Ras oncogene at 85D


Effects of Mutation or Deletion (part 3/3)

Ras pathway and tracheal development

The involvement of Breathless, a Drosophila FGF receptor tyrosine kinase homolog, in border cell migration has prompted an inquiry as to whether RAS, a downstream effector for receptor tyrosine kinases, contributes to receptor tyrosine kinase-mediated motility. A dominant-negative RAS protein inhibits cell migration when expressed specifically in border cells during the period when these cells normally migrate. When expressed prior to migration, dominant-negative RAS promotes premature initiation of migration. Conversely, expression of constitutively active RAS prior to migration results in a significant delay in the initiation step. The defect in initiation of border cell migration found in slbo1, a mutation at the locus that encodes Drosophila C/EBP homolog, is largely rescued by reducing RAS activity in border cells prior to migration. Taken together, these observations indicate that RAS activity plays two distinct roles in the border cells: (1) reduction in RAS activity promotes the initiation of that migration process and (2) RAS activity is required during border cell migration. The possible involvement of two downstream effectors of Ras in border cell migration was also examined. Raf activity was dispensable to border cell migration while reduced Ral activity inhibited initiation. Ra1 is a small GTPase that is activated by RAS. RAS appears to play a critical role in the dynamic regulation of border cell migration via a Raf-independent pathway. It is believed that reducing RAS activity bypasses the normal requirement for SLBO expression for cell migration. The alternative explanation, that SLBO activates the expression of specific receptor tyrosine kinases is held as not tenable (Lee, 1996).

Breathless, a Drosophila FGF receptor homolog, is required for the migration of tracheal cells and the posterior midline glial cells during embryonic development. Deregulated receptors containing the cytoplasmic domains of DFGF-R2, DER, torso, and Sevenless were all able to partially rescue the migration defects. Consistent with the notion that these RTKs share a common signaling pathway, constructs containing the activated downstream elements Dras1 and Draf were also able to rescue tracheal migration, demonstrating that these two proteins are key elements in the DFGF-R1 signaling pathway (Reichman-Fried, 1994).

Rhomboid 3 orchestrates Slit-independent repulsion of tracheal branches at the CNS midline

EGF-receptor ligands act as chemoattractants for migrating epithelial cells during organogenesis and wound healing. Evidence suggests that Rhomboid 3/EGF signalling, which originates from the midline of the Drosophila ventral nerve cord, repels tracheal ganglionic branches and prevents them from crossing the midline. rho3 acts independently from the main midline repellent Slit, and originates from a different sub-population of midline cells: the VUM neurons. Expression of dominant-negative Egfr or Ras induces midline crosses, whereas activation of the Egfr or Ras in the leading cell of the ganglionic branch can induce premature turns away from the midline. This suggests that the level of Egfr intracellular signalling, rather than the asymmetric activation of the receptor on the cell surface, is an important determinant in ganglionic branch repulsion. It is proposed that Egfr activation provides a necessary switch for the interpretation of a yet unknown repellent function of the midline (Gallio, 2004).

The morphogenesis of the embryonic tracheal network depends on the charted migration of ~2000 epithelial cells deriving from 20 epidermal invaginations. These cells undergo three successive rounds of branching to generate a tubular network that extends along stereotyped paths towards specific target tissues. The last branching event produces thin, unicellular terminal branches that associate with distinct organs. The ventral nerve cord (VNC) is invaded by 20 ganglionic branches (GBs), which sprout from the lateral trunk of the trachea. GB migration towards and inside the CNS is highly stereotyped. Each GB initially tracks along the inter-segmental nerve and toward the CNS. GB1, the leading cell of the ganglionic branch, enters the nerve cord and changes substrate to track along the segmental nerve, proceeding ventrally on top of the longitudinal fascicles and towards the CNS midline. Finally, after reaching the midline, GB1 takes a sharp turn and migrates dorsally through the dorsoventral channel and then turns posteriorly on the dorsal side of the VNC. At the end of embryogenesis, GB1 will have trailed a remarkable 50 µm inside the CNS. Genetic analysis has uncovered a number of factors that are necessary for this fixed migratory path: the FGF homolog Branchless is required to guide the GBs towards the CNS and to induce them to enter it, in part by inducing the expression of the nuclear protein Adrift. Once inside the CNS, Slit, the main repulsive cue for axons at the midline, becomes a key guiding cue for the migrating GBs. Slit controls several, distinct aspects of ganglionic branch pathfinding into the CNS: it is first required to attract GBs toward the CNS, an effect mediated by Slit's receptor Robo2, and then to prevent GBs from crossing the midline once they reach it: this is also mediated by Robo (Gallio, 2004 and references therein).

A collection of 2640 P-element insertions was screened for mutants affecting the pathfinding of the ganglionic branch (GB) into the CNS. One of the recovered mutants, named inga (from ingen återvändo, meaning 'no turning back' in Swedish) was characterised by a specific midline-cross phenotype: at stage 16.3-4, upon approaching the CNS midline, a significant number of inga GBs failed to turn posteriorly and dorsally at the midline and crossed to the other side, or remained lingering on it. No other defect was detected in the tracheal system of inga embryos. Sequence analysis of the genomic region surrounding the transposon in inga mutants showed that the P-element was inserted in roughoid/rho3, and all available ru/rho3 alleles as well as inga/Df(3L)Ar14-8 embryos (a chromosomal deficiency removing the 61-62 region). These showed the same tracheal phenotype as inga. Therefore, it was concluded that inga is an allele of rho3 and subsequent analysis focused on a previously characterised null allele (Gallio, 2004),

The essential components of the Egfr signalling pathway were associated with ventral nerve cord development soon after their discovery. rhomboid, spitz and pointed mutants were originally identified for their effect on the ventral ectodermal region. Egfr signalling also plays a central role in the development of the VNC midline, where it is first required for cell differentiation and positioning of midline glia and later for their survival during the late stages of embryogenesis (Gallio, 2004)

