream regulators (e.g., ex and mer) (Genevet, 2010).

Several transcriptional targets of the SWH network have been identified, such as the Drosophila Inhibitor of Apoptosis 1 (DIAP1) gene, the cell cycle regulator cycE, the miRNA bantam, as well as ex. In kibra mutant wing or eye discs, no strong change in DIAP1, CycE, or ex-lacZ reporter levels could be detected. Since overgrowth of kibra mutant cells in the wing is subtle compared to wts mutants, it is possible that Kibra plays a relatively minor role in SWH signaling in the wing. Accordingly, using an anti-Kibra antibody, it was noted that Kibra staining in the wing disc is weak and consists of a punctate apical staining which can clearly be observed when kibra is overexpressed in a stripe of cells. Thus, the extent to which Kibra is required may vary in different tissues (Genevet, 2010).

Ovarian posterior follicle cells (PFCs) are particularly sensitive to SWH loss of function, leading to a study the kibra^Δ32 phenotype in the ovary. First, it was noted that Kibra protein levels are higher in follicle cells than in the wing discs. Kibra staining is mainly apical and is severely reduced in kibra^Δ32 clones. Similarly to hpo or wts loss of function, kibra loss of function in the PFCs induces an upregulation of the ex-lacZ reporter. hpo or wts mutant PFCs also show a misregulation of the Notch (N) pathway and ectopic cell divisions. The N target Hindsight (Hnt) is normally repressed in all follicle cells up to stage 6 and switched on from stage 7 to stage 10B. Cut, which is repressed by Hnt, presents an opposite pattern of expression. In kibra mutant PFCs from stage 7-10B egg chambers, Hnt expression is lost, while Cut is ectopically expressed. This indicates that N signaling is downregulated in kibra mutant PFCs. Loss of kibra also leads to perturbation of epithelial integrity, as mutant PFCs show an accumulation of the apical polarity protein aPKC and the N receptor as well as multilayering of the follicular epithelium. Ectopic mitotic divisions are also observed in PFCs clones after stage 6, as detected by phospho-histone H3 (PH3) staining. Together, these phenotypes are identical to those observed in hpo or wts loss of function, suggesting that Kibra is indeed a member of the SWH network (Genevet, 2010).

To further explore the role of Kibra in the SWH network, genetic interaction and epistasis experiments were performed. Overexpressing kibra in the eye under the GMR (Glass Multimer Reporter) promoter elicits the formation of a small rough eye with frequent ommatidial fusions. This phenotype can be partially rescued by removing one copy of the hpo gene. In contrast, overexpressing kibra could not rescue the hpo-like overgrowth phenotype induced by yki overexpression, suggesting that Kibra may be an upstream regulator of the pathway (Genevet, 2010).

To conduct epistasis experiments between kibra and yki, the MARCM system was used to generate clones of mutant cells while simultaneously overexpressing or depleting other pathway components. MARCM clones expressing yki RNAi generated with eyFLP lead to the formation of a normal eye, because yki-depleted cells are eliminated by apoptosis and replaced by WT cells. As expected, eyFLP kibra MARCM clones cause eye overgrowth. This overgrowth is rescued by yki depletion in the mutant cells, indicating that the kibra overgrowth phenotype is yki dependent. Furthermore, overexpressing kibra in the eye under the GMR promoter induces apoptosis in third instar eye discs, which is suppressed by loss of hpo. Together, these epistasis experiments are consistent with Kibra being a member of the SWH network acting upstream of Yki and Hpo (Genevet, 2010).

Genetic interactions between kibra, mer, and ex, upstream members of the SWH network, were then investigated. Expressing a kibra, an ex, or a mer RNAi line in the eye under the GMR promoter induces eye overgrowth. Combined depletion of either Ex/Kibra or Mer/Kibra shows stronger phenotypes than individual depletion of these proteins. The MARCM technique was used to evaluate epistatic relationships between those three genes. hsFLP MARCM clones of various genotypes were generated and scored according to the severity of the wing overgrowth phenotypes, with type 0 representing normal wings and type 4 the strongest overgrowth. Overexpressing ex or mer in kibra mutant clones significantly rescues the overgrowth of kibra mutant clones. Reciprocally, kibra overexpression was also able to suppress the ex overgrowth phenotype. Thus, a strict epistatic relationship between kibra, ex, and mer could not be determined, consistent with a model whereby kibra, ex, and mer cooperate to control SWH pathway activity (Genevet, 2010).

As well as being an upstream regulator of the SWH network, ex is also one of its transcriptional targets, as are other upstream regulators (e.g., mer, four-jointed, dachsous). Since epistasis experiments place Kibra at the level of Mer and Ex, it was of interest to test whether this is also the case for kibra. Kibra levels were highly upregulated in mer;ex or hpo clones, showing an apical localization. The same is true in hpo clones in follicle cells. Similarly, hpo-depleted cultured Drosophila S2R+ cells have increased Kibra levels. To determine whether kibra is a transcriptional SWH network target, quantitative RT-PCR experiments were performed on yki-overexpressing and control wing imaginal discs. As expected, ex mRNA levels were increased in yki-expressing discs compared to control discs. Interestingly, kibra mRNA levels were also upregulated in yki-expressing discs, confirming that kibra is a Yki transcriptional target and suggesting the existence of a possible negative feedback loop regulating Kibra expression (Genevet, 2010).

Because hpo clones present increased levels of Kibra as well as Mer and Ex, these constitute a good system to evaluate the colocalization of those proteins. Indeed, Kibra colocalizes with Mer in the wing disc. As expected, Mer and Ex also colocalize. Thus, Kibra, Mer, and Ex colocalize apically in imaginal disc cells, but are dispensable for each other's apical sorting, because Kibra is still apical in mer;ex clones and Mer/Ex are normally localized in kibra clones (Genevet, 2010).

Because Kibra colocalizes with Mer/Ex, a possible association between those proteins was examined by conducting coimmunoprecipitation (co-IP) assays in S2R+ cells. Kibra was found to co-IP with Ex and Mer, but not with Hpo or with the negative regulator of Hpo, dRASSF. Kibra possesses two WW domains, which are predicted to mediate protein-protein interactions by binding to PPXY motifs. Furthermore, the first WW domain of human KIBRA was shown to recognize the consensus motif RXPPXY in vitro. In flies, Mer does not contain any PPXY sites, while Ex has two PPXY sites (P⁷⁸⁶PPY and P¹²⁰³PPY) and an RXPPXY site (R⁸⁴²DPPPY). The association between Kibra and Ex was investigated by mutating amino acids that are known to be required for WW domains and PPXY sites to interact. A Kibra protein mutant for its first WW domain (P85A) could no longer co-IP WT Ex. Reciprocally, WT Kibra could not co-IP an Ex protein deficient for its RXPPXY site (P845A). Thus, Kibra associates with Ex through its first WW domain and the Ex RXPPXY motif (Genevet, 2010).

Because Kibra complexes with Ex and a Yki/Ex interaction has recently been described, attempts were made to determine whether Kibra can affect Yki activity. S2R+ cells were treated with RNAi against several SWH pathway components, and Yki phophorylation on Ser168 was monitored by western blotting. The phosphorylation of Yki by Wts at Ser168 leads to Yki inactivation and sequestration in the cytoplasm, where it has been reported to bind Ex, Wts, Hpo, and 14.3.3. lacZ RNAi-treated cells show a high basal level of phospho-Yki (P-Yki). As expected, Yki phosphorylation is abolished when Wts is depleted, and mildly reduced when the Wts cofactor Mats is depleted. In wts treated RNAi cells, a Yki downward shift can also be observed using a pan-Yki antibody. ex RNAi treatment has only a mild effect on P-Yki levels. Interestingly, kibra depletion leads to a marked reduction in P-Yki. When depleted in conjunction with ex, the P-Yki signal becomes even further reduced (Genevet, 2010).

This suggests that Kibra and Ex are required for Wts activity on Yki, which prompted an investigation of whether Kibra could associate with Wts. Co-IP assays reveal that Kibra interacts with Wts. Wts does not seem to compete with Ex for Kibra association, because it could still complex with a form of Kibra mutant for its first WW domain. Because Kibra associates with Wts and Ex interacts with Yki, whether Wts requires Kibra/Ex to bind Yki was investigated. Endogenous IPs between Yki and Wts were performed in S2 cells treated with various dsRNAs. In these conditions, the effect of kibra and ex depletion on Yki phosphorylation can also be observed. In control cells, Wts binding to Yki is detected after immunoprecipitating Yki. This endogenous interaction is unaffected by the individual or combined depletion of ex and kibra. These results suggest that Ex and Kibra are required to activate the SWH pathway by nucleating an active Hpo/Wts kinase cassette, rather than promoting the Wts/Yki interaction (Genevet, 2010).

These data identify Kibra as a regulator of the SWH network that associates with Ex and Mer, with which it is colocalized apically and transcriptionally coregulated. Given that the apical surface of epithelial cells is instrumental in both cell-cell signaling and tissue morphogenesis, it is speculated that Kibra may cooperate with Ex and Mer to transduce an extracellular signal, or relay information about epithelial architecture, via the SWH network, to control tissue growth and morphogenesis (Genevet, 2010).

Recent data have suggested that an apical scaffold machinery containing Hpo, Wts, and Ex recruits Yki to the apical membrane, facilitating its inhibitory phosphorylation by Wts. Since Kibra associates with Ex and is also apically localized, it is hypothesized that Kibra is also part of this scaffold and participates in nucleating an active Hpo/Wts complex and recruiting Yki for inactivation. This view is supported by the finding that Kibra complexes with Wts and that combined depletion of Kibra and Ex leads to a strong decrease in Yki phosphorylation, but does not disrupt the Wts/Yki interaction. The data also suggest that the importance of Kibra may be tissue-specific since robust phenotypes were observed in ovaries and hemocyte-derived S2R+ cells, but weaker effects in imaginal discs. Thus, considering the relative levels of expression of Ex, Mer, and Kibra may be important in determining pathway activation. Finally, since mammalian KIBRA complexes with the NF2/MER tumor suppressor, these findings raise the possibility that human KIBRA may contribute to tumor suppression in human neurofibromas and potentially other tumors (Genevet, 2010).

The WW domain protein Kibra acts upstream of Hippo in Drosophila

The conserved Hippo kinase pathway plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. Whereas the function of the core kinase cascade, consisting of the serine/threonine kinases Hippo and Warts, in phosphorylating and thereby inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling by the atypical cadherin Fat may act via Expanded, but how Merlin is regulated has remained elusive. This study shows that the WW domain protein Kibra is a Hippo signaling component upstream of Hippo and Merlin. Kibra acts synergistically with Expanded, and it physically interacts with Merlin. Thus, Kibra predominantly acts in the Merlin branch upstream of the core kinase cascade to regulate Hippo signaling (Baumgartner, 2010).

Overexpression of Drosophila Kibra in the developing eye has been shown to decrease the size of the adult organ. Four different loss-of-function alleles of Kibra were generated to define its function in growth control. Deletion of the first exon (harboring the translational start site) by imprecise excision of a P element resulted in the alleles Kibra¹ and Kibra². Kibra³, a mutation in the initiating ATG, was generated by means of an EMS reversion mutagenesis of the EP-mediated Kibra overexpression phenotype. Finally, the entire Kibra locus was removed by the hybrid element insertion (HEI) technique. All alleles were lethal when homozygous and failed to complement each other but were complemented by the precise P element excision used as a control throughout this study. All mutants displayed the same growth phenotypes, and homozygous mutant animals died as first-instar larvae. It is concluded that all Kibra alleles are genetically null (Baumgartner, 2010).

Kibra mutant heads were enlarged in comparison to controls. Similarly, wings containing posterior compartments largely mutant for Kibra were larger than control wings. The presence of a UAS-Kibra overexpression construct, without any Gal4 driver, rescued the lethality of Kibra homozygous mutant flies as well as the size defects of Kibra mutant organs, proving that the growth alterations are caused by the loss of Kibra function. Thus, Kibra is a general regulator of growth that is required to restrict organ size (Baumgartner, 2010).

To determine the cause of the Kibra mutant overgrowth phenotypes, a clonal analysis in wing imaginal discs was performed. Clones of Kibra mutant cells were larger than their corresponding wild-type sister clones. The number of cells per clone was increased in Kibra mutant clones compared to wild-type clones but not to the same extent as the clone size. However, FACS analysis revealed that cell size was unchanged in Kibra mutant cells, suggesting a change in cellular architecture in cells devoid of Kibra function. It is concluded that Kibra mutant clones in the wing imaginal disc were enlarged because Kibra mutant cells exhibit a proliferative advantage over wild-type cells (Baumgartner, 2010).

Tangential sections were analyzed of mosaic compound eyes consisting of Kibra mutant cells surrounded by heterozygous cells. The mutant ommatidia were normally structured and the different cell types properly differentiated, but the interommatidial regions were enlarged compared to the control. The increased distance between mutant ommatidia was due to more cells, because clones of Kibra mutant cells in the pupal retina displayed an increase in the number of interommatidial cells. Supernumerary interommatidial cells are a hallmark of inactivation of the Hippo pathway. Whereas a complete loss of Hippo signaling causes a pronounced excess of interommatidial cells, a mild extra interommatidial cell phenotype is observed in mutants that reduce but do not abrogate Hippo signaling, such as ex or Mer (Baumgartner, 2010).

A reduction in Hippo signaling activity results in extra interommatidial cells because the developmental apoptosis in pupal retinae is largely eliminated. Conversely, overexpression of hpo or ex induces apoptosis in third instar eye discs. Overexpression of Kibra in clones in the wing imaginal disc reduced clone size. Kibra-overexpressing clones contained fewer cells than control clones. To investigate whether overexpression of Kibra induces apoptosis, Kibra overexpression clones were generated in the third instar eye disc by using the Gene-Switch system. Indeed, the Kibra-overexpressing clones located anterior to the morphogenetic furrow (MF) showed an increase in programmed cell death as judged by staining for cleaved Caspase-3 and TUNEL staining, suggesting that overexpression of Kibra induces inappropriate apoptosis of proliferating cells. Consistently, co-overexpression of Diap1, a direct Yorkie transcriptional target, partially rescued the small eye phenotype associated with Kibra overexpression. Co-overexpression of CycE, another target of the Hippo pathway, also resulted in a partial rescue of the small eye. The size of Kibra-overexpressing eyes was further restored by concomitant overexpression of Diap1 and CycE. These results suggest that the effects elicited by Kibra overexpression are at least partly due to a reduction in the expression of the Hippo pathway target genes Diap1 and CycE (Baumgartner, 2010).

