Interactive Fly, Drosophila

REGULATION

DEVELOPMENTAL BIOLOGY

See the embryonic expression pattern of Mer at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

In contrast to the largely complementary pattern observed for Merlin and Moesin-like during oogenesis, the two appear to be coexpressed in most cells during embryogenesis. Merlin and Moesin-like are present from cellularization throughout embryonic development. Merlin expression is found to be enhanced in the early mesoderm of the germband extended embryo, whereas Moesin-like is expressed uniformly throughout the embryo at this stage. Late in embryogenesis, both proteins are expressed ubiquitously throughout the tissues of the embryo, including the epidermis, salivary glands, foregut, midgut, hindgut, and the embryonic nervous system. At this stage Merlin expression is enhanced in the midgut (McCartney, 1996).

In the embryonic central nervous system (stage 15), Merlin and Moesin-like staining is detected in the neuropil, a structure composed of the developing axonal bundles of the ventral nerve cord and in the developing brain. Merlin and Moesin-like localization is also observed in the neuronal cell bodies of the CNS, with Moesin-like expression enhanced in these tissues. During stage 11 of embryogenesis, the neurons of the embryonic peripheral nervous system develop from specialized regions within the epidermis termed the proneural clusters. The cell bodies of the differentially bipolar sensory neurons can be observed in a regular pattern within the epidermis of a stage 17 embryo. At this stage, Moesin-like is enriched at the membranes of these cells, whereas Merlin is found to localize to an intensely staining spot within the cell body (McCartney, 1996)

Larval

In the developing eye Moesin-like is localized at the membranes of the cone cells, secondary and tertiary pigment cells, and in the bristle precursor cells. In contrast, Merlin is localized primarily in the cytoplasm of the secondary and tertiary pigment cells, and is greatly enhanced in the bristle precursor cells, which are interspersed between outer pigment cells. Both Merlin and Moesin-like are more intensely expressed in the center of each ommatidium. This corresponds to the region of the rhabdomeres, the photosensitive microvilli of the photoreceptors, which project into the center of each ommatidium (McCartney, 1996).

Adult

During oogenesis, Merlin and Moesin-like display strikingly different tissue distributions: this distinction is clearly observed as early as the germarium, the location of the germline stem cells. Merlin is expressed predominantly in the germline, while Moesin-like is expressed at greater levels in the follicle cells. In addition, Merlin expression becomes enhanced in the developing oocyte at approximately stage 6 of development and persists until the end of oogenesis. Lower levels of Merlin expression are also detected at the apical ends of the follicle cells at stage 10, late in oogenesis. In contrast, Moesin expression is found at the apical and basolateral ends of the follicular epithelium, although some expression is detected in the germline of early egg chambers and in the nurse cells at stage 10. Fully developed oocytes (stage 14) clearly display membrane associated Merlin, while no Moesin-like expression is detected at this stage (McCartney, 1996).

Effects of Mutation or Deletion

Neurofibromatosis-2 is an inherited disorder characterized by the development of benign schwannomas and other Schwann-cell-derived tumors associated with the central nervous system. The Neurofibromatosis-2 tumor suppressor gene encodes Merlin, a member of the Protein 4.1 superfamily most closely related to Ezrin, Radixin and Moesin. This discovery suggested a novel function for Protein 4.1 family members in 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, another Protein 4.1 superfamily member in Drosophila. 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. Further genetic analyses suggest a role for Merlin and Expanded specifically in Decapentaplegic-mediated differentiation events. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for understanding the functions of other Protein 4.1 superfamily members (McCartney, 2000).

Hypomorphic alleles of either Mer (Mer³) or ex (ex⁶⁹⁷) result in very similar adult wing and eye phenotypes. In both cases, mutant adults displayed 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 was 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 and Mlodzik, 2000, in press, cited by McCartney, 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).

Mammalian Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). As is the case for NF2 tumors in vertebrates, Drosophila cells lacking Merlin function will overproliferate, relative to their neighbors. Using in vitro mutagenesis, functional domains within Merlin have been defined that are required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane-associated NH2-terminal 350 amino acids of Merlin. Removal of a seven-amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. Evidence is provided that the COOH-terminal region of Merlin has a negative regulatory role, as has also been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene (LaJeunesse, 1998).