The expression of rho3 in VUMs and its function in GB1 guidance away from the midline identifies a new role for Egfr signalling in the VNC. Unlike rho1, rho3 mutants have a normal VNC pattern in which longitudinal connectives and glial populations appear normal, suggesting that rho3 is specifically required for GB1 guidance. Expression of dominant-negative forms of the EGF receptor or Ras in GB1 phenocopies the rho3 guidance phenotype. In addition, overactivation of Egfr signalling in the trachea is sufficient to redirect GB1 and induce early turn phenotypes. Finally, rho3 is required in parallel to slit, the main repulsive cue deriving from midline glia. Taken together, these results suggest that rho3 mutant GB1s are misrouted because of reduced levels of Egfr/Ras signalling in GB1 cells, rather than to indirect, subtle defects of midline patterning or signalling capacity in rho3 mutants. This led to the proposal of a simple model in which Rho3 activates one or more Egfr ligands secreted by the midline cells. Reception of this signal by migrating GBs is mediated by Egfr and Ras, and promotes turning away from the midline (Gallio, 2004)

Three Drosophila Egfr ligands are activated by Rhomboid proteases: Gurken (which is only present in oocytes), Spitz and Keren, the latter expressed in embryos below the detection level of in situ hybridisation or antibody staining. Thus, the ligand activated by Rho3 to guide GB1 migration is very likely Spitz; it is expressed and is functional at the midline, but a contribution by Keren cannot be firmly excluded (Gallio, 2004)

The mammalian EGF receptors regulate migration in a variety of contexts, but in all known examples they appear to promote responses to chemoattractants. They do so by directly affecting cytoskeletal organisation, mainly through the PI3K, PKC or PLC pathways. The proper activation of the fly Egfr is also necessary for the migration of border cells toward the source of Gurken in the dorsal part of the oocyte. During this migration Egfr activation is coordinated with the activation of the fly PDGF/VEGF receptor homologue and requires the conserved adaptor protein Mbc (Dock 180/CED-5). Mbc provides a link to activated Rac and actin re-arrangements, which leads to the stereotyped attraction of the border cells towards the oocyte. It is, however, unclear whether Egfr provides the necessary spatial information for border cells during their pathfinding, or if it is required for the interpretation of positional cues provided by Pdgf/Vegfr or other receptors (Gallio, 2004 and references therein)

Egfr signalling is mediated by a number of downstream effectors in different cell types. In order to determine which one is used in GB1 pathfinding, a panel of mutants and dominant-negative constructs of known downstream effectors were analysed for their effect on GB migration. myoblast city (mbc) is a conserved adaptor necessary for the chemo-attractant function of Gurken during border cell migration in the ovary. mbc alleles had no defects in GB pathfinding. Since mbc has negligible maternal contribution and is not readily detected in tracheal tissues, it is concluded that it is unlikely to have a role in Egfr-mediated GB repulsion from the midline. Two additional effectors were tested that have been implicated in Egfr-elicited migratory responses in other systems: PLCgamma and PI3K. The fly PLCgamma is encoded by the small wing (sl) locus. small wing embryos had extra terminal sprouts emanating from the primary tracheal branches but show no specific defects in GB migration inside the VNC. Deltap60 is a deletion variant of the adaptor p60, which has dominant-negative effects on PI3K activity in vivo and in vitro. SRF-Gal4 driven expression of Deltap60 resulted in a stalling phenotype of 19% of the GBs but not midline crosses. This may reflect a requirement of PI3-K in the early extension of the GBs toward the midline, which is also impaired by the expression of the dominant-negative form of Egfr in GB1 (Gallio, 2004).

The activation of Ras is a necessary step in many of the cellular responses induced by Egfr signalling in Drosophila. It leads to the activation of Raf, and culminates with activation of the Ets-transcription factor Pointed and the nuclear export of Yan, another Ets protein that antagonises Pnt in the activation of target genes. SRF-Gal4-directed expression of a dominant-negative form of Ras results in stalled branches inside or outside the VNC. Importantly, a significant number of GBs was grossly misrouted (8%) or crossed the midline (4%) suggesting that Ras is required in the GB1 cells for their turn away from the midline. The large proportion of arrests in cell migration observed in these experiments might reflect a broader requirement for these common effectors in tracheal cell migration and sprouting (Gallio, 2004).

To analyse whether Egfr mediated repulsion of GB1 from the midline requires Raf or downstream pathway components, a dominant-negative form of Raf and an activated form of Yan were expressed under the control of SRF-Gal4. These constructs caused many of the branches to stall or misroute but in neither case could any branches that crossed the VNC midline be found. As an example, expression of the activated Yan construct stalled the migration of 45% of the GBs, and misrouted an additional 7%, but not a single midline cross was observed (Gallio, 2004).

In summary, activation of Ras appears to be required for repulsion of GB1 from the midline, whereas the remaining components of the pathway are required for tracheal cell extension inside the VNC but not for the decision to cross the midline barrier (Gallio, 2004).

There are substantial differences in the ways by which Egfr controls migration in GB1 and in border cells. This analysis indicates that Egfr signalling is not a chemotactic cue for tracheal pathfinding -- rather, it reveals a surprising role in mediating repulsion from the signalling source. In addition, mbc mutants do not show any midline crossing phenotypes that would resemble the phenotypes of rho3 or the ones generated by inactivation of the receptor. Furthermore, the increase of signalling levels in GB1, either by the expression of Rho1, activated receptor or activated Ras, results in a significant phenotype opposite that of the rho3 mutants: such treatments induced GBs to turn early before reaching the midline. This suggests that at the appropriate distance from the midline, Egfr activation becomes a switch to initiate the turn of GB1 away from it. Hence, an experimental increase of signalling levels can shift the crucial switch further away from the midline, while decreased signalling causes midline crosses. In essence, migrating GBs use Egfr activation to efficiently compute their relative distance from the midline, fine-tuning their response to the repulsive and attractive cues originating from it (Gallio, 2004)

Migration in general, and axonal pathfinding at the midline in particular, is known to rely on a number of guidance signals, at times redundant ones. The major midline repulsive signal for GB1 is Slit, yet a genetic test shows that rho3 acts in parallel to Slit. It is hypothesized that Egfr works in an analogous manner by activating a second, yet undiscovered, signalling system for GB repulsion. Such a guidance cue may be specific for GB1 migration, since axonal fascicles are not affected in rho3 mutants. Alternatively, the activation of Egfr in GB1 provides an epithelial specific regulation of a common repulsive signal used by both axons and GB1 (Gallio, 2004)