The striking similarities of the Kibra, ex, and Mer phenotypes prompted a genetic test of whether Kibra restricts tissue size via Hippo signaling. Interaction studies were started at the level of the transcriptional coactivator yki, which induces target genes promoting cell proliferation and cell survival and is inactivated by Hippo signaling. Three lines of evidence suggest that Kibra acts via inactivation of Yki. First, the coexpression of Kibra and yki during eye development suppressed the eye size reduction caused by Kibra and resulted in the same overgrowth phenotype as observed in eyes overexpressing yki alone. Second, the growth advantage of Kibra mutant cells was completely abolished by the concomitant loss of yki function. Third, a pupal lethal hypomorphic combination of Kibra alleles was rescued to viability by removal of a single copy of yki (Baumgartner, 2010).

To determine whether (and at which level) Kibra acts in the Hippo pathway to inactivate Yki, a series of epistasis tests were performed. It was found that the loss-of-function phenotypes of hpo, sav, and wts were epistatic to the Kibra overexpression phenotype, indicating that Kibra acts upstream of Hpo (Baumgartner, 2010).

Next, interaction with the upstream components Ex and Mer was tested. Overexpression of ex in a Kibra mutant background resulted in an intermediate phenotype. Vice versa, overexpression of Kibra also yielded an additive effect in an ex mutant head. Conversely, Kibra overexpression failed to reduce organ size in a Mer mutant head, indicating that Kibra requires Mer to exert its function. The eyFlp/FRT recombination system (without cell lethal) was used to generate mosaic animals with heads largely homozygous for ex and Mer mutations, as well as ex Kibra and Mer Kibra double mutations, respectively. Both ex and Mer mosaic heads showed only mild overgrowth. Strikingly, pupae with mosaic heads doubly mutant for ex and Kibra did not eclose, and normal head structures were displaced by overgrown tissue. In contrast, flies with Mer Kibra mosaic heads were viable. However, Mer Kibra double mutant clones showed stronger overgrowth than Mer clones. Reducing ex function during eye development by the expression of a hairpin RNAi construct did not alter the wild-type eye size but resulted in a severe enhancement of the Kibra loss-of-function phenotype, and the resulting eyes resembled those of hpo mutants. Reducing Mer function caused subtle overgrowth but enhanced the Kibra mutant phenotype much less (Baumgartner, 2010).

Whereas single mutants for ex and Mer cause a mild overgrowth phenotype, ex Mer double mutants display strong synergistic effects, suggesting that the two FERM domain proteins act in separate branches to activate Hippo signaling. These findings suggest that Kibra acts primarily upstream of Mer. However, since Mer Kibra double mutant clones show stronger overgrowth than Mer mutant clones and a reduction of Mer function enhances the Kibra loss-of-function phenotype, Kibra also contributes to Mer-independent regulation of Yki activity (Baumgartner, 2010).

To confirm that Kibra acts via Hippo signaling, whether Kibra mutant clones upregulated the expression of a Diap1 enhancer element (diap1-GFP4.3) that had been published to be a minimal Hippo responsive element (HRE) was tested. A pronounced upregulation of diap1-GFP4.3 was evident in clones of hpo mutant cells posterior and, to a weaker extent, anterior to the MF in eye imaginal discs. Cells lacking Kibra function also upregulated diap1-GFP4.3 expression, although to a lesser degree and with restriction to the differentiating tissue posterior to the MF. Clones of ex mutant cells, in resemblance to hpo clones, upregulated diap1-GFP4.3 strongly behind and somewhat weaker before the MF, whereas Mer mutant cells, like Kibra mutant cells, upregulated diap1-GFP4.3 expression weakly and solely posterior to the MF. Thus, the loss of Kibra results in an upregulation of a Hippo signaling reporter gene. The similar response of diap1-GFP4.3 to loss of Kibra or Mer suggests that Kibra and Mer act in the same way on Hippo signaling to regulate the HRE (Baumgartner, 2010).

This study provides genetic and biochemical evidence that the WW domain protein Kibra is a Hippo signaling component. Several lines of evidence indicate that Kibra acts predominantly in the Mer branch. First, the mild overgrowth phenotype caused by loss of Kibra function is akin to the Mer phenotype. Second, genetic epistasis experiments place Kibra upstream of Mer. Third, the effects of Kibra and Mer loss-of-function on a reporter for Hippo signaling activity are very similar. Fourth, Kibra and Mer synergise with ex in a similar fashion. Fifth, Kibra physically interacts with Mer. However, since the genetic analysis of Kibra also revealed a synergism with Mer, Kibra also acts on Yki activity in a Mer-independent manner (Baumgartner, 2010).

FERM domain proteins, such as Mer, have been suggested to connect membrane proteins with the underlying cortical cytoskeleton in order to integrate signals from the membrane and initiate intracellular signaling cascades. Thus, it is conceivable that Mer, together with as yet unknown proteins, assembles downstream cytoplasmic components of the Hippo pathway at the membrane and that controlled assembly and stabilization of such multiprotein complexes regulates the activity of the Hippo kinase cascade. In such a scenario, adaptor proteins providing multiple protein-protein interaction domains are of special interest (Baumgartner, 2010).

The WW domain protein Kibra binds Mer and could enable signaling events at the membrane/cytoskeleton interface that activate the Hpo kinase cascade. Since a truncated Kibra protein lacking the WW domains interacts more fiercely with Mer, it is likely that the physical association of Kibra and Mer is modulated by binding of other factors to the WW domains of Kibra (Baumgartner, 2010).

Interestingly, the effects caused by the concomitant loss of ex and Kibra functions are more severe than those elicited by mutated Hippo signaling core components. In addition to massively overgrowing, clones of ex Kibra double mutant cells round up, a behavior that was never observed in clones of hpo mutant cells. Furthermore, the diap1-GFP4.3 reporter indicates higher Yki activity in proliferating ex Kibra mutant eye imaginal disc cells as compared to hpo mutant cells. It thus appears that Yki activity is unleashed in cells lacking both ex and Kibra functions. Since Ex has been shown to directly bind Yki, it is tempting to speculate that Kibra participates in a distinct (Mer-independent) mechanism to prevent nuclear Yki localization (Baumgartner, 2010).

Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway

Recent studies have shown that the Hippo-Salvador-Warts (HSW) pathway restrains tissue growth by phosphorylating and inactivating the oncoprotein Yorkie. How growth-suppressive signals are transduced upstream of Hippo remains unclear. This study shows that the Sterile 20 family kinase, Tao-1, directly phosphorylates T195 in the Hippo activation loop and that, like other HSW pathway genes, Tao-1 functions to restrict cell proliferation in developing imaginal epithelia. This relationship appears to be evolutionarily conserved, because mammalian Tao-1 similarly affects MST kinases. In S2 cells, Tao-1 mediates the effects of the upstream HSW components Merlin and Expanded, consistent with the idea that Tao-1 functions in tissues to regulate Hippo phosphorylation. These results demonstrate that one family of Ste20 kinases can activate another and identify Tao-1 as a component of the regulatory network controlling HSW pathway signaling, and therefore tissue growth, during development (Boggiano, 2011).

During development, organisms must determine the overall size and shape of their individual organs through mechanisms not fully understood. The recent discovery of the evolutionarily conserved Hippo-Salvador-Warts (HSW) signaling pathway has revealed a unique mechanism to regulate proliferation independent of developmental patterning. The core members of the HSW pathway, Hippo (Hpo) and Warts (Wts), together with their scaffolding partners Salvador (Sav) and Mats, phosphorylate and inactivate the transcriptional co-activator Yorkie (Yki). Phosphorylation prevents Yorkie from translocating to the nucleus where it binds to TEAD-family transcription factors and drives the transcription of genes that promote growth and inhibit apoptosis. Loss of HSW pathway function in Drosophila leads to increased cellular proliferation resulting in tumor-like overgrowths in epithelial tissues. Similarly, knockout mouse models of HSW homologs grow tumors, and human HSW homologs have been implicated in cancers. These studies suggest that HSW signaling is a crucial part of an organism's ability to regulate cell proliferation and overall tissue size (Boggiano, 2011 and references therein).

A central, unanswered question regarding HSW function is how the activity of Hpo, the most upstream kinase in the pathway is regulated. The atypical cadherin Fat and its ligand Dachsous can function at the plasma membrane to initiate HSW signaling, however the extracellular cues that trigger signaling and the mechanism by which Fat activates Hpo remain elusive. In addition, genetic evidence strongly suggests that another source of Hpo activation functioning in parallel to Dachsous-Fat activation must exist. At least three different cytoplasmic proteins are believed to act upstream of Hpo to initiate signaling through the pathway, Expanded (Ex), Merlin (Mer), and Kibra. Ex and Mer are members of the Four-point-one, Ezrin, Radixin, Moesin (FERM) family and Kibra is a WW-domain containing protein. Though these three proteins are thought to physically interact with each other in varying complexes, only Ex can form a complex with Hpo and it is unclear how this interaction leads to activation of Hpo. Moreover, there is strong genetic evidence that Ex, Mer, and Kibra act in parallel to each other, implying that other mechanisms for activating Hpo independently of Ex must exist (Boggiano, 2011).

This study sought to identify genes that might function upstream of Hpo to activate the pathway using a candidate gene approach and discovered that the Sterile 20 kinase Tao-1 is a member of this signaling pathway. Tao-1 previously has been shown to destabilize microtubules and has been implicated in apoptosis in the Drosophila germline. This study shows that loss of Tao-1 function results in increased cellular proliferation and upregulation of Yki target gene expression. It was further demonstrated that Tao-1 regulates HSW pathway activity by phosphorylating Hpo at a critical activating residue. Thus, these results identify Tao-1 as a member of the HSW pathway and provide a molecular mechanism for Hpo activation (Boggiano, 2011).

In an effort to identify additional regulators of HSW signaling, this study examined the role of Tao-1 in growth control during development. Tao-1 depletion in either the eye or wing epithelium results in overgrowth phenotypes as well as transcriptional upregulation of HSW targets. Using a combination of genetic epistasis, experiments in cultured S2 cells, and in vitro biochemistry, it was demonstrated that Tao-1 directly phosphorylates the critical T195 regulatory residue in the activation loop of Hpo to promote HSW pathway activation. The observation that a mammalian orthologue of Tao-1, TAOK3, can phosphorylate MST kinases at the same residue further suggests that this regulatory function is conserved in mammals. Taken together, these results implicate Tao-1 as a component of HSW signaling (see A model for Tao-1's function in the HSW pathway) and reveal a mechanism for regulation of Hpo activity (Boggiano, 2011).

While Tao-1 depletion results in overgrowth phenotypes that are similar to mutations in other HSW pathway genes, these phenotypes are less severe than those of core components such as hpo and wts. One likely explanation for this is that the RNAi transgenes that were used in these studies do not completely remove Tao-1 function. It is also possible that there are multiple mechanisms for activating HSW signaling, including, but not limited to, Tao-1 phosphorylation of Hpo. Indeed, previous studies have demonstrated that the upstream components Mer, Ex, and Kibra act, at least in part, in parallel to activate Hpo. Biochemical evidence indicates that two of these proteins, Mer and Ex, function with Tao-1 to activate HSW signaling. While it is probable that Kibra functions upstream of Tao-1, it cannot be ruled out that Kibra functions independently of Mer and Ex to activate HSW signaling in a Tao-1-independent manner. Further genetic analysis using a Tao-1 null allele would be helpful in defining Tao-1's role relative to other HSW components, but unfortunately the deletions associated with the sole existing Tao-1 null allele, Tao-150, also appear to affect an adjacent gene. In addition, Tao-1 maps very close to the most proximal FRT element on the X chromosome, making it difficult to generate recombinant chromosomes for somatic mosaic analysis (Boggiano, 2011).

How do Mer, Ex and Tao-1 cooperate to regulate Hpo phosphorylation? Given that Ex has been shown to interact with Hpo, one possibility is that Mer and Ex function to scaffold Tao-1 together with Hpo, thereby promoting the ability of Tao-1 to phosphorylate and activate Hpo. However, despite repeated attempts it has not been possible to detect Tao-1 in a complex with either Mer or Ex, and knockdown of Mer, ex or kibra does not diminish the ability of Tao-1 to promote Hpo phosphorylation in S2 cells. For these reasons, the possibility is favored that Mer and Ex indirectly affect Tao-1 function, perhaps by interacting with other proteins that in turn directly regulate Tao-1. For example, Tao-1 activity could be directly regulated by an unknown receptor at the cell surface whose localization or activity is controlled by interaction with Mer and Ex. This notion is consistent with the fact that both Mer and Ex have FERM domains, which are known to interact with the cytoplasmic tails of transmembrane proteins. Previous studies have suggested that Ex interacts with the transmembrane protein Crumbs, though the mechanistic significance of this interaction is unclear. It is not currently known whether Drosophila Merlin has transmembrane binding partners (Boggiano, 2011).

Two additional ideas related to Tao-1 function are suggested by the current data. In S2 cells, Tao-1 kinase activity is required for normal levels of Hpo phosphorylation at T195 in the kinase activation loop, suggesting that Tao-1 could function to maintain constant, low levels of pathway activation. In turn, this low level of Hpo activation might be necessary so that other, regulated inputs into HSW activity can quickly transition cells away from actively dividing and into a differentiated state following periods of growth. Alternatively, it is possible that Tao-1 activity itself is dynamically regulated during development, allowing it to rapidly alter levels of HSW pathway activity via its effect on Hpo phosphorylation. In either case, phosphorylation by Tao-1 at T195 is likely to promote Hpo's known ability to undergo autophosphorylation, thus amplifying the effect of even a small change in Tao-1 activity. Further studies will be required to answer these questions and to determine if, and how, Tao-1 activity is regulated (Boggiano, 2011).

An interesting aspect of the discovery that Tao-1 regulates HSW signaling is that Tao-1, and its mammalian orthologues TAOK1-3, have been shown to regulate MT stability. The current results indicate that this effect on MT stability is not mediated through HSW signaling, since mutations in other HSW pathway components do not display similar MT phenotypes. However, it is interesting to speculate that Tao-1's association with MTs might affect its ability to regulate HSW pathway activation. More work will be required to determine whether the function of Drosophila Tao-1 in HSW signaling is entirely independent of its role in microtubule dynamics, though a recent study in mammalian cultured cells found that microtubule disruption did not affect localization of Yap, a mammalian Yki ortholog, suggesting that in mammalian cells these roles might be independent (Boggiano, 2011).