Reverse genetic analysis in Drosophila has been greatly aided by a growing collection of lethal P transposable element insertions that provide molecular tags for the identification of essential genetic loci. However, because the screens performed to date primarily have generated autosomal P-element insertions, this collection has not been as useful for performing reverse genetic analysis of X-linked genes. A reverse genetic screen has been designed that takes advantage of the hemizygosity of the X chromosome in males together with a cosmid-based transgene that serves as an autosomally linked duplication of a small region of the X chromosome. The efficacy and efficiency of this method is demonstrated by the isolation of mutations in Drosophila homologs of two well-studied genes, the human Neurofibromatosis 2 tumor suppressor and the yeast CDC42 gene. The method describe should be of general utility for the isolation of mutations in other X-linked genes, and should also provide an efficient method for the isolation of new alleles of existing X-linked or autosomal mutations in Drosophila (Fehon, 1997).

Merlin, the Drosophila homologue of neurofibromatosis-2, is specifically required in posterior follicle cells for axis formation in the oocyte

In Drosophila, the formation of the embryonic axes is initiated by three signals -- one from Gurken, a transforming growth factor; another signal from the oocyte to the posterior follicle cells, and an unknown polarizing signal back to the oocyte. Drosophila Merlin is specifically required only within the posterior follicle cells to initiate axis formation. Merlin mutants show defects in nuclear migration and mRNA localization in the oocyte. Merlin is not required to specify posterior follicle cell identity in response to the Gurken signal from the oocyte, but is required for the unknown polarizing signal back to the oocyte. Merlin is also required non-autonomously, only in follicle cells that have received the Gurken signal, to maintain cell polarity and limit proliferation, but is not required in embryos and larvae. These results are consistent with the fact that human Merlin is encoded by the gene for the tumor suppressor neurofibromatosis-2 and is a member of the Ezrin-Radixin-Moesin family of proteins that link actin to transmembrane proteins. It is proposed that Merlin acts in response to the Gurken signal by apically targeting the signal that initiates axis specification in the oocyte (MacDougall, 2001).

To identify new genes required for axis specification, a screen was carried out of a collection of X-linked ts lethal mutations generated by selecting for male lethality at 29°C and viability at 21°C. Homozygous female progeny were collected at 21°C from 73 viable ts lethal lines, shifted to 29°C for 3 days and GRK RNA in situ hybridization on ovaries was performed. In wild type at 29°C or in all strains at 21°C, the oocyte nucleus migrates correctly to the antero-dorsal corner of the oocyte with GRK mRNA localiz ing between the nucleus and the overlying future dorsal follicle cells. In one mutant line, l(1)ts594, identified as a mutation in Merlin (termed here Mer^ts1), 55% of oocyte nuclei fail to migrate and GRK mRNA localizes at the posterior after stage 8. The remaining 45% of cases were similar to wild type (MacDougall, 2001).

To test whether the defects in the oocyte are primarily due to a defect in MT organization, MT polarity was examined. Kin:betagal, a plus end-directed MT motor fusion that leads to betagal accumulation at the posterior of the oocyte, was used. Nod:betagal, a MT motor fusion that leads to betagal accumulation at the anterior, where the minus ends of MTs are thought to localize, was also used. The betagal motor fusions indicate that prior to stage 7, there is an MTOC at the posterior. In wild-type oocytes after stage 7, the posterior MTOC disassembles, a diffuse MTOC appears at the anterior with MT plus ends at the posterior. Prior to stage 7, Mer^ts1 mutant oocytes show a similar MT organization to wild type, but after stage 7, the MTOC fails to disassemble at the posterior and a second diffuse MTOC forms at the anterior. This leads to a symmetric organization of MTs, with their plus ends at the center of the oocyte and minus ends at the anterior and posterior. The overall distribution of MTs was examined using a maternally expressed TauGFP line showing the highest concentration of MTs at the anterior cortex of wild-type oocytes. A similar Tau-GFP distribution was observed in Mer^ts1 oocytes at 21°C. In Mer^ts1 at 29°C Tau-GFP shows an abnormally high level at the posterior, consistent with a failure to disassemble the posterior MTOC. It is concluded that the mislocalization of mRNA and failure of the oocyte nucleus to relocate in Mer^ts1 oocytes are due primarily to defects in MT organization (MacDougall, 2001).

Merlin protein is expressed in the oocyte and in posterior follicle cells. To determine where Merlin functions in egg chambers, homozygous Mer^ts1 germline clones were generated using X-rays in females raised at the restrictive temperature (29°C). Ten Mer^ts1 oocytes surrounded by Mer^ts1/+ follicle cells were examined, and they all showed normal mRNA localization and led to normal eggs. It is concluded that Merlin is not required in the germline derived nurse cells or oocyte (MacDougall, 2001).