What could this repulsive signal be? Likely candidates fall in the short list of conserved signals repelling axons and non-neural cells in different systems: Netrins, Semaphorins and Ephrins. Netrins are involved in the repulsion of motor axons in both vertebrates and invertebrates and both Drosophila Netrins are expressed at the CNS midline, where they mediate attraction of commissural axons. Semaphorins and Ephrins are also capable of repelling axons and non-neural cells in different contexts, and they therefore represent possible guiding cues for GBs. Intriguingly, each family uses receptor tyrosine kinases as receptors (in the case of Ephrin) or co-receptors (in the case of Semaphorins). Most of these signals are bi-functional, they can elicit both attractive and repulsive responses on the receiving cells depending on context. Egfr activation in GB1 may lead to the post-translational modifications that activate a repellent receptor or inactivate an attractant one and may represent a general 'switch' mechanism for changing the orientation of cell migration depending on the strength of RTK signalling (Gallio, 2004)

Fgf and Egf signaling require Ras for tracheal morphogenesis

Branching morphogenesis is a widespread mechanism used to increase the surface area of epithelial organs. Many signaling systems steer development of branched organs, but it is still unclear which cellular processes are regulated by the different pathways. The development of the air sacs of the dorsal thorax of Drosophila was used to study cellular events and their regulation via cell-cell signaling. Two receptor tyrosine kinases play important but distinct roles in air sac outgrowth. Fgf signaling directs cell migration at the tip of the structure, while Egf signaling is instrumental for cell division and cell survival in the growing epithelial structure. Interestingly, Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct migration, suggesting that both cytoskeletal and nuclear events are downstream of receptor activation. Ras and the Mapk pathway are also needed for Egf-regulated cell division/survival, but Pointed is dispensable (Cabernard, 2005).

The air sac of the dorsal thorax grows from a bud that arises during the third larval instar from a wing disc-associated tracheal branch. To illustrate the development of the air sac, a GFP trap line was used that rather ubiquitously expressed membrane bound GFP; tracheal cells were counterstained with an mRFP1-moesin construct under the direct control of the trachea-specific breathless (btl) enhancer. From the early to late third instar stage, a bud-like structure grows out of the transverse connective and spreads on the wing imaginal disc epithelium; this outgrowth corresponds to the primordium of the air sac of the dorsal thorax (Cabernard, 2005).

It has been proposed that the air sac of the dorsal thorax forms de novo from a small group of wing imaginal disc cells, and that the resulting sac subsequently generates a tracheal lumen by an unknown process (Sato, 2002). Since, in the early Drosophila embryo, the lumen arises from an epithelial invagination via cell migration, it was asked whether the cells in the growing air sac are epithelial in nature with a clear apical/basal polarity. For this purpose, a Dα-Catenin-GFP (Dα-Cat-GFP) fusion construct was expressed in the developing air sac and the distribution of GFP from early to late third instar larvae was analyzed. Dα-Cat-GFP labels the adherens junctions (AJs) of epithelial cells. Clearly, the growth of the air sac was accompanied from the early stages onward by an out-bulging of an AJ network, suggesting that most or all of the cells in the growing bud were epithelial in nature, and that a luminal space was generated at the apical side of the epithelial tracheal cells during outgrowth. To confirm this interpretation, use was made of the recent identification of a protein, Piopio (Pio), which is apically secreted into the tracheal lumen in the embryo (Jazwinska, 2003). Indeed, the prospective luminal space in the outgrowing air sac is filled with Pio protein, demonstrating that the air sacs consist of a sac-like epithelial sheet, generating a luminal space as they grow (Cabernard, 2005).

To test whether all cells maintained an apical-basal polarity during air sac budding, single tracheal cells were labelled by using a recently developed assay system that allows for the visualization (and manipulation) of individual tracheal cells in vivo (Ribeiro, 2004). When this scenario was used in the presence of a UAS-Dα-Catenin-GFP chromosome, it was found that, in virtually all cases, such individually labeled air sac cells contacted the lumen and formed AJs with neighboring cells, even when these cells were located at the tip of the outgrowing air sac. The same conclusion was reached when the expression of GFP-moesin was analyzed in single air sac cells; cells at the tip made clear contact with the lumen. Therefore, it is concluded that the air sac is sculpted from an epithelial cell layer, which expands and at the same time generates an apical luminal space filled with secreted proteins (Cabernard, 2005).

It was of interest to better understand how Ras can be used in the same tissue at the same time for different cellular processes. egfr mutant cells can contribute to the tip of the growing air sac, although the clones are relatively small. In the stalk of the air sac, cells lacking Egfr often appeared fragmented, a sign of cell death. Indeed, when egfr mutant cells were sustained with anti-Drice, a marker for apoptotic cells, a strong accumulation of this protein was found. When p35, a viral antiapoptotic protein, was expressed in egfr mutant cell clones, these clones grew to larger sizes and were able to populate the air sac tip at a significantly higher frequency than in the absence of p35. These experiments establish that Egfr is dispensable for migration, and that migration is exclusively triggered by one of the two RTKs, Btl/Fgfr. The experiments also demonstrate that, during the growth phase, Btl/Fgfr signaling is dispensable for cell division; clones can grow to large sizes, although they fail to populate the tip. This same result was obtained with two other components, which are exclusively used by the Fgfr signaling pathway in the air sac (and not the Egfr pathway), namely, Dof and Pointed. Thus, migration and cell division are controlled by two different RTKs, but both RTKs signal via the activation of Ras and the Map kinase pathway to regulate these different cellular outcomes (Cabernard, 2005).

How does Ras control cell migration in the tip and cell division in the remaining air sac? To start to address these questions, whether high levels of constitutive active Ras were compatible with directional cell migration was tested and RasV12 was expressed in wild-type tissue in small cell clones. Interestingly, such clones expanded considerably and grew to large sizes in the center of the air sac or in the stalk, resulting in bulgy outgrowths; however, clones expressing RasV12 never contributed to the tip of the air sac. This finding suggests that unrestricted levels of Ras in a cell perturb its capacity to read out the migratory cues (presumably the Bnl/Fgf ligand); wild-type cells were apparently much better in taking up the leading position. In line with this interpretation, expression of an activated version of Btl (Torso-Btl/Fgfr) also resulted in bulky outgrowths. In addition, cells expressing the chimeric Btl receptor never populated the tip. Quite in contrast, activated Egfr (Egfr fused to a lambda dimerization site) was not able to perturb air sac guidance, but it also triggered higher division rates in clones, generating bulgy outgrowths (Cabernard, 2005).

To test whether single cells expressing activated receptor constructs changed their behavior with regard to cytoskeletal dynamics, the expression of either the activated version of Fgfr or Egfr was induced in early third instar stages and the behavior of such cells was analyzed with live imaging of cultured discs. Cells in the stalk of the air sac expressing activated Fgfr showed extremely dynamic cytoskeletal activity and formed large lamelipodia extending away from the air sac, similar to cells at the tip. Quite in contrast, cells expressing activated Egfr did not show increased lamelipodia formation, and their basal side remained relatively inactive (Cabernard, 2005).