An additional possible mechanistic link between Tao-1 and HSW signaling is suggested by studies in flies and in mammalian cells indicating that Par-1, a polarity protein, is positively regulated by Tao-1. Par-1 has been shown to promote basolateral polarity in the Drosophila follicular epithelium and to regulate the stability and organization of MTs in these cells. Recent studies have implicated components of both apical and basolateral polarity in the regulation of HSW signaling. Conversely, HSW signaling also seems to feed back onto Crumbs, an apical determinant, and perhaps other components to regulate apical-basal polarity. Whether Tao-1 plays a role in the linkage between cell polarity and growth control remains to be established, but the ability to both directly activate Hpo function through phosphorylation and control cytoskeletal organization and cell polarity through microtubule organization potentially places Tao-1 in a unique position to coordinate these important cellular processes (Boggiano, 2011).

Crumbs promotes expanded recognition and degradation by the SCFSlimb/beta-TrCP ubiquitin ligase

In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. This study shows that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. Skp/Cullin/F-boxSlimb/beta-transducin repeats-containing protein (SCFSlimb/beta-TrCP) was identifed as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function (Ribeiro, 2014).

Recent work in flies and mammals has implicated the apical polarity determinant Crb as a transmembrane receptor for Hpo signaling. Accordingly, clonal loss of crb function leads to Yki derepression and increased growth. However, the observation that Crb is required for Ex membrane localization apparently conflicts with the finding that Crb^intra overexpression reduces Ex levels and, like crb loss of function, leads to increased Yki activity. This study reconciles these findings by showing that Crb is not only required for Ex tethering at the apical membrane but also for promoting its degradation via the SCF^{Slimb/β-TrCP} E3 ubiquitin ligase. Indeed, immediately downstream of its FERM domain, Ex contains a sequence that conforms to the D/S/TSGφXS consensus sequence for canonical Slmb targets, which is conserved in Ex orthologs from arthropod species but absent from related FERM domain proteins such as Moe and Mer. In addition, loss of Slmb increases Ex levels in vivo, whereas Slmb depletion prevents Crb^intra-induced Ex degradation in cell culture. Thus, in ^crb mutants, Ex no longer reaches the apical membrane and is protected from degradation in the cytoplasm, where it accumulates but is presumably unable to repress Yki. When Crb^intra is overexpressed, Ex turnover at the membrane (or in an endocytic compartment if Ex degradation occurs after Crb internalization) is accelerated, leading to its depletion and consequent Yki activation. Therefore, in both cases, the outcome is Yki derepression, albeit for different reasons (Ribeiro, 2014).

How is the Slmb:Ex association regulated by Crb? Previous observations point to the involvement of a phosphorylation-dependent degradation mechanism, because Crb induces Ex phosphorylation. Accordingly, mutation of the phosphodegron (Ser⁴⁵³) or the putative priming site (Ser⁴⁶²) leads to loss of Slmb:Ex association and Ex stabilization. The most obvious candidate Ex kinase in this context is aPKC, which is known to interact with the Crb polarity complex and to phosphorylate Crb at its FBM. However, aPKC is thought to be recruited to the Crb complex via Par-6, which interacts with Crb through the PBM at the C terminus of the Crb intracellular domain. This is inconsistent with the fact that the PBM is dispensable for Crb to promote both Ex phosphorylation and degradation. Moreover, a dominant-negative version of aPKC is unable to rescue the overgrowth phenotype of Crb^intra overexpression in the wing. The Hpo downstream kinase Wts is another attractive candidate, because mammalian LATS1/2 promotes YAP phosphorylation on Ser³⁸¹, providing priming for phosphorylation by CK1δ/ε and triggering degradation by SCF^β-TrCP. However, neither Ser⁴⁶² nor Ser⁴⁵³ conform to the Wts/LATS consensus. The identity of the kinases therefore remains open (Ribeiro, 2014).

Regulation of tissue growth during development and adult life depends on the maintenance of tissue architecture, which, in turn, relies on cell-cell and cell-matrix interactions. Due to its intimate coupling to polarity and cytoskeletal regulators, the Hpo signaling pathway is thought to sense epithelial integrity and couple tissue architecture to growth control. In particular, YAP has been shown to sense contact inhibition in cell culture, such that its progrowth activity is silenced as cultured epithelial cells reach confluence. This is thought to depend on the assembly of tight junctions, leading to repression of YAP by the mammalian Crb complex. Recent work in flies and zebrafish has suggested that Crb can form homodimers in trans. This suggests that Crb mediates local cell-cell communication in epithelial tissues. Indeed, clonal loss of Crb leads to Crb depletion in the junctions of wild-type cells abutting the crb mutant tissue. This loss of Crb at the clone boundary is mirrored by loss of Ex, which is dependent on Crb for its apical localization. Thus, loss of Crb caused by loss of polarity or cell death can be transmitted to neighboring cells. This has been proposed as a means of cell- cell communication to induce regenerative proliferation through Yki activation. Indeed, genetic induction of epithelial wounds in imaginal discs has been shown to up-regulate Yki activity in cells neighboring the wound. In addition, Yki/ YAP is activated and promotes tissue regeneration upon injury in the vertebrate and fly intestine, as well as in the mouse liver (Ribeiro, 2014).

These findings suggest that Crb (and perhaps other polarity proteins) functions as a sensor of cell density and tissue integrity during development. In this model, disruption of Crb function would lead to Yki/YAP derepression, which, upon tissue injury, would allow regenerative growth to ensue. Another interesting question is whether liganded Crb behaves differently to unliganded Crb with respect to regulation of Ex stability. For example, it is possible that unliganded Crb promotes Ex turnover faster than its liganded counterpart, which might provide a sensitive means of responding to the status of neighboring cells. Further work will be needed to resolve this issue. The present work indicates that Crb fulfills a dual function in Hpo signaling, both recruiting Ex apically to repress Yki activity and promoting Ex turnover through phosphorylation and Slmb-dependent degradation. This mechanism could ensure constant turnover of Ex at the apical membrane, allowing Yki activity to rapidly respond to changing environmental conditions. This dynamic equilibrium could be particularly important to promote fast tissue regeneration upon injury (Ribeiro, 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).

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).

DEVELOPMENTAL BIOLOGY

Larval

Antibodies generated against the Ex protein were used to detect its expression in imaginal discs. The Ex protein is detected by early third instar. Expression is relatively uniform throughout leg discs, but is intensified in the presumptive wing pouch relative to elsewhere in wing disc. Sections of stained mature third instar discs show that the protein is localized to the extreme apical cell surface. EX mRNA also appears to be apically localized in whole-mount and sectioned discs that were hybridized with digoxigenin-labelled probe from the first ex exon (Boedigheimer, 1993).

The Drosophila expanded gene encodes a product that shares homology with the Protein 4.1 family of proteins, many of which are enriched at specific lateral cell junctions and the apical cellular domain. Ex colocalizes with actin in the apical domain of imaginal disc epithelial cells, where it partially overlaps the distribution of phosphotyrosine (PY)-containing proteins. This suggests that Ex is present in or associated with adherens junctions (Boedigheimer, 1997).

EFFECTS OF MUTATION

The expanded gene was first identified by a spontaneous mutation that causes broad wings. An enhancer-trap insertion has been identified within expanded and it was used to generate additional mutations, including one null allele. expanded is an essential gene, necessary for proper growth control of imaginal discs and, when mutant, causes either hyperplasia or degeneration depending on the disc. Wing overgrowth in expanded hypermorphs is limited to specific regions along the anterior-posterior and dorsal-ventral axis (Boedigheimer, 1993).

The ex mutant phenotype is incompletely penetrant and expressive. Weak mutant alleles, such as ex¹, generally show only a broad wing phenotype; occasionally, the wings have an upward or downward arc. Intermediate mutants such as ex⁶⁹⁷ or mutant combinations such as ex^l(2)ey/ex⁶⁹⁷ display phenotypes affecting legs, thorax, head and wings. Occasionally, entire legs are missing, but more commonly, legs are kinked or have swollen distal tarsal segments, with completely separated internal vesicles of unknown origin and composition. In intermediate mutant combinations such as ex^l(2)ey/ex⁶⁹⁷, about 50% of the individuals display leg defects. In these flies, only one or two legs are typically affected. The thoracic defects include duplication of scutellar bristles and sensilla on the wing. Defects in the head capsule are variable. Eyes are reduced in size and occasionally split. Duplicated antennae or vibrissae occur in about 50% of ex^l(2)ey/ex⁶⁹⁷ heterozygotes, apparently at the expense of peripheral eye tissue. In addition to being broad, the wings are arced down and have incomplete crossveins (Boedigheimer, 1993).

Compared to the intermediate alleles, ex^e1 pharate adults display a highly penetrant and expressive phenotype. ex^e1 mutants survive until the pharate adult stage and show massive head, wing and leg defects. All ex^e1 pharate adults have leg defects and usually all legs of an individual are defective. Some legs are entirely missing, but the most common defect is missing distal tarsal segments including the claw organs. The legs usually terminate beyond the second tarsal segment. The remaining proximal tarsal segments have supernumerary bristles. The antennae are enlarged and are missing aristae, but otherwise appear normal. The reduction of eye tissue seen in intermediate mutant alleles is exaggerated in ex^e1 pharate adults, such that eyes fail to differentiate ommatidia. Unlike intermediate mutants, antennal duplications do not generally occur (Boedigheimer, 1993).

To understand better the function of ex, the phenotype of null mutants was examined at earlier stages. No visible defects are seen in ex^e1 mutant embryos and they hatch at a rate comparable to wild type. The first obvious defects occur in the imaginal discs at about mid-third instar, by which time the wing disc is noticeably enlarged. The wing disc continues to grow, mainly in the presumptive wing pouch region and, by day five, begins to form an extra fold approximately at the anterior-posterior compartment boundary. Growth continues during an extended larval period (2-3 extra days in uncrowded conditions), usually until the wing disc reaches double the wild-type size, although occasionally continuing until it is many times the size of wild type. Overgrowth also occurs in the haltere disc. In the eye-proper and the leg discs, degeneration is visible. The eye region of the eye-antennal disc is largely atrophied, presumably due to a lack of ommatidial development, as evidenced by the lack of a morphogenic furrow or organized photoreceptor cells and by the missing eye in pharate adults. The presumptive head capsule is still intact. The leg discs appear to be lacking distal segments. All discs examined retained a single-cell layer epithelium (Boedigheimer, 1993).

Since each cell in the wing blade is associated with a single hair, the hairs can serve as markers for cell number and position. An examination of ex hypomorphic mutant wings reveal that the increased size of the wing is due to hyperplasia in two regions of the wing. In one of these regions, there is an increase in cell density, an elongation of cells in the broad axis of the wing and a greater elongation in dorsal surface than ventral surface. To analyze their shape, the wings were aligned along a wing vein and scaled to the same size. After scaling, all mutant wings were essentially superimposable (as are wild type), showing that the mutant wing phenotype is highly reproducible. This was true regardless of the heteroallelic combinations tested, allowing a comparison of wing shape to be made between scaled mutant and wild-type wings. Moreover, the overgrowth phenotype is completely recessive, indicating that overgrowth is not due to dominant allele-specific affects. The shape of ex mutant wings is broader than wild type, and rounder at the tip. The distal tip of wild-type wings occurs where L3 meets the margin, whereas the distal tip of ex wings is at the margin between L3 and L4. The sizes of intervein regions were compared separately between wild-type and ex mutant wings. This analysis reveals that only specific regions of the wing are larger in expanded mutants. The area between L1 and L2, and between L4 and L5 are increased the most. The area bounded by L3, L4 and the anterior crossvein is not significantly different in the mutant. The posterior crossvein is about the same length in mutants and wild type and does not span the intervein region in ex mutants. Thus, it is possible that the overgrowth is limited to a region anterior to the incomplete crossvein. From this analysis, it cannot be determined whether overgrowth is limited in the proximal distal axis. To determine whether overgrowth is spatially limited in null mutants, adult wings were dissected from pharate adults and carefully flattened. There is considerably more variation in shape among mutant pharate wings. The mutant pharate wings are generally larger than wild type with excessive growth between L1 and L2, and between L4 and the margin. These results indicate that regional hypertrophy occurs in complete as well as partial loss-of-function ex mutations (Boedigheimer, 1993).

Hair distribution was used to examine the shape and distribution of cells in wild type and ex^l(2)ey/ex⁶⁹⁷ in the most severely affected region, between L4 and L5. The density of hairs on mutant wings is increased and the hairs are more disorganized than on wild-type wings. The ratio of the average distance between hairs in the broad and long axis of the wing is larger in the mutant than in wild type. The ratio in wild-type wings is the same for the dorsal and ventral wing blade surfaces. However, in mutants the ratio is larger on the dorsal surface than on the ventral surface. The abnormal hair distribution indicates that, in this region, the cells in ex mutants are elongated along the broad axis of the wing. This type of cell elongation does not occur in the region between L5 and the margin in ex^l(2)ey/ex⁶⁹⁷ mutants (Boedigheimer, 1993).

An average increase of 0.7 cell divisions per cell occurs in the region between L4 and L5. This is a minimum estimate, since it is quite possible that increased cell death occurs in ex mutants, as has been described for other tumor suppressor mutants. Also, ex^l(2)ey/ex⁶⁹⁷ is a viable allelic combination, and a qualitative difference in size exists between mutant wing discs from this allelic combination and null alleles. Based on visual examination of mutant wing discs from null mutants and not accounting for possible cell death, it is estimated that the increase in cell divisions is slightly greater than 1 (Boedigheimer, 1993)

Genetic studies show that Ex is necessary for proper regulation of final cell number in adult wings and for the formation of eyes, distal leg, and distal antennal segments. Mitotic clones that lack Ex were generated using the twin spot technique, and it was demonstrated that the primary function of Ex is to regulate cell proliferation. Overexpressing Ex protein results in a decrease in final cell number in wings, suggesting a direct relationship between Ex function and proliferation rate (Boedigheimer, 1997).

Hypomorphic alleles of either Mer (Mer³) or ex (ex⁶⁹⁷) result in very similar adult wing and eye phenotypes. In both cases, mutant adults display enlarged wings due to an increase in cell number rather than an increase in cell size. In fact, the cell size in the mutants appears to be slightly decreased. Furthermore, this expansion in wing area is often accompanied by the disruption or complete absence of the posterior cross vein. In addition, the anterior cross vein is sometimes disrupted in ex⁶⁹⁷ adults. Mer and ex mutants have smaller, weakly roughened eyes. Histological sections of Mer³ eyes reveal only minor perturbations in interommatidial organization and no obvious disruptions in ommatidial polarity. Concomitant with the reduction in eye size is an apparent expansion of the ventral peripheral head cuticle and the development of ectopic vibrissae. Although stronger alleles of both Mer and ex (ex^e1, a null allele) result in lethality, Mer mutant larvae do not develop the hyperplastic discs characteristic of ex mutant larvae (McCartney, 2000).