To test whether Merlin is required within the somatically derived posterior follicle cells to receive the Grk signal from the oocyte, the expression of different follicle cell markers were examined in Mer egg chambers. The results show that Mer^ts1 posterior follicle cells receive the Grk signal correctly; they express posterior and not anterior markers. It is concluded that Merlin is not required for any aspect of Grk signaling or its reception in the posterior follicle cells. Merlin is also not required for Notch signaling among the posterior follicle cells, which is required to specify the correct number of posterior cells (MacDougall, 2001).

The posterior follicle cells in fixed and living Mer egg chambers often have a slightly disrupted morphology. To study these defects in more detail, actin and DNA were covisualized to highlight each cell and its boundaries. Posterior follicle cells in controls have a uniform columnar appearance characteristic of epithelial sheets. However, after stage 6, Mer^ts1 egg chambers have a double layer of follicle cells only at the posterior where follicle cells are in contact with the oocyte. To determine whether the double layer of posterior follicle cells is due to overproliferation, the number of cells was counted using three-dimensional microscopy and a twofold increase in the number of posterior follicle cells was found, but no changes in other follicle cells. To determine whether the overproliferation of posterior follicle cells is accompanied by polarity defects, MT polarity was studied by covisualizing DNA, the nuclear envelope and centrosomes. In control egg chambers, most centrosomes lie on the apical side of each nucleus, where the minus ends of MTs are found. In contrast, Mer^ts1 posterior follicle cells mostly lose the apical-basal polarity of their MTs (MacDougall, 2001).

To investigate whether other aspects of the apical-basal polarity of the posterior follicle cells are also disrupted, the distribution of beta-spectrin heavy chain (betaH-spectrin) was examined in Mer mutants. betaH-spectrin is normally restricted to the apical side of follicle cells within a Spectrin-based membrane skeleton. In Mer^ts1 mutants, betaH-spectrin is apically localized in the cells adjacent to the oocyte, but not detected in the second layer of follicle cells. These results suggest that in Mer mutants, the apical surface of posterior follicle cells contacts the oocyte correctly, and is probably competent to send and receive signals to the oocyte (MacDougall, 2001).

To determine whether the defects in cell proliferation and polarity in Mer egg chambers are dependent on receiving the Grk signal, the follicle cells of Mer;grk double mutants was examined. Even a hypomorphic allele of grk suppresses the Mer posterior follicle cell phenotype entirely. It is concluded that Merlin is required only in cells that receive the Grk signal and is not a constitutive factor required for cell polarity and proliferation. Additional experiments show that Merlin is required only in posterior follicle cells. Also, Merlin has been shown to acts cell non-autonomously among the posterior follicle cells (MacDougall, 2001).

These results show that Merlin is required for two distinct processes involving signalling, but whether the two processes depend on a single signal or two distinct signals cannot be distinguished. The restriction of posterior follicle cell proliferation could require the same unknown signal that initiates MT repolarization in the oocyte. Both processes could depend on the same signal secreted into the space between the follicle cells and oocyte. Indeed, it is intriguing that Merlin egg chambers have MT polarity defects in both the oocyte and the posterior follicle cells. However, further progress awaits the identification of the signal or signals involved (MacDougall, 2001).

The identity of the polarizing signal is unknown, but some genes are known to be required for the signal, including PKA, Mago and Laminin A. Merlin is unlikely to be required for PKA and Mago functions because they are required in the oocyte. In contrast, Laminin A is expressed and required in posterior follicle cells as a component of the extracellular matrix (Deng, 2000). It is tempting to speculate that Merlin and Laminin A could be functionally linked as specialized structural components required specifically in the posterior follicle cells for the transduction of the polarizing signal (MacDougall, 2001).

A systematic screen for dominant second-site modifiers of Merlin/NF2 phenotypes reveals an interaction with blistered/DSRF and scribbler

Merlin, the Drosophila homolog of the human tumor suppressor gene Neurofibromatosis 2 (NF2), is required for the regulation of cell proliferation and differentiation. To better understand the cellular functions of Merlin, recent work has concentrated on identifying proteins with which Merlin interacts either physically or functionally. Genetic screens designed to isolate second-site modifiers of Merlin phenotypes are described from which five multiallelic complementation groups have been identified that modify both loss-of-function and dominant-negative Merlin phenotypes. Three of these groups, Group IIa/scribbler (also known as brakeless), Group IIc/blistered, and Group IId/net, are known genes, while two appear to be novel. In addition, two genes, Group IIa/scribbler and Group IIc/blistered, alter Merlin subcellular localization in epithelial and neuronal tissues, suggesting that they regulate Merlin trafficking or function. Mutations in scribbler and blistered display second-site noncomplementation with one another. These results suggest that Merlin, blistered, and scribbler function together in a common pathway to regulate Drosophila wing epithelial development (LaJeunesse, 2001).