Since the expression of constitutive active versions of the two different RTKs during air sac growth had different effects, whether the endogenous receptors activated the Ras/Mapk pathway to different levels in wild-type air sacs was investigated. In order to monitor the strength of Mapk signaling, an antibody recognizing the double-phosphorylated form of Erk, dpErk were used. Indeed, high levels of dpErk was detected in the nucleus of tip cells; lower dpErk levels were found in the cells in the center of the air sac, and dpErk was mostly cytoplasmic (Cabernard, 2005).

From all of the above-mentioned data, it is concluded that air sac development makes use of two distinct RTKs to control directed organ extension via cell migration (Fgfr) and organ growth via cell division (Egfr). This study carefully analyzed air sac outgrowth from early to late stages, by using a number of different markers labeling either membranes or AJs of individual air sac cells, or the apical luminal compartment. It was found that the thoracic air sac is modeled out of the existing tracheal epithelium, and that a luminal space is generated by the migration of a few cells away from the cuticle of the existing tracheal branch; the luminal space is then expanded by increasing the cell number in the sac-like epithelial structure via cell division. During this process, all cells remain within the epithelium and only round up when they divide. Even those cells that send out filopodia and lamelipodia and migrate in the direction of Bnl/Fgf remain embedded within the epithelium, contact the lumen, and form AJs with their neighbors. Thus, the directed outgrowth of the thoracic air sac during larval development is very similar to the budding of tracheal branches in the early embryo, in that epithelial cells form extensions from the basal side, ultimately resulting in cell movement toward the Fgf ligand. In contrast, during tubule formation of MDCK cells in culture, cells initially depolarize and migrate to form chain-like structures before they repolarize and form the luminal cavity; tubulogenesis is thus accompanied with partial epithelial-to-mesenchymal as well as mesenchymal-to-epithelial transitions. The tube-forming process has been subdivided into different stages such as cyst, extension, chain, cord, and tubule. In the case of the MDCK model system, growth factors have been proposed to trigger branching by inducing a dedifferentiation that allows the monolayer to be remodeled via cell extension and chain formation. Similar to the MDCK system, it was found in Drosophila that growth factor signaling induces the formation of cellular extensions, the first sign of outgrowth. Also, in both systems, cell division is an integral part of the process, but it occurs randomly throughout the structure and not locally at the point of outgrowth. However, two different RTKs are used in the air sac to control extension (migration) and cell division, and chain and chord stages are not observed. It thus appears that both similarities and differences exist between these different cellular systems (Cabernard, 2005).

It has already been reported that cells divide during air sac formation. The cell division rates have been semiquantified and it was found that the elongating structure does not grow preferentially at the tip. The genetic analysis demonstrates that the Egfr is essential for cells to divide and survive efficiently in the air sac. Egfr signals via Ras and the Mapk pathway, but it does not require the Pnt transcription factor to regulate cell division. It is not yet known which ligand activates Egfr, and whether expression of this ligand is induced at early stages of development by Fgf signaling. As shown before (Sato, 2002), the complete lack of Fgfr signaling results in the absence of air sacs; Fgf signaling might thus be used at the onset of the budding process to initiate or trigger cell division, but it is clearly dispensable in later stages. Since cells in the tracheal branch, which gives rise to the air sac primordium, also divide in the absence of Fgf signaling, it is possible that the role of Fgf signaling consists in generating an outgrowth via directed cell movement, triggering cell division indirectly (Cabernard, 2005).

Interestingly, a recent study addressing the role of GDNF/Ret signaling in kidney branching morphogenesis in vivo has shown that ret mutant cells (which are unable to respond to GDNF) can contribute to the primary outgrowth of the ureteric bud, but are excluded from the ampulla that forms at its tip. Apparently, a Ret-dependent proliferation of tip cells under the influence of GDNF controls branch outgrowth. This study found that in Drosophila, in the developing air sac, cells lacking Fgfr are also excluded from the tip. However, evidence is provided that Fgf signaling is translated into directed migration in the leading structure and not into a local increase in cell proliferation. The isolation and cultivation of wing imaginal discs allows for using 4D imaging to document cell behavior during air sac growth. It was found that numerous tip cells extend long filopodia and lamelipodia, similar to the findings reported earlier (Sato, 2002). Tip cells not only produce extensions, but they indeed change their respective position with time, and move forward over the substrate in the direction of the filopodia/lamelipodia. Thus, tip cells are clearly motile and migrate in the direction of Bnl/Fgf. Cell clones incapable of responding to different families of ligands were produced and marked and were examined with regard to their capacity to populate the air sac tip. Among the receptors analyzed, only Btl/Fgfr was strictly required for cells to populate the leading tip of the air sac. Considering the observation that cells in the tip actively migrate, that Btl/Fgfr signaling is required for tracheal cell migration in the embryo, that tracheal cells migrate to ectopic sources of Bnl/Fgf in the embryo and the larva (Sato, 2002), and that cells form numerous filopodia and lamelipodia upon constitutive activation of the Fgf signaling pathway, it is concluded that Fgf steers cell migration in the tip of the air sac and leads to its directional outgrowth on the surface of the wing imaginal disc. The demonstration that the MARCM system can be used to analyze gene function with regard to cell migration in the developing air sac prompted an investigation of the role of Ras and the Mapk pathway in Fgf-directed cell movement (Cabernard, 2005).

Using the MARCM system, it was found that Ras activation is essential for cells to migrate at the tip of the air sac. The requirement for Cnk and Ksr strongly suggests that one important branch downstream of Ras in the control of cell migration is the Mapk pathway. This interpretation is supported by the somewhat surprising finding that the transcription factor Pnt is also strictly required for cell migration. In the Drosophila embryo, genes regulated by Fgf signaling at the transcriptional level and essential for migration have not been identified so far; although both pnt itself and blistered/DSrf are targets of Fgf signaling with important functions in tracheal morphogenesis, they are not required for migration. One possible target of Fgf signaling in the dorsal air sac cells might be the btl/fgfr gene itself. Attempts were made to rescue the pointed defects in air sac development by supplementing a btl transgene under the control of UAS sequences. It was found that even when Btl/Fgfr is provided by the transgenes, pnt mutant clones do not reach the tip. A second gene that might have been a transcriptional target of Pointed is dof; however, it was found that Dof protein is still present in pnt mutant clones (Cabernard, 2005).