Dose-sensitive genetic interactions have been shown to be a reliable indicator of functional interactions between genes. Reduction of ex function in the Mer³ hemizygous background with either ex⁶⁹⁷ or ex^e1 results in an enhancement of the Mer³ head phenotypes; the adult eye is reduced in size, more ectopic head cuticle and vibrissae are observed, and the bristles normally found between ommatidia are often duplicated and disorganized. Those ommatidia that form contain the normal complement of photoreceptors, however. In addition, Mer³ wing area and the frequency of posterior cross vein disruptions increase when ex function is reduced. Consistent with these observations, reduction of Mer function in ex⁶⁹⁷ mutants causes an increase in ex⁶⁹⁷ wing size (McCartney, 2000).

Previous studies indicate that loss of function of either Mer or ex in clones results in a 2- to 3-fold overproliferation of the mutant tissue compared with the wild-type twin spot. In the wing, loss of either Mer or ex alone in clones has no apparent effect on the differentiation and morphology of the affected tissue. Similarly, in the eye, loss of function of Mer results in overproliferation without obvious changes in the underlying morphology. In contrast, loss of ex function in the eye results in defects in planar polarization, in addition to proliferation defects (Blaumueller, 2000). Because the loss of function of either Mer or ex results in overproliferation, the consequences of loss of function of both genes were examined using somatic mosaic analysis. In the adult wing, both vein and intervein cells differentiate in mutant tissue. In contrast, clones that intersect the position of the posterior cross vein disrupt its development, consistent with the variably penetrant disruption of the posterior cross vein observed in hypomorphic alleles of Mer or ex. Clones in the position of the anterior cross vein differentiate normally. Within the mutant intervein and vein clones, apparent defects were observed in proliferation control. In the proximal region of the wing, clonal vein tissue forms a raised protrusion. In other regions of the wing, bulges in the veins are also observed, although more frequently vein clones are merely broadened when compared with the surrounding vein. In the intervein regions, the clonal tissue appears to bulge and crinkle within the confines of the normal tissue, suggesting overproliferation. Similar cuticular bulges or protrusions have been reported for mutant clones of genes that have tumor suppressor phenotypes, such as warts. Thus this phenotype is interpreted to indicate that the Mer;ex double mutant clones in the wing proliferate at a greater rate than the single mutant clones, though this could not be confirmed directly. Based on general morphology, the cells within the clone appear to differentiate as intervein cells, however, the cuticle deposited at the base of each wing hair within the mutant clone appears to be thickened and is distinct from cuticle produced by either the heterozygous intervein or vein cells. Clones that develop within the eye appear either as small scars with associated clusters of bristles, or as elongated scars and associated indentations running from within the eye field toward the anterior margin. Although these clones do not differentiate ommatidia, the position of the twin spot was used to indicate the position of the mutant clone. Mutant clones are often associated with overproliferated head cuticle (McCartney, 2000).

Reduction of dpp function in the eye imaginal disc (dpp^blk) results in reduction of the eye along the dorsoventral axis such that the ventral portion of the eye is replaced by head cuticle. A similar, yet less severe, phenotype is observed in Mer³ hemizygotes and ex⁶⁹⁷ homozygotes. To ask whether dpp expression is disrupted in Mer mutants, a dpp-lacZ transgene was ovexpressed in the Mer³ background. In the wild-type eye-antennal complex, dpp is expressed at the lateral margins and in the morphogenetic furrow of the eye disc and in a ventral wedge of tissue in the antennal disc. In the Mer³ mutants, the ventral portion of the disc is significantly enlarged. The expression pattern of dpp is disrupted such that the cells expressing dpp at the margin are displaced to the outer tip of the overproliferated tissue. In some cases, this dpp staining is associated with an ectopic furrow and developing photoreceptors. To better understand the functional relationship between dpp, Mer and ex, genetic interactions were examined between these genes. Reduction of dpp dose in Mer³ hemizygotes enhances the severity of the Mer³ eye phenotype, resulting in a smaller, more roughened eye and expansion of the head cuticle. Similar reduction of dpp function in ex⁶⁹⁷ homozygotes results in enhanced eye phenotypes and variably penetrant truncated leg phenotypes, reminiscent of those observed in pharate adults null for ex function. Although it is appealing to think that the effects of Mer and ex on patterning and proliferation are both mediated through the DPP pathway, this seems unlikely given that loss of either gene seems to negatively affect DPP patterning functions, but simultaneously causes overproliferation of mutant cells. It therefore seems more likely that the proliferation phenotypes of Mer and ex loss-of-function mutations are mediated through effects on one or more other pathways that regulate proliferative events (McCartney, 2000).

In addition to leading to subtle patterning defects, loss of ex results in dramatic growth abnormalities in the eye. This is evident both in clones and in discs from homozygous null animals. Homozygous mutant clones of ex^e1 tissue have a significant growth advantage over their wild-type counterparts in larval discs. Similar effects are evident in pupal discs and in adult eyes. In the cases of large mutant clones, the tissue protrudes out of the plane of the disc. Whereas it was not possible to do cell counts in these clones, the increased number of ommatidia within the mutant clone relative to the wild-type twin-spot and the relatively normal architecture of the ommatidia implies an increase in cell number within clonal tissue. In discs isolated from homozygous mutant larvae, the effects on growth are even more extreme. The eye discs are disproportionately large in comparison with the antennal discs of the same complex, reaching several times the size of those of wild-type larvae. Anterior regions of homozygous mutant discs also tended to lose their 'flat' character, leading to the formation of additional tissue flaps. These data demonstrate that ex acts as a negative regulator of growth in the eye as in the wing. As is the case in clones, eye patterning initiates relatively normally in discs from ex null animals. Stainings with markers for the morphological differentiation of eye tissue and for neuronal differentiation demonstrate that the morphogenetic furrow moves across mutant tissue, and that cell fate determination takes place. In summary, loss-of-function data from the developing eye disc indicate that ex plays an important role as a negative regulator of growth in the eye disc, and also affects patterning and differentiation at the levels of establishing cell fate and planar polarity (Blaumueller, 2000).

Delineation of a Fat tumor suppressor pathway

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, 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).

The tumor suppressors Merlin and Expanded function cooperatively to modulate receptor endocytosis and signaling

The precise coordination of signals that control proliferation is a key feature of growth regulation in developing tissues. While much has been learned about the basic components of signal transduction pathways, less is known about how receptor localization, compartmentalization, and trafficking affect signaling in developing tissues. This paper examines the mechanism by which the Drosophila Neurofibromatosis 2 (NF2) tumor suppressor ortholog Merlin (Mer) and the related tumor suppressor expanded (ex) regulate proliferation and differentiation in imaginal epithelia. Merlin and Expanded are members of the FERM (Four-point one, Ezrin, Radixin, Moesin) domain superfamily, which consists of membrane-associated cytoplasmic proteins that interact with transmembrane proteins and may function as adapters that link to protein complexes and/or the cytoskeleton. Merlin and Expanded function to regulate the steady-state levels of signaling and adhesion receptors, and loss of these proteins can cause hyperactivation of associated signaling pathways. In addition, pulse-chase labeling of Notch in living tissues indicates that receptor levels are upregulated at the plasma membrane in Mer; ex double mutant cells due to a defect in receptor clearance from the cell surface. It is proposed that these proteins control proliferation by regulating the abundance, localization, and turnover of cell-surface receptors and that misregulation of these processes may be a key component of tumorigenesis (Maitra, 2006).

Merlin's tumor suppressor function is conserved from humans to flies, but the cellular basis for this function remains unclear. Genetic studies in Drosophila suggest that Mer regulates signaling pathways that control proliferation, and cell biological experiments indicate that Merlin may play a role in endocytic processes. In addition, Merlin physically interacts with Expanded, a distantly related member of the FERM superfamily, and these proteins colocalize in the apical junctional region of epithelial cells. Furthermore, genetic studies have shown that while mutations of each gene produce modest overproliferation phenotypes in the eye and wing, double mutant Mer; ex cells display severe overgrowth and differentiation defects that are not seen in either mutation alone. Thus, Mer and ex are partially redundant in regulating proliferation and differentiation (Maitra, 2006).

Given these observations, it was reasoned that the difficulty in identifying precise cellular functions for Merlin might stem from its redundancy with Expanded and that this difficulty could be overcome by examining tissues from double mutant animals and double mutant cell clones generated by somatic recombination. Overproliferation of Mer; ex wing imaginal discs is more extreme than that observed with either mutation alone. Surprisingly, however, Mer⁴; ex⁶⁹⁷ eye-antennal imaginal discs have severely reduced eye primordia with a substantial reduction in or total absence of photoreceptors, although the antennal portion is normal or slightly larger than normal and occasionally is duplicated. Apoptosis does not appear to be enhanced in double mutant eye-antennal discs, suggesting that loss of the eye primordium is not due to cell death. Thus, loss of Mer and ex function has a tissue-specific defect in the developing eye that is very different from its effects on proliferation in the wing imaginal disc (Maitra, 2006).

Why does the combined loss of two tumor suppressors cause reduction rather than hypertrophy of eye tissue? Previous studies have shown that initiation of the morphogenetic furrow, which organizes development of the eye, is regulated by a complex network of signals at the posterior and lateral margins of the eye-antennal disc. Mutations that affect these signals not only block furrow initiation, but also may significantly reduce the size of the eye field and disrupt photoreceptor differentiation. For example, ectopic Wingless expression either at the posterior and lateral margins or throughout the eye primordium results in dramatic losses of eye tissue that closely resemble the Mer; ex phenotype just described. Similar effects are seen from reduction in Decapentaplegic (DPP) or Hedgehog signaling in the same cells (Maitra, 2006).

If Merlin and Expanded affect initiation of the morphogenetic furrow rather than differentiation of photoreceptors, then Mer; ex double mutant somatic clones should block ommatidial development only when present at the posterior or lateral margins of the eye field. Indeed, Mer; ex clones could differentiate photoreceptors, but only when located in the middle of the eye field. In contrast, clones in contact with the posterior or lateral margin of the eye fail to produce photoreceptors. It is inferred from these observations that one or more of the signaling pathways that control initiation of the morphogenetic furrow are likely disrupted in Mer; ex double mutant cells (Maitra, 2006).

Given that Merlin is associated with the plasma membrane and may function in endocytic processes, it was asked if Merlin and Expanded play a role in regulating localization and/or abundance of transmembrane receptors that function in eye development. For these studies, Mer; ex somatic mosaic cell clones were examined to allow side-by-side comparisons of wild-type and mutant cells in the wing and eye imaginal discs. Immunofluorescence staining with specific antibodies then allowed comparison of the steady-state levels of receptors between adjacent wild-type and mutant cells. Intriguingly, Notch, the EGF receptor, Patched, and Smoothened all displayed increased antibody staining in double mutant cells relative to their wild-type neighbors. Notch, which is primarily localized to the apical junctional domain in wild-type cells, showed not only increased junctional staining in mutant cells, but also more diffuse staining. Similarly, preparations with anti-EGFR display more abundant membrane-associated and cytoplasmic staining in mutant than in wild-type cells. Patched staining, which is less obviously junctional than Notch or EGFR, appeared more punctate in Mer; ex cells. Thus, simultaneous loss of Merlin and Expanded results in increased abundance of receptors for multiple signaling pathways, though the precise localization defect seems to be specific to each receptor. Two adhesion-related receptors, E-cadherin and Fat, a cadherin superfamily member, were examined; both are similarly upregulated in Mer; ex cells. However, Coracle, a membrane-associated cytoplasmic protein, is not affected. In addition, the localization of markers for apical-basal polarity, including DLG, PATJ, and aPKC, was unaffected in the double mutant cells, indicating that epithelial polarity is not disrupted. In contrast to the double mutant cells, clones lacking just Merlin show no apparent difference in receptor localization or abundance, and ex^e1 cells display only a slight increase in staining. Taken together, these results indicate that Merlin and Expanded are required to reduce the steady-state abundance of a variety of signaling and adhesion receptors in developing epithelia (Maitra, 2006).

Membrane trafficking was examined in Mer; ex double mutant cells. Antibodies were used against the extracellular domain of Notch (anti-ECN) to label protein on the surface of living cells in imaginal discs bearing somatic mosaic clones. Side-by-side comparisons of wild-type and Mer; ex mutant cells show increased cell-surface Notch labeling, consistent with what was observed with fixed tissue and indicating that there are increased levels of receptor at the plasma membrane in mutant cells. In addition, in double mutant cells, the junctional band of Notch staining is broader, indicating that Notch localization to the junctional region also may be affected. Similar differences in junctional staining were observed with the same antibody on fixed and permeabilized tissues, indicating that surface labeling of live cells does not affect Notch localization (Maitra, 2006).

To ask if the increased abundance is due to a defect in turnover, a pulse-chase approach was used to label Notch receptor at the plasma membrane and then its removal from the cell surface was followed. To restrict analysis to Notch that remains at the cell surface, tissues were fixed but not permeabilized at the end of the chase period. A progressive loss was observed of Notch staining at the cell surface during the chase period that appeared more rapid in wild-type than in mutant cells, suggesting a defect in trafficking off the plasma membrane. Quantitative fluorescence analysis was used to determine the relative quantities of Notch on wild-type and mutant cells at the various chase time points. The results indicate that the ratio of cell-surface Notch fluorescence in mutant versus wild-type cells increases significantly between 0 and 10, 30, or 60 min postlabeling. Therefore, Notch protein is cleared more rapidly from the surface of wild-type than mutant cells (Maitra, 2006).

It is worth noting that current models for Notch receptor activation require cleavage and release of its extracellular domain in response to ligand binding. Because an antibody was used that recognizes this domain, it follows that these studies examined only ligand-independent trafficking of the receptor. In support of this inference, the pattern of Notch internalization in pulse-chase experiments was unaffected in Delta⁻ clones. These observations suggest that Merlin and Expanded function in steady-state, ligand-independent clearance of receptors from the plasma membrane, rather than internalization and degradation that occurs in response to ligand binding (Maitra, 2006).

