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).
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).
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 exl(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 exl(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 exl(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, exe1 pharate adults display a highly penetrant and expressive phenotype. exe1 mutants survive until the pharate adult stage and show massive head, wing and leg defects. All exe1 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 exe1 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 exe1 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 exl(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 exl(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, exl(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 (exe1, 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 exe1 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 (dppblk) 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 exe1 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).
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 exe1 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).
Blaumueller, C. M. and Mlodzik, M. (2000). The Drosophila tumor suppressor expanded regulates growth, apoptosis, and patterning during development. Mech. Dev. 92(2): 251-62. Medline abstract: 10727863
Boedigheimer, M. and Laughon, A. (1993). Expanded: a gene involved in the control of cell proliferation in imaginal discs. Development 118(4): 1291-301. Medline abstract: 8155582
Boedigheimer, M. J., Nguyen, K. P. and Bryant, P. J. (1997). Expanded functions in the apical cell domain to regulate the growth rate of imaginal discs. Dev Genet 1997;20(2):103-10. Medline abstract: 9144921
Hamaratoglu, F., et al. (2006). The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis. Nat. Cell Biol. 8(1): 27-36. 16341207
McCartney, B. M., et al. (2000). The Neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation. Development 127: 1315-1324. Medline abstract: 10683183
Maitra, S., Kulikauskas, R. M., Gavilan, H. and Fehon, R. G. (2006). The tumor suppressors Merlin and Expanded function cooperatively to modulate receptor endocytosis and signaling. Curr. Biol. 16(7): 702-9. 16581517
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date revised: 15 December 2006
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