In this screen seven new alleles of sbb were identified; allelism was based on noncomplementation with a null sbb allele and the presence of nonsense mutations in two sbb alleles identified. Null and strong hypomorphic mutations in scribbler result in aberrant axon guidance and behavioral phenotypes. However, none of the sbb alleles identified in this screen display either of these phenotypes. In addition, none of the previously identified P-element insertional mutations in sbb modify Merlin phenotypes, although the null sbb⁴ allele and Df(2R)PC4 do enhance Merlin phenotypes. These data suggest that sbb has two distinct functions, one in axon guidance of photoreceptor cells and the other in regulation of proliferation in epithelial cells, and that these functions are independent. Consistent with this model, previous studies showed that sbb encodes two novel proteins of unknown function, SBB-A and SBB-B. Although it was shown that the two SBB isoforms are functionally redundant in axon guidance, the presence of a zinc finger domain and a novel Region B in the larger SBB-B isoform suggests that it may have functions distinct from SBB-A. Sequence analysis indicates that two of the alleles isolated as Merlin modifiers correlate with nonsense mutations that affect the SBB-B product but leave the BSKA product intact (LaJeunesse, 2001).

The identification of SBB-B mutations that specifically modify Merlin phenotypes but do not affect photoreceptor axon guidance supports a model where SBB-B has distinct functions in the proliferation and differentiation of wing tissue. Both SBB isoforms are reported to be nuclear proteins and the presence of a zinc finger in SBB-B suggests that it may be involved in transcriptional regulation. How SBB proteins interact with Merlin, a membrane-associated cytoplasmic protein, is unclear. The observation that Merlin subcellular localization is disrupted in sbb mutant cells makes this question particularly intriguing and suggests that sbb may play a role in a cellular pathway that regulates Merlin function. The identity of this pathway is currently unknown (LaJeunesse, 2001).

While sbb encodes novel proteins with unknown function, the bs gene product, also known as the Drosophila serum response factor (BS/DSRF), is a well-characterized transcription factor. bs is required for formation of terminal tracheal branches and differentiation of the adult wing. BS/DSRF activity, like that of its mammalian homolog, is regulated by the epidermal growth factor receptor (Egfr) signaling pathway. During development of the wing imaginal disc, cells can adopt one of two fates; most cells form wing blade (intervein tissue), while a subset form the characteristic longitudinal veins. BS/DSRF is believed to promote the intervein cell fate -- loss-of-function bs mutations result in wings in which all cells develop as vein tissue. Activity of the Egfr pathway is believed to promote the vein cell fate by downregulating BS/DSRF function in the vein primordia and promoting the expression of vein-specific genes. Thus interactions between the Egfr pathway and BS/DSRF play a crucial role in wing development (LaJeunesse, 2001 and references therein).

The identification of bs as a dominant modifier of Merlin phenotypes suggests that Merlin, like Blistered, is involved in Egfr signaling. Specifically, the observation that bs mutations enhance Merlin dominant-negative and loss-of-function phenotypes suggests that Merlin may function antagonistically to Egfr pathway function. Although this hypothesis should be considered as tentative, several lines of evidence support this notion: (1) developing wing cells that have lost both Merlin and expanded, which appear to function redundantly, produce abundant ectopic vein material adjacent to endogenous veins; (2) net, which was also identified as a Merlin modifier, has been shown to modify phenotypes of components of Egfr signaling in the wing; (3) a role for Merlin in negatively regulating Egfr function is consistent with the observation that Merlin mutations result in overproliferation phenotypes and (4) a hypermorphic Egfr mutation called Ellipse enhances phenotypes expressed by dominant-negative and hypomorphic Merlin alleles. However, despite these intriguing indications that Merlin may function to regulate Egfr pathway activity, it should be noted that Merlin does not interact genetically with several other known pathway members (Star, asteroid, and rhomboid), nor does it interact with hypomorphic Egfr mutations. In addition, because other signaling pathways, including dpp, wingless, and Notch, are involved in vein specification, it is possible that Merlin functions to regulate one or more of these either instead of or in addition to the Egfr pathway. In support of this notion, Merlin and expanded have both been shown to genetically interact with dpp. Further experiments are required to determine the significance of these genetic interactions. Nonetheless, the identification of Merlin modifiers suggests testable hypotheses regarding Merlin cellular functions and opens new avenues for further investigation of the molecular basis of the NF2 disorder (LaJeunesse, 2001).

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).

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Merlin: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation

date revised: 10 December 2006

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