Increased receptor abundance may be expected to result in increased signaling output, if receptor quantity is a limiting factor. In addition, even if overall receptor quantity is not limiting, alterations in subcellular localization or the dynamics of receptor trafficking may have dramatic effects on receptor function. To ask if loss of Merlin and Expanded result in increased output from signaling pathways that regulate eye development and cell proliferation, markers specific for downstream activation of the EGFR, Wingless, and Notch signaling pathways were used. First, double mutant clones were stained with an antibody that recognizes the phosphorylated, activated form of MAP kinase (anti-dpERK), a downstream effector of the EGFR pathway. In addition to the normal anti-dpERK pattern in the wing imaginal disc, increased staining was observed in Mer; ex clones relative to their wild-type neighbors, suggesting upregulation of EGFR pathway activity. Similarly, output from the Wingless pathway was monitored by looking at expression of Distalless, a target of Wingless signaling and it was found to be dramatically higher in the double mutant wing clones. In contrast, similar experiments with the mAb323 antibody to E(spl) bHLH proteins, a marker for Notch pathway activity, did not show upregulation of Notch signaling. This result is consistent with the observation that overexpression of Notch in a wild-type genetic background has little or no phenotype. To examine this further, a genetic context was analyzed in which Notch receptor quantities are known to be limiting, that is, in animals that are heterozygous for a null Notch mutation. Such animals display a dominant, haploinsufficient phenotype characterized by notching along the wing margin. To ask if reduction in Merlin and Expanded in this context can cause upregulation of Notch pathway output, animals triply heterozygous for Notch, Merlin, and expanded were generated and it was found that the characteristic Notch wing phenotype was strongly suppressed (Maitra, 2006).

Taken together, these results are consistent with the observation that the steady-state level of multiple receptors is elevated in Mer; ex cells and indicate that, depending on the precise developmental or genetic context, loss of Merlin and Expanded can result in increased output from the corresponding signaling pathways. In Mer; ex eyes, upregulation of Wingless signaling may be a primary contributor to the observed defect in ommatidial development. Previous studies have shown that ectopic Wingless signaling produces remarkably similar eye phenotypes, and preliminary data suggest that inhibiting Wingless signaling partially suppresses the Mer; ex eye phenotype. In the wing, the dramatic overproliferation of Mer; ex cells may be the combined result of upregulation of several pathways, including EGFR and Wingless (Maitra, 2006).

Merlin and Expanded are associated with the apical junctional region in imaginal epithelia and with endocytic vesicles in cultured cells. Results shown in this study indicate that loss of these proteins affects abundance, cell-surface localization, and endocytic trafficking of Notch, EGFR, and other signaling and adhesion receptors in epithelial cells. Recent studies of endocytic trafficking in receptor/ligand regulation suggest aspects of endocytosis that could relate to Merlin and Expanded function. For example, it is possible that Merlin and Expanded function at the plasma membrane to recruit or anchor transmembrane proteins at sites on the membrane from which they are endocytosed or in the sorting between recycling endosomes and lysosomal degradation by promoting receptor degradation. Both possibilities are consistent with observations of increased receptor levels at the plasma membrane in Mer; ex mutant cells and colocalization of Merlin and Expanded with Notch in punctate structures at the plasma membrane. In addition, a partial colocalization was observed of Merlin and Expanded with Rab 11, a marker for recycling endosomes, and with EEA-1, which labels early endosomes. Intriguingly, it has been suggested that the closely related ERM protein Ezrin functions to promote recycling rather than degradation of the β2-adrenergic receptor via its interactions with filamentous actin. Understanding the exact relationship of Merlin and Expanded to endocytosis and recycling of receptors, as well as their possible relationship to ERM proteins in this process, will require further analysis (Maitra, 2006).

A recent study has proposed that Merlin and Expanded function upstream of Hippo in the Warts signaling pathway, which regulates proliferation. Merlin and expanded mutants display similar phenotypes to those seen in hippo mutants. However, there are significant phenotypic differences between Mer; ex and hippo mutations, most notable of which is that hippo mutations have not been reported to block induction of eye morphogenesis. In addition, there is no evidence to suggest that the Hippo pathway regulates output of the EGFR, Wingless, or Notch signaling pathways. Thus, the relationship of Merlin and Expanded to the Hippo pathway may be more complicated than the linear pathway proposed. One possibility is that Hippo activation is a downstream consequence of Merlin and Expanded's effects on output of multiple signaling pathways (Maitra, 2006).

More than a decade after its molecular characterization, the precise cellular functions of Merlin in regulating cell proliferation remain unclear. Based on the current studies, it is proposed that Merlin's tumor suppressor phenotype results from defects in endocytic trafficking of signaling receptors and accompanying hyperactivation of associated signaling pathways. Recent studies highlight the importance of endocytosis in regulation of signaling pathways. Based on the results presented in this study, it is suggested that proper regulation of membrane trafficking also may have important implications for understanding the cellular basis of tumor suppression in flies and mammals (Maitra, 2006).

The salvador-warts-hippo pathway is required for epithelial proliferation and axis specification in Drosophila

In Drosophila, the body axes are specified during oogenesis through interactions between the germline and the overlying somatic follicle cells. A Gurken/TGF-alpha signal from the oocyte to the adjacent follicle cells assigns them a posterior identity. These posterior cells then signal back to the oocyte, thereby inducing the repolarization of the microtubule cytoskeleton, the migration of the oocyte nucleus, and the localization of the axis specifying mRNAs. However, little is known about the signaling pathways within or from the follicle cells responsible for these patterning events. It study shows that the Salvador Warts Hippo (SWH) tumor-suppressor pathway is required in the follicle cells in order to induce their Gurken- and Notch-dependent differentiation and to limit their proliferation. The SWH pathway is also required in the follicle cells to induce axis specification in the oocyte, by inducing the migration of the oocyte nucleus, the reorganization of the cytoskeleton, and the localization of the mRNAs that specify the anterior-posterior and dorsal-ventral axes of the embryo. This work highlights a novel connection between cell proliferation, cell growth, and axis specification in egg chambers (Meignin, 2007).

Multicellular organisms develop through an orchestrated temporal and spatial pattern of cell behavior, which is controlled by cell-to-cell signaling. In Drosophila melanogaster, the establishment of the embryonic axes occurs in the oocyte and depends on a sequence of signals between the germline and the somatic cells. First, Gurken (Grk) signals from the oocyte to the adjacent follicle cells (FCs), in which Torpedo (Top, EGFR) is activated, and this signal instructs them to adopt a posterior identity. The posterior FCs (PFCs) then send an unidentified signal back to the oocyte, leading to the movement of the nucleus from the posterior to the dorsoanterior (DA) corner and the repolarization of the microtubule (MT) cytoskeleton, with the minus ends at the anterior and lateral cortex and the plus ends at the posterior. This repolarization results in the localization of the mRNAs that encode key patterning factors. grk mRNA is next to the nucleus at the DA corner of the oocyte. At this corner, Grk instructs the overlying FCs to adopt dorsal fates. In contrast, oskar (osk) and bicoid (bcd) mRNAs are localized at the posterior and anterior pole, respectively, thus defining the anterior posterior (AP) embryonic axis and the germ cells. Although several genes are required in the FCs to control these events, little is known about the signaling pathways within and from the FCs (Meignin, 2007).

One of the genes required for axis formation during oogenesis is the tumor suppressor merlin (mer). However, it is not known whether Mer influences axis specification directly or what signaling pathways lie downstream of Mer. In other tissues, Mer is known to activate the Salvador Warts Hippo (SWH) pathway, which is a tumor-suppressor pathway. Inhibition of the SWH pathway leads to a characteristic overgrowth phenotype in adult organs because of an overproliferation of cells, increased cell growth, and defects in apoptosis. To test whether the SWH pathway is required in the function of Mer in axis formation, the localization of grk, bcd, and osk mRNA was examined in egg chambers with warts (wts) and hippo (hpo) mutant FCs. wts and hpo encode two serine/threonine kinases that are core components of this pathway. In both cases, grk mRNA is mislocalized at the posterior, osk mRNA is mislocalized at the center, and bcd mRNA is mislocalized at the posterior and anterior poles. The mislocalization of these mRNAs could be due to failure of the MTs to repolarize, as has been previously shown in grk/EGFR and mer mutants. In wild-type oocytes, the MTs are organized in an AP gradient. In contrast, in egg chambers with hpo mutant FCs, the MTs are distributed diffusely all over the oocyte cytoplasm. Considering these results, together with previous characterizations of similar phenotypes, it is concluded that the oocyte cytoskeleton in mutant egg chambers for the SWH pathway is disorganized with the MT plus ends at the center and the minus ends at the anterior and posterior poles. These defects resemble those described in oocytes lacking the Grk signal. In wts mutants, however, Grk protein is detected at the posterior pole, where grk mRNA is mislocalized. This demonstrates that the axis-specification defects in wts mutant egg chambers are not a consequence of the absence of Grk protein (Meignin, 2007).

It was shown that mer is required in the FCs for the repolarizing signal back to the germline and consequently for the migration of the oocyte nucleus from the posterior to the DA corner. Similarly, when mutant FC clones were generated for wts, hpo, and expanded (ex), an activator of the SWH pathway, the oocyte nucleus fails to migrate to the anterior. Another protein that is upstream of the SWH pathway is the giant atypical cadherin fat (ft). However, egg chambers with ft mutant FCs show no defects in oocyte polarity, and both the nucleus and Staufen (Stau) [a marker for osk mRNA] are always properly localized. In other epithelia, hpo and wts are required to repress the activity of Yorkie (Yki) and overexpression of yki phenocopies loss-of-function mutations of hpo and wts. Similarly, it was found that overexpression of yki in the FCs also causes the mislocalization of Stau and the oocyte nucleus. These results indicate that the SWH pathway, with the exception of Ft, might be required for the repolarizing signal back from the FCs to the oocyte (Meignin, 2007).

Because this signal is sent by the PFCs, whether the SWH pathway is required only in these cells was analyzed. In egg chambers with wild-type PFCs within an otherwise hpo or wts mutant epithelium, as well as in hpo, wts, and ex germline clones, the oocyte polarity is unaffected. However, in egg chambers with hpo mutant PFCs in an otherwise wild-type epithelium, the oocyte nucleus is mislocalized. It was also observed that when only a few cells at the posterior are mutant, Stau localizes in the region of the oocyte that faces the posterior wild-type cells. The SWH pathway is not required in the polar cells for axis determination because egg chambers with hpo or wts mutant PFCs and wild-type polar cells show oocyte polarity defects. It is concluded that the SWH pathway is required only in the PFCs to induce axis specification in the oocyte (Meignin, 2007).

In contrast to the monolayered wild-type epithelium, anterior and posterior, but not lateral, hpo and wts mutant cells form a bilayered, and occasionally a multilayered, epithelium. Given that the SWH pathway is required to control proliferation in epithelia of imaginal discs, whether the bilayered epithelium is a result of overproliferation was analyzed. At stage 6 of oogenesis, wild-type FCs undergo a Notch-dependent switch from a mitotic cell cycle to an endocycle. For this reason, phosphohistone 3 (PH3), a marker for mitotic cells, is detected only until that stage and never later. In contrast, hpo mutant anterior and posterior FCs are often positive for PH3 at stage 7-10B, indicating that these cells are still dividing. Similar results are obtained in yki overexpressing FCs. Taken together, these findings show that the SWH pathway is required for the control of proliferation at the anterior and posterior FCs (Meignin, 2007).

The formation of a multilayered epithelium was also observed in stage 3-5 mutant FCs, although the number of dividing cells is similar to that of the wild-type. It has been recently shown that the aberrant orientation of the mitotic spindle in the FCs results in the formation of a multilayered epithelium. Therefore the orientation of the mitotic spindle was analyzed in wild-type and hpo mutant cells. It was observed that, contrary to wild-type cells, the mitotic spindle in mutant FCs is often at an angle or perpendicular to the membrane. This aberrant orientation disrupts the remaining daughter cells within the same plane, thereby resulting in a bilayered epithelium (Meignin, 2007).

Often, tumor suppressors are important for the polarity of the epithelia. To determine whether this is the case for the SWH pathway, the atypical (novel) Protein Kinase C (nPKC), an apical marker, and Disc large (Dlg), a lateral marker, were examined in the FCs. In wild-type cells, as well as in hpo mutant FCs that maintain a monolayer epithelium, nPKC and Dlg localize at the apical and lateral membrane, respectively. However, when the mutant epithelium forms several layers of cells, nPKC and Dlg are often mislocalized, with a reduction of the nPKC staining and an expansion of the Dlg-positive membrane. Nevertheless, a certain degree of the polarity in these cells is maintained because nPKC is always apical in the cells that are in contact with the oocyte (Meignin, 2007).

Because SWH pathway mutant cells do not exit mitosis and keep dividing, it is possible that their differentiation is impaired. To address this question, the expression of Fasciclin III (FasIII) and eyes absent (eya) were analyzed in wild-type and wts and hpo mutant FCs. FasIII and Eya are downregulated in a Notch-dependent manner in the main-body FCs after stage 6 of oogenesis. However, the levels of FasIII in hpo mutant PFCs and Eya in wts mutant PFCs remain high after stage 6. To further assess the effect of the SWH pathway on the Notch-dependent maturation of the FCs, the expression of Hindsight (Hnt), a transcription factor that is upregulated by Notch signaling in all FCs was examined.. In hpo posterior FC clones, this Hnt upregulation is blocked. Contrary to notch clones, however, hpo lateral and anterior clones do not show defects in FasIII, Eya, or Hnt expression. Furthermore, border, centripetal, and stretched cells that are mutant for hpo migrate normally. Considering all these results together, it is concluded that the SWH pathway is essential for the PFCs to fully differentiate (Meignin, 2007).

The findings described above, together with the proliferation defects in hpo and wts mutant cells, suggest that the SWH pathway is required for Notch signaling. To test whether this is the case, the expression of universal Notch transcriptional reporters was analyzed in wild-type and hpo mutant FCs. In wild-type egg chambers, the Notch reporter E(spl)mß7-lacZ is expressed in all FCs upon Notch activation at stage 6 of oogenesis. In contrast, it was found that in hpo mutant cells, the levels of E(spl)mß7-lacZ are weakly reduced in 53% of the clones and normally expressed in the rest. It has been shown that in wing imaginal discs, mer and ex are required to control Notch localization in the cell and consequently its activity. Similarly, the subcellular distribution of Notch is affected in hpo mutant FCs. Contrary to the wild-type, in which Notch accumulates in the apical membrane, Notch expands to other membranes and is often detected in clusters in hpo clones. The results point out that hpo is essential in the PFCs for the Notch-dependent expression of several differentiation markers, such as FasIII, Eya, and Hnt, and for Notch subcellular localization. These observations and the weak defects on the Notch reporters support a function of the SWH pathway in modulating Notch signaling (Meignin, 2007).