The results demonstrate that the outgrowth of the dorsal air sac along the underlying wing imaginal disc is controlled by Btl/Fgfr and Egfr. Fgf signaling is required for directional outgrowth via cell migration, and Egf signaling is required for organ size increase sustaining cell division/cell survival. Both signals use the Ras/Mapk pathway to elicit their cellular responses. To what extent these two pathways regulate different downstream targets is not known at present. However, this study shows that Pointed is only required downstream of Fgf signaling in the control of directed cell migration, and not downstream of Egf signaling in the control of cell division/survival. Since the activation of the Map kinase pathway is much stronger in the cells at the tip as compared to the cells in the central portion or in the stalk of the air sac (according to the levels of dpErk), it is thought that the local availability of the Bnl/Fgf ligand results in a local signaling peak. Egf signaling in more central and proximal cells does not result in a strong activation of the Map kinase pathway, yet this activation is apparently sufficient to control cell division and survival. The independent regulation of cell migration and cell division by two different RTKs might be even more important in later stages of dorsal air sac development, when the growing tip is yet farther away from the main body of the air sac. It will be interesting to find whether other growing branched tissues use similar mechanisms to uncouple directional expansion and size increase (Cabernard, 2005).

Ras and dorsal closure

The Rho subfamily of Ras-related small GTPases participates in a variety of cellular events including organization of the actin cytoskeleton and signaling by c-Jun N-terminal kinase and p38 kinase cascades. These functions of the Rho subfamily are likely to be required in many developmental events. A study has been performed of the participation of the Rho subfamily in dorsal closure (DC) of the Drosophila embryo, a process involving morphogenesis of the epidermis. Expression of dominant negative Ras causes partial loss of the leading edge cytoskeleton, and constitutively active Ras increases Pak levels at the leading edge. Thus, Ras1 has phenotypic effects similar to those of Dcdc42 and Drac1. There is mounting evidence that the Rho subfamily proteins lie downstream of Ras, contributing to the ability of Ras to cause transformation and regulate the actin cytoskeleton. The results of this study are consistent with Ras1 activating Dcdc42 and/or Drac1 during DC, although given that Ras1 expression has milder phenotypic effects than Dcdc42 or Drac1 during DC, it is not proposed that Ras1 is a chief activator of either of these p21s. The finding that constitutively active Ras1 can increase Pak levels at the leading edge is the first demonstration of Ras having an effect on the behavior of a Pak family member. Interestingly, it has recently been shown in mammals that kinase deficient PAK1 mutants can inhibit Ras transformation, indicating that PAK may be a component of Ras signaling. Although constitutively active Ras1, like constitutively active Cdc42, elevates Pak levels at the leading edge, it does not cause the loss of leading edge components seen following constitutively active Cdc42 expression. Looking at these results in the context of the model for Pak function, it may be that constitutively active Ras1 does not increase Pak accumulation at the leading edge to a level sufficient to cause down-regulation of the cytoskeleton (Harden, 1999).

Ras pathway and eye development

In the developing eye of Drosophila, Ras performs three temporally separate functions. In dividing cells, it is required for growth but is not essential for cell cycle progression. In postmitotic cells, it promotes survival and subsequent differentiation of ommatidial cells. The different roles of Ras during eye development have been analyzed by using molecularly defined complete and partial loss-of-function mutations of Ras. The three different functions of Ras are mediated by distinct thresholds of MAPK activity. Low MAPK activity prolongs cell survival and permits differentiation of R8 photoreceptor cells while high or persistent MAPK activity is sufficient to precociously induce R1-R7 photoreceptor differentiation in dividing cells (Halfar, 2001).

How does Ras control growth? One possibility is that Ras directly binds and activates PI3K. Clones mutant for components in the insulin receptor/PI3K pathway also have a growth disadvantage compared to wild-type cells. Although in vertebrates, H-RasG12V,Y40C activates PI3K, no evidence was found that the corresponding mutant activates PI3K in Drosophila. Partial loss-of-function mutations in genes coding for Raf and MAPK, respectively, show similar growth defects as Ras mutants. Furthermore, Ras D38E shows a significant rescue of the growth disadvantage of Ras minus clones. Thus, it is proposed that while cell growth depends on the activities of the MAP kinase as well as the PI3K pathway, the activation of the MAP kinase pathway represents the only Ras function. It has recently been proven that cooperation between the Ras/MAP kinase and the PI3K/PKB pathway is required in order to induce growth in cultured cells. In fibroblasts, activation of Raf and PI3K is required for cyclin D1 expression and entry into S-phase. Induction of DNA synthesis by activation of the platelet-derived growth factor (PDGF) receptor requires an early activation of MAPK and a late phase PI3K activity (Halfar, 2001 and references therein).

Ras mutant cells located behind the morphogenetic furrow die by programmed cell death (PCD). Ras controls the PCD inducer Hid, by repressing its expression and by modifying its activity through phosphorylation by MAPK. In mammalian cells, PI3K promotes survival via PKB-mediated phosphorylation of the pro-apoptotic protein Bad. Thus, survival could at least in part be mediated by the activation of PI3K. Indeed, a partial suppression of Hid-induced apoptosis in the eye by the expression of RasG12V,Y40C (providing high levels of Ras activation) has been taken as evidence that PI3K supports survival in the developing eye. This is unlikely in the light of the data presented here, since it has been shown that RasG12V,Y40C is unable to activate PI3K. Furthermore, the RasD38E transgene, providing low levels of Ras activation, rescues Ras minus cells posterior to the morphogenetic furrow from PCD. Thus it appears that the function of Ras in survival is mediated exclusively through the activation of MAPK. In the adult, however, Ras mutant cells were never observed. This may be due to an exclusive role of Ras in promoting cell survival of ommatidial cells at later stages or due to an additional role in cell fate specification. Several lines of evidence argue against an exclusively anti-apoptotic role of Ras during the later stages of eye development: (1) reduced Ras activity in the R7 precursor cell in the absence of the Sev receptor tyrosine kinase results in a change in cell fate rather than death of this progenitor cell; (2) constitutive activation of Ras in cone cell precursors is sufficient to induce R7 differentiation in these cells; (3) ectopic expression of an activated EGF receptor or RasG12V results in precocious induction of photoreceptor cell differentiation anterior to the furrow. Thus in the case of R1-R7 differentiation, high levels of Ras activity are required for a choice in cell fate rather than mere survival of the cells. The differentiation of R8 cells, which depends on Ras activity, however, may be different in that Ras may be required in the R8 photoreceptor for survival only. R8 cell differentiation is rescued by RasD38E, concomitant with the survival of the mutant clones. Therefore it is possible that Ras-mediated survival is sufficient for R8 cell differentiation. Interestingly, loss of EGF receptor function still allows the formation of R8 cells, suggesting that the low levels of Ras activity required for R8 differentiation are achieved by another receptor system (Halfar, 2001).

