Merlin

REGULATION
Drosophila PI4KIIIalpha is required in follicle cells for oocyte polarization and Hippo signaling

DEVELOPMENTAL BIOLOGY

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).

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).

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

date revised: 5 October 2020

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