Because the SWH pathway is required for the polarization of the oocyte, as well as for the differentiation of the PFCs, whether the mutant cells are competent to respond to Grk and indeed adopt a posterior fate was analyzed. Dystroglycan (DG) is expressed in all FCs at early stages of oogenesis, but upon Grk signaling, DG forms an AP gradient with lower levels at the PFCs. The fact that this Grk-dependent gradient of DG is also observed in the hpo mutant epithelia suggests that the mutant cells are responsive to the Grk signaling. Similarly, when hpo clones affect only a portion of the PFCs, the posterior fate marker pointed is expressed as in the wild-type in 40% of the cases. However, in 60% of the egg chambers with partial hpo posterior clones, and in all cases when all the PFCs are mutant, the expression of pointed is abolished. These results illustrate that hpo is required to fully process the Grk/EGFR signal in the PFCs. Conversely, in grk mutant egg chambers, the Hpo-dependent expression of Hnt is not affected, suggesting that the EGFR pathway is not required for the activation of the SWH pathway in the PFCs (Meignin, 2007).

Considering all these results together, it is concluded that the SWH pathway is involved in the Notch- and Gurken-dependent maturation of the PFCs. Whether the SWH pathway modulates this maturation directly or indirectly, for example by affecting membrane properties, needs to be further investigated (Meignin, 2007).

To study whether the oocyte polarity defects in egg chambers with FCs mutants for the SWH pathway are a consequence of the FCs proliferation and differentiation defects, egg chambers with ex and ft mutant PFCs were analyzed. Egg chambers with ft PFCs occasionally form a bilayer, although they never have defects in oocyte polarity, suggesting that the morphological disruption of the epithelia in itself does not block the repolarizing signal. Egg chambers with ex PFCs show weak defects in the epithelium, with a bilayer rarely formed and restricted to only a few mutant cells, but Stau is never properly localized. However, Hnt is not properly expressed in stage 7 ex mutant FCs, suggesting that the mislocalization of Stau is a consequence of the ex mutant cells being undifferentiated at the stage when the repolarizing signal is sent to the oocyte. These results suggest that the defects in oocyte polarity are probably due to a lack of proper differentiation of FCs in SWH mutant egg chambers (Meignin, 2007).

This study has analyzed the requirement of the SWH pathway during oogenesis. Several of the components of this pathway, but not ft, are required in the PFCs to induce the axis specification in the germline. The defects in oocyte polarity, however, are probably due to a lack of proper differentiation of the PFCs in SWH mutant egg chambers. In addition, the pathway is required in the terminal cells to control their proliferation. It has already been shown that terminal follicle cells are different from lateral follicle cells. The distinct spatial requirement of the SWH pathway for differentiation and proliferation is another feature that distinguishes the terminal from the lateral FCs, and the posterior from the anterior FCs. These results point out that this dual function of the SWH pathway might be achieved by modulation of the Notch and EGFR signals. In conclusion, the SWH pathway lies at the intersection of two signaling pathways and is permissive for the signal that is sent from the follicle cells to repolarize the oocyte (Meignin, 2007).

Salvador-warts-hippo signaling promotes Drosophila posterior follicle cell maturation downstream of notch

The Salvador Warts Hippo (SWH) network limits tissue size in Drosophila and vertebrates. Decreased SWH pathway activity gives rise to excess proliferation and reduced apoptosis. The core of the SWH network is composed of two serine/threonine kinases Hippo (Hpo) and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). Two band 4.1 related proteins, Merlin (Mer) and Expanded (Ex), have been proposed to act upstream of Hpo, which in turn activates Wts. Wts phosphorylates and inhibits Yki, repressing the expression of Yki target genes. Recently, several planar cell polarity (PCP) genes have been implicated in the SWH network in growth control. This study shows that, during oogenesis, the core components of the SWH network are required in posterior follicle cells (PFCs) competent to receive the Gurken (Grk)/TGFβ signal emitted by the oocyte to control body axis formation. These results suggest that the SWH network controls the expression of Hindsight, the downstream effector of Notch, required for follicle cell mitotic cycle-endocycle switch. The PCP members of the SWH network are not involved in this process, indicating that signaling upstream of Hpo varies according to developmental context (Polesello, 2007).

Body axis formation is a critical stage of development in most multicellular organisms. In Drosophila melanogaster, the anteroposterior (AP) body axis is determined by the polarization of the developing oocyte. The egg chamber is composed of 16 germ cells (15 nurse cells plus the oocyte) and the follicular epithelium. Specification of the AP axis requires active transport of several mRNAs along the microtubule network, thereby resulting in asymmetric mRNA and protein localization inside the oocyte. For example, bicoid (bcd) and oskar (osk) mRNAs localize to and control the formation of the anterior and posterior poles, respectively. This process is initiated through bidirectional signaling between the oocyte and the adjacent follicle cells. In midoogenesis egg chambers, grk mRNA is localized between the oocyte nucleus and the plasma membrane at the presumptive posterior pole and targets the Grk signal to the posterior follicle cells (PFCs) only. Grk is believed to be the ligand for the Torpedo/DER (EGFR) signaling pathway, which controls PFC identity. Once they are specified, the PFCs send an unknown signal back to the oocyte; this signal is required to establish oocyte posterior polarity (Polesello, 2007).

Mer, which has recently been proposed to be part of the SWH network in tissue-size control, has been suggested to play a role in signal back. Therefore whether other members of this network could play a role in body axis formation was addressed. It was tested whether hpo, like mer, is required in PFCs to control oocyte polarity by generating FLP/FRT mitotic clones of mutant cells in the egg chamber was tested with either a kinase-dead (hpo^JM1) or a truncating (hpo^BF33) allele of hpo. These two alleles behave similarly in all subsequent experiments (Polesello, 2007).

In wild-type egg chambers, the RNA-binding proteins Staufen (Stau) and Osk are localized in a crescent at the posterior pole of the oocyte. When the PFCs were mutant for hpo (visualized by the lack of GFP), both Osk and Stau are mislocalized. If all PFCs were mutant, both Stau and Osk were found in the middle of the oocyte or were absent in some cases for Osk. When hpo clones affected only a portion of the PFCs, Stau was mislocalized almost exclusively in the mutant part, showing the importance of the crosstalk between PFCs and the oocyte (Polesello, 2007).

In hpo germline clones, Stau localization is unaffected if the PFCs are wild-type, suggesting that Hpo is not required for secretion of the Grk signal by the oocyte. Similarly, hpo activity in polar cells is not sufficient to rescue hpo PFC phenotypes because chambers with mutant PFCs and wild-type (GFP-positive) polar cells show disrupted Stau localization. Together, these data suggest that hpo is required in the PFCs to control oocyte polarity (Polesello, 2007).

By using Stau localization as a readout, it was found that like mer and hpo, ex, sav, mats, wts, and yki are playing a role in PFCs to control oocyte polarity, suggesting that 'canonical' Hpo signaling is responsible for the observed phenotype. In contrast, fat (ft) and discs overgrown (dco) are not required in PFCs to control oocyte polarity. This suggests that the core components of the SWH network but not the SWH-associated PCP genes are required for anteroposterior axis formation (Polesello, 2007).

The microtubule cytoskeleton plays an active role in the correct localization of posterior determinants such as Osk mRNA and Stau. Therefore, whether the microtubules are normally organized when the PFCs were mutant for hpo was tested. The oocyte nucleus is initially positioned at the posterior pole (up to stage 6) and migrates to an anterodorsal localization in a microtubule-dependent manner after the signal back from the PFCs (stages 7-14). The oocyte nucleus fails to migrate to an anterodorsal position in 50% of egg chambers with PFC hpo clones. The expression of a tubulin-GFP fusion protein was drived in the germline to visualize the microtubule network. In control oocytes, tubulin-GFP forms a regular network of filaments with a stronger accumulation at the anterior pole corresponding to the nucleation site. Egg chambers with hpo mutant PFCs present ectopic Tubulin-GFP accumulation at the posterior pole of the oocyte. Apart from this defect, the general aspect of the microtubule network is normal in egg chambers with hpo PFC clones, even when the oocyte nucleus has failed to migrate to the anterior end. Finally, microtubule polarity was examined by using both Nod-βGalactosidase (Nod-βGal, minus end marker-anterior) and Kinesin-βGalactosidase (Kin-βGal, plus end marker-posterior) fusion proteins. When the PFCs were mutants for hpo, Nod-βGal was present at both poles or only at the posterior of the oocyte when the nucleus failed to migrate. When all PFCs were hpo mutant, Kin-βGal localization was in a diffuse cloud in the middle of the ooplasm. As for Stau, only half of the Kin-βGal was normally localized when only part of the PFCs were hpo mutant. Together these data support the idea that core components of the SWH pathway are required in the PFCs to build oocyte polarity, controlling microtubule-network orientation (Polesello, 2007).

Because the SWH network is known to control cell number, a phosphorylated Histone 3 (PH3) antibody was used to follow cell division in the follicle cells. During egg-chamber development, follicle cells undergo normal mitotic divisions up to stage 6, thereby giving rise to ~650 follicle cells surrounding the germ cells. Follicle cells then switch from mitotic cycles to three rounds of endoreplication cycles (endocycles) during stages 7-10A. Thus, follicle cells normally stop proliferating after stage 6, as assayed by the absence of PH3-positive cells. hpo PFC clones still contained PH3-positive cells until stage 10B. This excess proliferation observed in hpo mutant cells gives rise to both a reduction of the size of follicle cell nuclei (reduced endocycling) and formation of double layers of cells at the posterior of the egg chamber. Formation of extra layers in the follicular epithelium has been reported to result from misorientation of the mitotic spindle. Normally, the mitotic spindle is parallel to the surface of the germline cells but appears randomly oriented in hpo mutant PFCs because both parallel and perpendicularly oriented spindles were observed. This defect in the mitotic-spindle orientation is probably responsible for the double-layer formation. The proliferation defect specifically affects PFCs because reduced nuclei, ectopic PH3 foci or double layers were not obvious elsewhere. Finally, it was found that loss of the core components of the SWH network, but not of ex for which the proliferation defect is weaker, produced a double cell layer (Polesello, 2007).

In imaginal discs, loss of SWH pathway genes leads to increased expression of Yki target genes. Whether this is also the case in PFCs was tested. As expected, disruption of SWH activity in PFCs gave rise to an increase in ex expression, although no changes were detected in DIAP1 or cycE expression. ex upregulation was restricted to the PFCs in both wts mutant cells and yki gain-of-function experiments. These results suggest that core components of the SWH network specifically control proliferation of a particular subset of follicle cells required for body axis establishment (Polesello, 2007).

Because hpo mutant PFCs were still dividing after stage 6, whether hpo loss of function could affect PFC polarity was assessed. Armadillo (Arm) and Discs large (Dlg) normally label the adherens junctions and the lateral region of the cell, respectively. In hpo mutant PFCs, these were found all around the cells. In addition, the level of Arm, atypical Protein Kinase C (aPKC), and phosphorylated Moesin (P-Moe) were increased. Nevertheless, some aspects of the polarity in these cells were preserved because aPKC was still localized in the apical domain facing the oocyte (Polesello, 2007).

Grk signals via the EGF receptor Torpedo (Top) and activates the Ras signaling pathway, specifying the PFC identity. The PFC fate can be followed by the expression of the Ras target pointed (pnt-LacZ). In the absence of hpo, pnt-LacZ expression was disrupted in most but not all PFC clones. Nevertheless, hpo mutant PFCs were still able to activate the Jak/STAT pathway in response to a signal emerging from the polar cells, (monitored with a STAT reporter suggesting that the polarity defect observed in hpo mutant PFCs does not affect their ability to receive secreted signals in general. wts mutant PFCs were negative for the dpp-LacZ reporter, a specific marker of the anterior follicle cell fate (stretch and centripetal cells), suggesting that when the SWH pathway is compromised, the PFCs are not merely transformed into anterior cells. In addition, it was found that hpo mutant PFCs present characteristics of immature cells such as maintenance of Fasciclin III (FASIII) and eyes absent (eya) expression. Normally, the level of these two genes is downregulated when the follicle cells switch from mitotic cycles to endocycles. It is noted that, when hpo mutant PFCs were FASIII positive, they did not express pnt-LacZ and vice versa. In addition, it was found that pnt-LacZ-positive hpo mutant PFCs have normal Stau localization. This suggests that the primary defect in hpo mutant cells is the failure to mature. In the rare cases where hpo mutant PFCs mature properly, they are competent to transduce the Grk signal, and oocyte polarity is normal (Polesello, 2007).

Notch (N) is required in the follicle cells for the mitotic-endocycle switch that occurs at stage 6 and for controlling follicle cell identity. N mutant follicle cells, like hpo mutant PFCs, keep proliferating because they are stuck in an immature state and continue to express undifferentiated markers such as FASIII. Recently, members of the SWH network were reported to modulate N activity by affecting its subcellular localization. N protein, which localizes to the apical part of the follicle cells, is downregulated at midoogenesis. This downregulation is delayed in wts and hpo mutant PFCs, possibly causing a defect in N signaling. Hindsight (Hnt), a target of N, which starts to be expressed in all follicle cells at stage 7 after N activation, was examined. Expression of Hnt in hpo mutant PFCs is compromised. In addition, it was found that the expression of Cut, which is normally inhibited by Hnt at stage 7, was maintained in hpo and wts clones up to stage 10. Finally, whether the modulation of N activity by the SWH network was direct was tested by looking at the expression of direct N reporters. no obvious reduction of the m7-LacZ reporter was found in hpo PFC clones. However, because of the perdurance of the β-galactosidase protein, this type of reporter is more suitable to follow increases rather than decreases in signaling. It therefore cannot be entirely rule out that the SWH network might directly affect Notch activity. Nevertheless, together these data show that inactivation of the SWH network compromises the regulation of downstream targets of Notch such as Hnt and Cut. As is the case for FASIII, misregulation of these genes is restricted to the PFCs in a SWH mutant background (Polesello, 2007).

Because of this spatial restriction of SWH activity to PFCs, whether the SWH network could be part of the Torpedo/Ras pathway acting downstream of the Grk signal was tested. ras, wts double loss-of-function clones were examined. ras, wts clones present characteristics of both ras and wts single-mutant clones, namely upregulation of Dystroglycan (DG), as observed in ras clones, and maintenance of FASIII protein, as observed in wts clones. In addition, grk mutant egg chambers present only DG upregulation but no FASIII modification and no substantial change in ex expression. It is therefore concludes that the SWH network and EGFR/Ras signaling are likely to act in parallel to control respectively PFC maturation and identity and that Grk is not the ligand that controls the SWH network activation (Polesello, 2007).