There are three different models for how specificity of Ras signaling is achieved: specificity may be controlled by (1) the cellular context, (2) the activation of distinct signaling pathways by Ras or (3) by different levels of Ras activity. The experiments presented here support the importance of the cellular context and the different levels of Ras activity but fail to provide evidence for the activation of different signaling pathways by Ras. All aspects of Ras signaling could be rescued by the activation of the Ras/MAPK pathway and no evidence was found (using the Ras effector site mutants) that constitutively active Ras activates the PI3K pathway directly (Halfar, 2001).

The cellular context in which Ras activates MAP kinase is clearly important. Expression of RasG12V in blastoderm cells triggers differentiation of head and tail structures, it triggers vein differentiation in wing disc cells and neuronal differentiation in eye disc cells. Different levels of Ras/MAPK activity appear to control distinct cellular responses within the same tissue. Low levels of Ras activity, provided by the RasD38E mutant, rescue R8 differentiation and survival but not R1-R7 differentiation. High levels of Ras/MAPK activity provided by wild-type Ras or by a combination of RasD38E and rolledSem are required for the differentiation of R1-R7 photoreceptor cells (Halfar, 2001).

There are two possibilities with regard to the nature of the activity thresholds that elicit the different cellular responses. The threshold may be quantitative. Cells could react to different activity levels within the cells. Alternatively, the threshold may be temporal and cells react to the difference in the duration of the signal. Staining of imaginal discs with an antibody that selectively recognizes activated MAPK (dpERK) was not sensitive enough to detect activated MAPK during normal photoreceptor cell recruitment or during ectopic neuronal differentiation in RasG12V-expressing clones anterior to the morphogenetic furrow (Halfar, 2001).

Therefore, it was not possible to distinguish between these two models. In the present case, however, the temporal model is favored because the highest levels of dpERK staining could not be detected behind the morphogenetic furrow during photoreceptor cell recruitment. In response to MAP kinase activation in the developing eye, a number of negative regulators of the pathway are induced. The EGF-related peptide Argos competes with the TGFalpha-like ligand Spitz for EGF receptor binding, and Sprouty, a cytoplasmic protein, associates with the EGF receptor to turn off the signaling pathway. Indeed, neuronal differentiation and ommatidial development in the RasD38E mutant is rescued by the prolonged activity of MAPK caused by the rlSem mutation. It is possible that the reduced activity of RasD38E towards Raf is caused by a more rapid inactivation, owing to increased GTPase activity. The observation that RasG12V,D38E is sufficient to induce neuronal differentiation ahead of the furrow, in conjunction with the G12V substitution, which inactivates the Ras GTPase activity, is consistent with the idea that D38E may stimulate GTP hydrolysis. Thus, neuronal differentiation in Drosophila may depend on the prolonged activation of Ras/MAP kinase, whereas transient activation is sufficient for survival upon exit from the cell cycle and differentiation of R8 photoreceptor cells. Therefore it appears that neuronal differentiation in response to Ras activation in the developing eye of Drosophila is similar to neuronal differentiation in PC12 cells: this also requires prolonged activation of MAPK. The modulation of levels and/or the duration of Ras/MAPK activity levels appear to be important determinants of cellular responses in multicellular organisms (Halfar, 2001).

Embryos lacking Jun activity exhibit a dorsal closure phenotype, very similar to that of basket and hemipterous mutants, indicating that Jun is a target of Hep/Bsk signaling. In eye and wing development Jun participates in a separate signaling pathway comprised of Ras, Raf, and the ERK-type kinase Rolled. In contrast to the strict requirement for Jun in dorsal closure, its role in the eye is redundant but can be uncovered by mutations in other signaling components. The removal of Jun function in the eye by mutation shows only minor defects. Occasionally, only one or two photoreceptors are lost in mutant ommatidia. Nevertheless, gain- and loss-of-function forms of Jun interfere specifically with the endogenously expressed wild-type protein, and Jun interacts genetically with the Sev/Ras/Raf/ERK signal transduction pathway. For example, when one copy of DJun is removed from transgenic lines expressing gain of function sevenless, ras and rolled mutations, a clear suppression of the mutant extra photoreceptor phenotype can be observed. The redundant function of Jun in eye development may contribute to the precision of photoreceptor differentiation and ommatidial assembly. Analysis of DJun mutants in the wing does not reveal any phenotypic defect characteristic of the Ras pathway. Nevertheless, removal of one copy of DJun suppresses the wing phenotypic defects of Ellipse gain-of-function alleles of the Epidermal growth factor receptor. It is concluded that DJun plays a role both in wing and eye development. It is suggested that the role of DJun in the wing and eye is not essential since other systems maintain proper morphogenesis in the absence of DJun. It is also concluded that DJun is a target of both JNK and MAP kinase in Drosophila (Kockel, 1997b).

The cellular functions of the Drosophila Src oncogene 1 (Dsrc) gene product, Dsrc, and of most vertebrate Src-family kinases, are unknown. The effects of over-expression of wild type and mutated forms of Dsrc were studied in transgenic Drosophila. Expression of both wild type Dsrc and a C-terminally truncated mutant at high levels during embryonic development induces extensive tyrosine phosphorylation of cellular proteins and causes considerable lethality, correlating with a block to germ-band retraction. Over-expression in the eye imaginal disc leads to excess production of photoreceptor cells in the adult ommatidia. In contrast, expression of a kinase-inactive form of Dsrc causes distinct nervous system abnormalities in embryos and decreases the numbers of photoreceptor cells in the adult eye ommatidia. This suggests that active forms of Dsrc alter development by phosphorylation. Both the lethality and the eye roughening caused by activated Dsrc are partially suppressed by mutations in the Drosophila Ras1 gene. These results suggest that over-expressed Dsrc may function through Ras1 to stimulate differentiation in the embryonic nervous system and eye imaginal disc, and that kinase-active Dsrc interferes with these processes (Kussick, 1993).

In Drosophila, Src oncogene 1 was considered a unique ortholog of the vertebrate c-src; however, more recent evidence has been shown to the contrary. The closest relative of vertebrate c-src found to date in Drosophila is not Dsrc64, but Dsrc41, a gene identified for the first time in this paper. In contrast to Src64, overexpression of wild-type Src41 causes little or no appreciable phenotypic change in Drosophila. Both gain-of-function and dominant-negative mutations of Src41 cause the formation of supernumerary R7-type neurons, suppressible by one-dose reduction of boss, sevenless, Ras1, or other genes involved in the Sev pathway. Dominant-negative mutant phenotypes are suppressed and enhanced, respectively, by increasing and decreasing the copy number of wild-type Src41. The colocalization of Src41 protein, actin fibers and DE-cadherin, as well as the Src41-dependent disorganization of actin fibers and putative adherens junctions in precluster cells, suggest that Src41 may be involved in the regulation of cytoskeleton organization and cell-cell contacts in developing ommatidia (Takahashi, 1996).

The onset of pattern formation in the developing Drosophila eye is marked by the simultaneous synchronization of all cells in the G1 phase of the cell cycle. These cells will then either commit to another round of cell division or differentiate into neurons. rux functions as a negative regulator of G1 progression in the developing eye. rux is suppressed by mutations in genes that promote cell cycle progression (i.e., cyclin A and string) and enhanced by mutations in genes that promote differentiation (i.e., Ras1 and Star) (Thomas, 1994).

The spitz gene is required for photoreceptor determination. Mosaic analysis suggests that spitz, which encodes a TGF alpha homolog, produces a diffusible signal during ommatidial development. Other members of the spitz group and the EGF receptor also interact with sevenless-rhomboid, in a pattern that suggests a model in which rhomboid can act as a mediator of a ligand-receptor interaction between spitz and Egfr in the developing eye. These data suggest that photoreceptors other than R7 use a Ras1 signaling pathway activated by the spitz/Egfr interaction, in a manner analogous to the Ras1 pathway activated by boss/sevenless in photoreceptor R7 (Freeman, 1994).

The photoreceptor cells R8, R2, and R5 are the first cells to initiate neuronal differentiation in the Drosophila eye imaginal disc. These three cells require Star gene function for proper ommatidial assembly. Star is also required for the formation of wing veins and is expressed in developing veins, suggesting that at least partially overlapping pathways may operate during photoreceptor cell differentiation and wing vein formation. The role of Star in cell-cell signaling is supported by the observation of genetic interactions between Star and mutations that reduce signaling through both sevenless and the Drosophila EGF-receptor homolog(s), including Ras1 and Son of sevenless (Heberlein, 1993).

lozenge exhibits a significant interaction with ras1. Sprite, a gain of function allele of lozenge was combined with ras1 mutation. Whereas only 5% of Sprite ommatidia contain fewer than the normal complement of R photoreceptor cells (albeit transformed into R7 fate), in a ras/Sprite mutant background, 83% of the ommatidia have lost at least one photoreceptor. It is unclear from this result whether both Lozenge and Ras pathway directly regulate Seven-up, or whether the Ras pathway acts on a downstream target of Seven-up (Daga, 1996).

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway that is essential very early in eye development, prior to facet assembly, to limit to eight the number of photoreceptor cells in each facet of the compound eye. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. The results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development (Q. Li, 1997).

Cone cells are lens-secreting cells in ommatidia, the unit eyes that compose the compound eye of Drosophila. Each ommatidium contains four cone cells derived from precursor cells of the R7 equivalence group which expresses the gene sevenless (sev). When a constitutively active form of Ras1 (Ras1V12) is expressed in the R7 equivalence group cells using the sev promoter (sev-Ras1V12), additional cone cells are formed in the ommatidium. Expression of Ras1N17, a dominant negative form of Ras1, results in the formation of 1-3 fewer cone cells than normal in the ommatidium. The effects of Ras1 variants on cone cell formation are modulated by changing the gene dosage at the canoe locus, which encodes a cytoplasmic protein with Ras-binding activity. An increase or decrease in gene dosage potentiates the sev-Ras1v12 action, leading to marked induction of cone cells. A decrease in cno+ activity also enhances the sev-Ras1N17 action, resulting in a further decrease in the number of cone cells contained in the ommatidium. In the absence of expression of sev-Ras1V12 or sev-Ras1N17, an overdose of wild-type cno (cno+) promotes cone cell formation, while a significant reduction in cno+ activity results in the formation of 1-3 fewer cone cells than normal in the ommatidium. It is proposed that there are two signaling pathways in cone cell development, one for its promotion and the other for its repression; Cno is thought to function as a negative regulator for both pathways. It is also postulated that Cno acts predominantly on a prevailing pathway in a given developmental context, thereby resulting in either an increase or a decrease in the number of cone cells per ommatidium. The extra cone cells resulting from the interplay of Ras1v12 and Cno are generated from a pool of undifferentiated cells, normally fated to either develop into pigment cells or undergo apoptosis (Matsuo, 1997).

The role of Ras signaling was studied in the regulation of cell death during Drosophila eye development. Overexpression of Argos, a diffusible inhibitor of the EGF receptor and Ras signaling, causes excessive cell death in developing eyes at pupal stages. The Argos-induced cell death is suppressed by coexpression of the anti-apoptotic genes p35, diap1, or diap2 in the eye as well as by the Df(3L)H99 chromosomal deletion that lacks three apoptosis-inducing genes, reaper, head involution defective (hid) and grim. Transient misexpression of the activated Ras1 protein (Ras1V12) later in pupal development suppresses the Argos-induced cell death. Thus, Argos-induced cell death seems to have resulted from the suppression of the anti-apoptotic function of Ras. Conversely, cell death induced by overexpression of Hid is suppressed by gain-of-function mutations of the genes coding for MEK and ERK. These results support the idea that Ras signaling functions in two distinct processes during eye development, first triggering the recruitment of cells and later negatively regulating cell death (Sawamoto, 1998).