A last concern was to test whether the SWH network is involved in the PFC signal back that controls oocyte polarity. To tackle this point, attempts were made to uncouple the possible signal back to the oocyte from the PFC maturation phenotypes. ex loss of function, which affects Stau localization but presents a very reduced proliferation rate and double-layer formation compared to other SWH members, was examined. Unfortunately, ex loss of function still affected Arm, FASIII, and Cut protein levels in the PFCs, in particular at midoogenesis, when both the N and Grk signals act. Therefore mer, cut double mutants were generated. In theory, this should force the cells to differentiate (lack of cut) and still affect SWH activity (lack of mer). As expected, whereas mer loss of function alone elicited both Cut upregulation and Stau mislocalization, mer/cut PFC clones were able to induce normal oocyte polarity, manifested by correct Stau localization. It is concluded that the activity of the SWH network is required to control PFC maturation, but this pathway is probably not involved in the signal-back process (Polesello, 2007).

In conclusion, this study has shown that the core components of the SWH network are required specifically to allow the maturation of the PFCs receiving the Grk signal, thus controlling AP body axis formation. The PFC defect is due to a lack of Hnt expression in response to Notch signaling. Because the function of the SWH network is restricted to the PFCs, one interesting speculation is that it is an added layer of Notch regulation specific to PFCs, which, given their crucial role in initiating body axis formation, need robust control of signaling. Placing this regulatory element in complement and in parallel to the signal that initiates PFC specification (Grk) would ensure, in cooperation with the Unpaired signal (Jak/STAT pathway) from the polar cells, a tight and robust boundary between the PFCs and the rest of the follicle cells (Polesello, 2007).

Finally the results make a clear distinction between the core components of the SWH network (hpo, sav, wts, mats, and yki) and mer, ex on one hand and the PCP genes (ft and dco) on the other. It is speculated that the core components are used in a variety of contexts during development, whereas the PCP genes are restricted to organ-size specification (Polesello, 2007).

The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth

The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Work in Drosophila has indicated indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. This study examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. It was found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells (Hamaratoglu, 2009).

The results show that the Hippo pathway regulates the amount of apical protein complexes and thereby the size of the apical domain and that this effect is independent of its growth control function. Importantly, the regulation of apical complexes is a specific effect of the Hippo pathway, since other growth control pathways do not regulate apical complexes. In addition, this effect of the Hippo pathway is a general effect, since upregulation of apical complexes was observed in multiple tissues and cell types. Although overexpression of Crb and aPKC are sufficient to drive extra growth, the results show that the upregulation of apical complexes is not required for the overgrowth phenotype and for the induction of Hippo target genes in wts mutant cells. It is thus concluded that the Hippo pathway regulates the amount of apical complexes in Drosophila imaginal disc cells in addition to and independently of its growth control function (Hamaratoglu, 2009).

It has been suggested that Mer and Ex regulate the levels of membrane receptors independently of the Hippo pathway. However, the current results show that the upregulation of DER, Ft and apical complexes was similar in hpo and wts mutant cells and mer;ex double mutant cells, and that this effect requires Yki. These results thus indicate that Mer and Ex act through the Hippo pathway to exert their effect and that they are bona fide members of the Hippo pathway. Similar conclusions have been drawn based on observations that overexpression of wts suppresses the lethality and overgrowth phenotypes of ex mutants (Hamaratoglu, 2009 and references therein).

How does the Hippo pathway regulate the size of the apical domain and the amount of the apical complexes? The observation that Yki is required and sufficient for the effect on the apical domain indicates that this effect of the Hippo pathway is mediated by transcriptional regulation. However, although the upregulation of Crb is necessary and sufficient for the expansion of the apical domain and for the accumulation of the other apical polarity complex proteins, it is not required for the upregulation of DER and Ft, which still accumulate in wts,crb double mutant cells. Thus a model is favored in which the Hippo pathway regulates the turnover of several apical membrane components, for example through regulation of endocytosis. Notably mer;ex mutant cells in wing imaginal discs have defects in Notch (N) endocytosis, which leads to accumulation of N. Moreover, the endosomal protein Hrs accumulates in hpo mutant follicle cells in Drosophila ovaries, and this study observed a similar accumulation of Hrs in wts mutant clones in imaginal discs. These observations thus support the hypothesis that Hippo signaling regulates the amount of endocytosis and membrane turnover, thereby affecting the amount of apical membrane proteins. The target of Yki that mediates these effects, however, is currently not known (Hamaratoglu, 2009).

Several other studies also demonstrated roles for the Hippo pathway beyond its function in growth control. For example, the Hippo pathway is required for the proper selection of photoreceptor subtypes in the Drosophila eye, and it is required in follicle cells to generate a signal that polarizes the underlying oocyte. For both of these functions, Hippo signals through Yki, but Yki may regulate different sets of target genes, since the phenotypic effects are different. In addition, the Hippo pathway regulates cellular behavior through pathways that may not require Yki and thus may not involve the regulation of gene expression. For example, Yki-independent functions of the Hippo pathway may regulate dendritic tiling of larval neurons and the death of salivary gland cells during metamorphosis. The finding that Hippo regulates apical polarity complexes in addition to and independently of its growth control function in imaginal discs cells thus further reveals the complex function of this pathway in the regulation of cellular behavior (Hamaratoglu, 2009).

Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration

An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. This study used a cricket model (the two-spotted cricket, Gryllus bimaculatus) to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. Knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. A model is presented to explain these results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg (Bando, 2009).

Regeneration-dependent RNAi is a type of RNAi that occurs specifically after leg amputation in cricket nymphs that have been injected with double-stranded RNA (dsRNA) for a target gene (Nakamura, 2008a). In this system, when the metathoracic (T3) tibia of the third-instar nymph is amputated, it takes approximately 40 days (six ecdyses) to restore the adult leg. The process begins with the covering of the amputated region by newly formed cuticle. A ligand of Epidermal growth factor receptor (Gb'Egfr) is then induced by Decapentaplegic (Gb'Dpp) and Wingless (Gb'Wg) in a blastema composed of epithelial stem cells, which begins to undergo rapid proliferation to restore the lost portion in the fourth instar (Mito, 2002; Nakamura, 2008b). In the fifth instar, the tibiae, tibial spurs, tarsi and tarsal claws are restored in miniature. In the seventh instar, the amputated legs restore the missing portion to regain a nearly normal appearance. As no leg regeneration was observed after amputation in the case of rdRNAi against Gb'armadillo (Gb'arm), the canonical Wnt pathway should be involved in the initiation of the regeneration (Nakamura, 2007; Bando, 2009 and references therein).

Using an RNAi knockdown approach against 23 candidate genes, this study identified 15 components of the Ds/Ft signalling pathway that are involved in cricket leg regeneration. Based on additional data from Gryllus and Drosophila, a model signalling cascade was proposed for the regulation of leg regeneration by the Ds/Ft signalling pathway. As the main components of the Ds/Ft signalling pathways are conserved in vertebrates, this signalling cascade may also be involved in vertebrate leg regeneration (Bando, 2009).

The most typical phenotypes were the short and thick legs induced by rdRNAi against Gb'ft or Gb'ds. It is known that the size of each leg segment normally scales with overall body size, a phenomenon known as allometry. Surprisingly, the size of the regenerated legs in the phenotypes that were observed did not scale with overall body size. Furthermore, the size of the regenerated legs depended upon the site of tibial amputation. It is noteworthy that, although the expression patterns of Gb'ft and Gb'ds were different, their short and thick phenotypes were similar. This is consistent with the fact that Drosophila mutant phenotypes of both ft and ds in adult legs are short and thick, despite the fact that ft and ds have distinct expression patterns in Drosophila imaginal discs. Thus, it is concluded that the activity of Ds/Ft signalling regulates leg segment size and shape during regeneration. Furthermore, it was shown that the Ds/Ft signalling pathway may regulate leg size during regeneration through the Hpo signalling pathway. This is also supported by the fact that the Hpo signalling pathway is involved in an intrinsic mechanism that restricts organ size and that the Ds/Ft signalling system defines a cell-to-cell signalling mechanism that regulates the Hpo pathway, thereby contributing to the control of organ size (Bando, 2009).

Meinhardt pointed out that two processes operate during leg regeneration. One, which operates during the restoration of distal structures, is instructed by a morphogen epidermal growth factor (Egf), which is itself induced by two morphogens, Dpp and Wg, at the amputated surface (Meinhardt, 1982; Mito, 2002; Nakamura, 2008a). The other, operating in intercalary regeneration, is directly controlled by neighbouring cells at the junction between host and graft, but not by long-range morphogens (Meinhardt, 2007; Nakamura, 2008a). It is likely that the Ds/Ft signalling pathway participates in both mechanisms, because rdRNAi against Gb'ft or Gb'ds affected leg regeneration after either distal amputation or intercalary transplantation. In the case of distal amputation, as the Gryllus tarsi and claws were not restored after tibial amputation in the Gb'Egfr rdRNAi nymphs (Nakamura, 2008a), it has been speculated that Gb'Egf functions as a morphogen in the leg regeneration, as found in Drosophila leg imaginal discs. Recently, it has been demonstrated in the Drosophila wing disc that the Fat signalling pathway links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth. Thus, it is speculated that the Ds/Ft system links the Egf-mediated establishment of gradients of positional values across regenerating blastemal cells to the regulation of regenerate growth (Bando, 2009).

As Gb'd rdRNAi legs exhibited the short-leg phenotypes, but not thick ones, and Gb'd (decapentaplegic) is epistatic of Gb'ft and Gb'ds, Gb'D may mediate the components of Ds/Ft signalling controlling leg size. The enlarged phenotype of Gb'wts RNAi nymphs was suppressed by RNAi against Gb'd in Gryllus, indicating that Gb'd is downstream of Gb'wts. This result differs from Drosophila data. A genetic analysis is necessary to confirm the difference, because the epistatic allele is not null in RNAi experiments. As the phenotype of rdRNAi treatment against Gb'ds was weaker than that against Gb'ft, as reported in the corresponding Drosophila mutants, Gb'Ft may interact with factors that are as yet unidentified. Although the effect of rdRNAi against Gb'fj on leg size was very mild, the possible involvement of Gb'fj in allometric growth cannot be excluded. The short phenotypes were observed in the Gb'sd RNAi nymphal legs, so it remains a possibility that Gb'sd is involved in allometric leg growth. However, the apparent contribution of the Hpo-Sav-Mats complex is as yet uncertain (Bando, 2009).

Regenerated legs of Gb'ex and Gb'Mer RNAi adults become longer than normal control legs, and Gb'ex and Gb'Mer regulate cell proliferation induced by the presence of positional disparity. These results suggest that Gb'ex and Gb'Mer are also involved in allometric growth of the leg segment. In Drosophila, Ex and Mer negatively regulate cell growth and proliferation through the Hpo/Wts pathway. In mammalian cells, Nf2 (merlin) is known to be a crucial regulator of contact-dependent inhibition of proliferation (Curto, 2008). Thus, it is concluded that activities of Ex and Mer may regulate contact-dependent inhibition of proliferation via the Wts signalling pathway to restore the proper leg segment size during regeneration (Bando, 2009).

A widely accepted model for leg regeneration is the intercalation model, based on positional information. This model is based on the intercalation of new structures so as to re-establish continuity of positional values during regeneration. However, on the basis of this model, it is difficult to explain the changes in leg size that were observed in the present study. Thus, the model need to be extended to include the control of growth and tissue size during regeneration. Several models have been proposed to explain how organ size is regulated. Lawrence (2008) proposed a model, which is referred to here as the Ds/Ft steepness model, to explain the mechanisms underlying the determination of organ size and PCP, including the Warts/Hippo pathway as the mechanism for controlling growth. In this model, it was hypothesized that: (1) the morphogens responsible for the overall pattern of an organ establish and orient the Ds/Ft system, which then forms a linear Ds/Ft gradient. The nature of the Ds/Ft gradient is unknown, although the number of Ds/Ft trans heterodimers is the key variable; (2) the steepness of the Ds/Ft gradient regulates Hpo target expression and cell proliferation, and its direction provides information used to establish the correct cellular polarity; (3) growth would be expected to cease when the slope of the gradient declines below a certain threshold value; and (4) the maximum and minimum limits of the system are conserved, while recently divided cells take up intermediate scalar values from their neighbours (Bando, 2009).

Using this model, a modified Ds/Ft steepness model is proposed for leg regeneration acting as follows. The results indicate that nymphal leg regeneration depends on two major processes: (1) proliferation and differentiation of blastemal cells and (2) growth of the pre-existing stump. In each of these processes, new positional identities are specified in relation to new segment boundaries. According to the Ds/Ft steepness model, in normal regeneration, a very steep gradient should be formed in the regenerating blastema. The regenerate may grow so as to restore the normal pre-existing steepness. Reassignment of positional identities after amputation will correlate with a similar re-setting of the minimum Ds/Ft scalar value, and the results are consistent with the steepness hypothesis (Bando, 2009).

Growth of the pre-existing stump is a normal component of leg growth, in which the pre-existing stump cells proliferate according to some allometric signals, which may be related to the maximum scalar value and a slope of the gradient, keeping their original positional information. This was observed in the truncated leg of Gb'arm rdRNAi adults (Nakamura, 2007; Bando, 2009).

In the absence of the proliferation and differentiation of blastemal cells, as observed in the Gb'ft rdRNAi leg, the minimum scalar value, which is the most distal positional value, would be established at the site of amputation, and the Ds/Ft gradient would be expected, in turn, to shift down with the same slope as the pre-existing one. The Ds/Ft steepness model provides an explanation for the observation that the final leg size depends on the amputated position, if it is assumed that the gradient shifts down with the same slope as that where cells at an amputated position have the minimum scalar value. Thus, the observed re-specification of regeneration legs induced in the legs treated with rdRNAi against Gb'ft or Gb'ds is as would be predicted by the Ds/Ft steepness model. Thus, it is likely that the Ds/Ft gradient functions to link positional and allometric information to the regulation of leg segment growth. Furthermore, if it is assumed that the activity of Ex/Mer is related to a threshold value of the slope of the gradient that determines when growth ceases, all rdRNAi phenotypes in the present study can be interpreted consistently with the Ds/Ft steepness model for regeneration (Bando, 2009).

Differential requirement of Salvador-Warts-Hippo pathway members for organ size control in Drosophila melanogaster

The Salvador-Warts-Hippo (SWH) pathway contains multiple growth-inhibitory proteins that control organ size during development by limiting activity of the Yorkie oncoprotein. Increasing evidence indicates that these growth inhibitors act in a complex network upstream of Yorkie. This complexity is emphasised by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue lacking the core SWH pathway components salvador, warts or hippo is highly overgrown and resistant to developmental apoptosis, whereas tissue lacking fat or expanded is not. This study explores the relative contribution of SWH pathway proteins to organ size control by determining their temporal activity profile throughout Drosophila eye development. Eye tissue lacking fat, expanded or discs overgrown displays elevated Yorkie activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. Fat and Expanded do possess Yorkie-repressive activity in the pupal eye, but loss of fat or expanded at this stage of development can be compensated for by Merlin. Fat appears to repress Yorkie independently of Dachs in the pupal eye, which would contrast with the mode of action of Fat during larval development. Fat is more likely to restrict Yorkie activity in the pupal eye together with Expanded, given that pupal eye tissue lacking both these genes resembles that of tissue lacking either gene. This study highlights the complexity employed by different SWH pathway proteins to control organ size at different stages of development (Milton, 2010).

The SWH pathway controls Drosophila eye size by limiting growth during the larval stage of development and by restricting proliferation and promoting apoptosis during pupal development. Eyes lacking core SWH pathway components (e.g. sav, wts or hpo) are significantly larger than eyes lacking the non-core components ft, ex, dco or Mer. Owing to this disparity, it has been hypothesized that ft and ex only partially affect SWH pathway activity, whereas sav, wts and hpo have stronger effects, or, alternatively, that non-core components affect pathway activity in a temporally restricted fashion. Analysis of tissue recessive for ft, ex or dco³ revealed that Yki activity was elevated during larval eye development when tissues are actively growing and proliferating, but not during pupal development when apoptosis ensues, supporting the idea that Ft, Ex and Dco influence SWH pathway activity in a temporally restricted fashion. However, when tissue lacking both Mer and ft, or Mer and ex, was analysed, Yki activity was found to be elevated during both larval and pupal development, similar to the Yki activity profile observed in tissue lacking core SWH pathway proteins. This is consistent with previous reports showing that Mer acts in parallel to both Ft and Ex, and that these proteins can compensate for each other to control SWH pathway activity. Therefore, Ft and Ex do contribute to SWH pathway regulation in the pupal eye to ensure appropriate exit from the cell cycle and developmental apoptosis, but these functions can be executed by Mer in their absence, suggesting a degree of plasticity in the regulation of Yki activity by non-core SWH pathway proteins. The ability of Mer to compensate for Ft or Ex cannot simply be explained by compensatory increases in Mer protein in pupal eye tissues lacking ft or ex, since Mer expression levels were found to be unaltered in these tissues (Milton, 2010).

Previous analyses of tissue lacking both ft and ex showed that these proteins function, at least in part, in parallel to control growth of larval imaginal discs. The current analysis of ft,ex double-mutant tissue suggests that these proteins are likely to function together to control Yki activity in the pupal eye. Yki activity was not elevated in tissue lacking ft, ex or both genes, showing that these genes cannot compensate for each other in the pupal eye. This is consistent with the notion that Ft influences the activity of downstream SWH pathway proteins by multiple mechanisms, an idea that is supported by THE analysis of the requirement of the atypical myosin, Dachs, for Ft signalling in the pupal eye. During larval imaginal disc development, Ft can influence Yki activity by repressing Dachs activity, which in turn can repress the core SWH pathway protein Wts. Analysis of pupal eye tissue that lacks both Mer and ft, or Mer, ft and dachs, showed that Yki activity was elevated in each scenario. This shows that in the pupal eye, the ability of Ft to compensate for Mer is not reliant on Dachs, and implies that Ft can employ different modes of signal transduction throughout eye development. However, because Ft and Mer can compensate for each other it is not possible to formally conclude that normal signal transduction by Ft in the pupal eye occurs independently of Dachs (Milton, 2010).

Expression of Ex is tightly controlled in response to alterations in SWH pathway activity at both the transcriptional and post-transcriptional levels. Interestingly, it was also found that Ex expression is controlled in a temporal fashion throughout eye development; Ex is expressed at relatively high levels in the larval eye, but at very low levels in the pupal eye. Despite the fact that Ex expression is very low in the pupal eye, it clearly retains function at this stage of development because it can compensate for loss of Mer to restrict Yki activity. The dynamic expression profile of Ex suggests that factors that influence its expression play an important role in defining overall eye size in Drosophila. At present, only two transcriptional regulatory proteins have been shown to influence the expression of ex: Yki and Sd. There are conflicting reports on whether Yki and Sd control basal expression of ex in larval imaginal discs. It is clear, however, that Yki and Sd collaborate to drive ex expression when the activity of the SWH pathway is suppressed, presumably as part of a negative-feedback loop. Despite the fact that basal ex expression is low in the pupal eye, the ex promoter is still responsive to Yki, as Ex expression is substantially elevated in pupal eye clones lacking hpo or Mer and ex. Future investigation of the ex promoter will help to clarify understanding of the complex fashion by which expression of the ex gene is controlled, and should aid understanding of eye size specification in Drosophila (Milton, 2010).

This study emphasises the complexity of the means by which the activity of core SWH pathway proteins is regulated by non-core proteins such as Ft, Ex, Mer and Dco. The signalling mechanisms employed by non-core proteins appear to differ at discrete stages of development in order to achieve appropriate organ size during the larval growth period of eye development, and to subsequently sculpt the eye by regulating apoptosis during pupal development (Milton, 2010).

The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements

In Drosophila, defects in asymmetric cell division often result in the formation of stem cell derived tumors. This study shows that very similar terminal brain tumor phenotypes arise through a fundamentally different mechanism. Brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. ChIP-seq was used to identify L(3)mbt binding sites, and it was shown that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting expression of the insulator protein gene mod(mdg4) results in upregulation of SWH-pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Therefore, very different primary defects result in the formation of brain tumors, which behave quite similarly in their advanced stages (Richter, 2011).

Drosophila nervous system recapitulates many steps in mammalian neurogenesis. Neurons in the adult fly brain arise from stem cells called neuroblasts which undergo repeated rounds of asymmetric cell division during larval stages. After division, one daughter cell remains a neuroblast while the other is called the ganglion mother cell (GMC) and divides just once more into two differentiating neurons. Most larval neuroblasts are inherited from the embryo but the so-called optic lobe neuroblasts (NB) located laterally on each brain lobe pass through a neuroepithelial (NE) stage and are therefore a particularly suitable model for mammalian neurogenesis. During early larval stages, the NE cells of the optic lobes (OL) proliferate and separate into the inner (IOA) and outer (OOA) optic anlagen. During late larval stages, NE cells switch to a neurogenic mode. On the medial side, they generate optic lobe neuroblasts (OL NBs), which generate the neurons of the medulla, the second optic ganglion. OL neurogenesis is controlled by a wave of lethal of scute (l(1)sc) expression passing through the neuroepithelium from medial to lateral. The activity of the Jak/STAT pathway inhibits neural wave progression and thereby controls neuroblast number. Differentiation of neuroepithelial cells also involves the Notch, Epidermal Growth Factor (EGF) and Salvador-Warts-Hippo (SWH) pathways (Richter, 2011).

Characterization of Drosophila genes identified in brain tumor suppressor screens has demonstrated that defects in neuroblast asymmetric cell division result in the formation of stem cell derived tumors that metastasize and become aneuploid upon transplantation. These screens also identified lethal (3) malignant brain tumor (l(3)mbt), a conserved transcriptional regulator that is also required for germ-cell formation in Drosophila. L(3)mbt binds to the cell cycle regulators E2F and Rb but the relevance of these interactions is unclear. This study shows that in Drosophila, L(3)mbt regulates target genes of the Salvador-Warts-Hippo (SWH) pathway that are important in proliferation and organ size control. The SWH-pathway is regulated by the membrane proteins Expanded (Ex) and Fat, which activate a protein complex containing the kinases Hippo and Warts to phosphorylate the transcriptional co-activator Yorkie. Yorkie acts together with the transcription factors Scalloped and Homothorax to activate proliferative genes like Cyclin E and the microRNA bantam (ban) and Drosophila inhibitor of apoptosis 1 (diap1: thread). Upon phosphorylation, Yorkie is retained in the cytoplasm and its target genes are not activated. In Drosophila the main role of the SWH-pathway is to limit proliferation in imaginal discs and its absence leads to tumorous overgrowth. In vertebrates, many homologs of key pathway members are tumor suppressors indicating that this function is conserved (Richter, 2011).

L(3)mbt contains three MBT domains which bind mono- or dimethylated histone tails. Biochemical experiments in vertebrates have suggested a role in chromatin compaction but whether this role is conserved is not known. Results published while this paper was under review have shown that germline genes are upregulated in l(3)mbt mutant brains and are necessary for tumor formation. The current data indicate that L(3)mbt is bound to insulator sequences, which affect promoter-enhancer interactions and influence transcription. In Drosophila, the proteins CTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF are found at insulator sequences but how these factors act is largely unknown (Richter, 2011).

The data presented in this study show that tumor formation in l(3)mbt mutants is initiated by the uncontrolled overproliferation of neuroepithelial cells in the optic lobes due to the upregulation of proliferation control genes normally repressed by the SWH-pathway. L(3)mbt is located at DNA sequences bound by chromatin insulators and we propose that the function of L(3)mbt as a chromatin insulator is essential for repressing SWH target genes and preventing brain tumor formation (Richter, 2011).

brat, lgl and dlg were previously identified as Drosophila brain tumor suppressors. In all cases, defects in asymmetric cell division cause a huge expansion of the neuroblast pool. In l(3)mbt mutants, however, the neuroblast pool is expanded because an upregulation of SWH target genes results in a massive expansion of neuroepithelial tissue. Why those neuroblasts proliferate indefinitely upon transplantation is currently not understood for any of those mutants (Richter, 2011).

While the SWH-pathway is essential for tumorigenesis in l(3)mbt mutants, its overactivation can not recapitulate the neuroblast tumor phenotype seen in l(3)mbt mutants (this study and Reddy, 2010). Similar to the multifactorial origin of mammalian tumors, therefore, the combined deregulation of several signaling pathways could be required. The Notch pathway could be involved as it regulates the formation of OL neuroblasts from neuroepithelia and Notch pathway gene insulator sequences are bound by L(3)mbt. Increased activity of the Jak/STAT pathway, a major regulator of OL development, was also observed. Finally, the deregulation of germline genes in l(3)mbt mutants that has been described while this manuscript was under review (Janic, 2010) could provide another exciting explanation (Richter, 2011).

The results indicate that L(3)mbt acts on insulator elements, which isolate promoters from the activity of nearby enhancers acting on other genes. the analysis showed that L(3)mbt binding sites overlap with CP190, CTCF and BEAF-32, placing the protein into what has been called the class I of chromatin insulators (Negre, 2010; Richter, 2011 and references therein).

The identification of a DNA consensus motif for a histone binding protein like L(3)mbt is highly unexpected as insulators are typically nucleosome free. Currently, the activity of these important transcriptional regulators could be explained in several ways. Either, they form physical barriers blocking the interaction between enhancers and promoters. Alternatively, they mimic promoters and compete with endogenous promoters for enhancer interaction. Finally, they could interact with each other or nuclear structures to form loop domains that regulate transcriptional activity. The data suggests another model in which insulators interact with histones on nearby nucleosomes and influence the structure of higher order chromatin. Importantly, in the regions flanking CTCF binding sites nucleosomes are enriched for histones that are mono- and di-methylated on H3K4 or mono-methylated on H3K9 or H4K20, the variants to which MBT domains can bind in vitro. As the human L(3)mbt homolog L3MBTL1 was shown to compact nucleosome arrays in vitro, a model becomes feasible in which simultaneous binding to insulators and the surrounding nucleosomes reduces flexibility and thereby restricts the ability of nearby enhancers to interact with promoters on the other side of the insulator. However, the data could equally well be worked into the other prevalent models for insulator activity. Since L(3)mbt is currently the only chromatin insulator besides CTCF that is conserved in vertebrates, analysis of its homologs will certainly allow to distinguish between those possibilities (Richter, 2011).

OL development resembles vertebrate neurogenesis. Both processes consist of an initial epithelial expansion phase followed by neurogenesis through a series of asymmetric divisions. Together with previous findings, these data demonstrate that l(3)mbt and the SWH-pathway are crucial regulators of the initial neuroepithelial proliferation phase. Interestingly, the SWH-pathway has been implicated in regulating neural progenitors in the chicken embryo and it will be exciting to test the role of mammalian L(3)mbt in this process. It is remarkable that YAP is upregulated and L3MBTL3 is deleted in a subset of human medulloblastomas. Medulloblastoma is the leading cause of childhood cancer death and investigating the role of the SWH-pathway might contribute to the progress in fighting this disastrous disease (Richter, 2011).

Alcohol interacts with genetic alteration of the hippo tumor suppressor pathway to modulate tissue growth in Drosophila

Alcohol-mediated cancers represent more than 3.5% of cancer-related deaths, yet how alcohol promotes cancer is a major open question. Using Drosophila, this study identified novel interactions between dietary ethanol and loss of tumor suppressor components of the Hippo Pathway. The Hippo Pathway suppresses tumors in flies and mammals by inactivating transcriptional co-activator Yorkie, and the spectrum of cancers associated with impaired Hippo signaling overlaps strikingly with those associated with alcohol. Therefore, these findings may implicate loss of Hippo Pathway tumor suppression in alcohol-mediated cancers. Ethanol enhanced overgrowth from loss of the expanded, hippo, or warts tumor suppressors but, surprisingly, not from over-expressing the yorkie oncogene. It is proposed that in parallel to Yorkie-dependent overgrowth, impairing Hippo signaling in the presence of alcohol may promote overgrowth via additional alcohol-relevant targets. Interactions between alcohol and Hippo Pathway over-activation were also identified. It is proposed that exceeding certain thresholds of alcohol exposure activates Hippo signaling to maintain proper growth control and prevent alcohol-mediated mis-patterning and tissue overgrowth (Ilanges, 2013).

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).

The Spectrin cytoskeleton regulates the Hippo signalling pathway

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. This study shows that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produced tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin bound to Ex and co-localised with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb were dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) were essential. Finally, the Spectrin cytoskeleton was required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. These findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors (Fletcher, 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 (Makhijani, 2007). The difference between cell size and shape becomes evident at late pupal stages (Roch, 2000). 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 (Roch, 2000) (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 (Crickmore, 2006, De Navas, 2006; Makhijani, 2007). Only when very large clones of Ubx^6.28Ubx^6.28 are generated, one can see increased growth compared to their twin spots (Crickmore, 2006). 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 (Weatherbee, 1998, Shashidhara, 1999; Prasad, 2003; Mohit, 2006; Crickmore, 2006, Pallavi, 2006; De Navas, 2006; Makhijani, 2007). 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 (Herranz, 2012). 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).

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