Local induction of patterning and programmed cell death in the developing Drosophila retina

The Drosophila retina represents a particularly accessible tissue to address issues of local cell-cell signaling. Correct pattern is achieved in the Drosophila retina in part through the temporal and spatial control of programmed cell death (PCD). The mature retina is composed of an organized array of some 750 unit eyes (ommatidia), each containing eight photoreceptor neurons, four cone cells, two primary pigment cells (1os), and a hexagonal lattice composed of secondary/tertiary pigment cells (2o/3os) and sensory bristle organules. With the possible exception of the cells of the bristle organule, cell fates in the retina are not determined through lineage-based restriction but instead rely on local signals passed between cells. These signals result in progressive recruitment of undifferentiated cells by their previously differentiated neighbors. Creation of the interommatidial lattice of 2o/3os is the result of the final cell fate decision in the retina: some cells are recruited as 2o/3os, while any remaining excess cells are removed by PCD. Two different cell types have been proposed to be the major regulators of cell death in the retina: 1os and cells of the bristle organule. 1os were implicated as potential regulators of PCD by experiments examining Notch loss-of-function alleles: reduction of Notch function led to loss of both 1os and PCD, leading to the suggestion that 1os direct PCD. Alternatively, bristles have been proposed as regulators of PCD in the retina due to clustering of apoptotic cells (detected by acridine orange staining) around bristle organules. More recent experiments indicate that cell death can occur in the absence of bristles, although their presence may still influence PCD. Evidence is provided that the cone cells and 1os provide a signal that promotes survival of cells in the interommatidial lattice. Further evidence is provided that this signal represents part of a balance between signals of the ras and Notch pathways, which appear to act in opposition to regulate the number of interommatidial cells permitted to remain (D. T. Miller, 1998).

The first cell types to emerge in the developing retina are the photoreceptor neurons and (non-neuronal) cone cells, which arise within the retinal neuroepithelium of the mature larva. The larva then undergoes pupation as the retina evaginates (disc eversion) and is repositioned to lie distally against the pupa’s cuticle. Soon after disc eversion, the 1os emerge to enwrap the cone cells (22-24 hours APF). They establish direct contact with the remaining undifferentiated cells which lie between ommatidia, and which are referred to as interommatidial precursor cells or ‘IPCs’. Finally, a hexagonal lattice is formed between ommatidia as IPCs are directed into one of two fates: 2o/3o or PCD. The result is a precise hexagonal array of ommatidia, each surrounded by nine 2o/3os and three bristles. Each cell type in the developing retina can be recognized by the position of its nucleus. Typically, nuclei are first found in the basal part of the neuroepithelium and rise apically as a cell begins its differentiation. During early pupal stages, cone cell nuclei are arranged as an apical ‘cloverleaf’ at the center of each ommatidium and several microns above the photoreceptor nuclei; two 1o nuclei form an apical ring around the cone cells; and the IPC nuclei are found basally between ommatidia (these nuclei are slow to rise apically except bristle nuclei, which are found at an intermediate level early in their differentiation). This stereotyped arrangement permits identification and ablation of each cell type. Experimentally induced ablation alters the arrangement and subsequent identity of cells in the retina, in order to understand the underlying mechanism of cell fate determination. Once the ablation is performed, pupae are permitted to develop for an additional 24 hours to allow for establishment of all cell types; retinae are then removed and stained with cobalt sulfide to highlight each cell type at the surface. In each experiment, the non-ablated partner is used as an internal control. The effects of ablation are limited to the target cell, with little apparent collateral damage to neighboring cells as assessed by their normal subsequent development (D. T. Miller, 1998).

Disc eversion is complete by 18 hours APF at 25oC; the first indication of 1o differentiation is the apical migration of its nucleus at 22-24 hours APF. In initial studies, laser ablation of a 1o at this stage results in its rapid replacement. 1o nuclei ablated after 24 hours APF are not replaced. With regard to establishment of the 1o fate, these results indicate: (1) several cells have the potential to differentiate as 1os; (2) this decision remains reversible for several hours; and (3) during this period, established 1os provide a signal inhibiting the 1o fate in their neighbors. Loss of pupal Notch activity blocks both 1o differentiation and PCD, leading to the suggestion that 1os promote PCD. Ras signaling promotes the 2o/3o fate at the expense of PCD. One pathway which can act in opposition to Notch is the Ras signal transduction pathway Ras is required for a variety of cell fate decisions in the developing retina. To test the role of Ras signaling during PCD, flies were used in which an inducible heat shock promoter was fused to the activated Ras form Dras1 Val12. A 1-hour pulse of Dras1 Val12 throughout the retina beginning at 26 hours APF rescues IPCs from PCD. Early removal of cone cells and 1os in four neighboring ommatidia has no effect on this rescue. This result indicates that Ras signaling acts to prevent PCD and/or promote the 2o/3o fate. With regard to PCD, therefore, Ras acts in opposition to Notch signaling (D. T. Miller, 1998).

This Ras-mediated rescue of cells is similar to, and epistatic to, the rescue provided by cone cells and 1os. Are the two signals linked? The Ras pathway has been demonstrated to be activated by a variety of extracellular stimuli, including signaling through receptor tyrosine kinases (RTKs). In the developing retina, the Egf receptor ortholog is an RTK that regulates a variety of cell fate determination steps including 2o/3o determination. Consistent with the results described above for activation of Dras1, loss of Egfr activity leads to a loss of 2o/3os, presumably due to an excess of PCD. To determine whether Egfr signaling is sufficient to block PCD, flies containing an activated form of Egfr (l-DER) fused to an inducible heat shock promoter received a 1-hour heat shock. Expression of l-DER throughout the young pupal retina results in a block in PCD. The loss of PCD is not complete, perhaps due to the relatively weak activation of Egfr provided by the l-DER protein. Egfr is a receptor that acts autonomously: Egfr expression in IPCs is anticipated in the cells where it is active during the stage of PCD. Consistent with this view, Egfr is found to be expressed primarily in the IPCs. These results suggest the possibility that IPCs receive a signal from their neighbors that activates their own Egfr signaling and represses PCD. The ablation results suggest this signal is derived from the 1os and perhaps the cone cells. Interestingly, the TGFalpha ortholog, Spitz, is expressed at high levels in the cone cells and bristles and can be detected at lower levels in the 1os. Spitz is a diffusible ligand of Egfr and may represent the 'life' signal provided by the ommatidium. Together, these observations suggest a model in which patterning requires local Spitz/Egfr signaling by (at least two) 1os to rescue neighboring IPCs from a Notch-imposed apoptotic fate. One important test of this model will require the removal of spitz function specifically in cones cells and 1os (D. T. Miller, 1998).

Role of the EGFR/Ras/Raf pathway in specification of photoreceptor cells in the Drosophila retina

The Drosophila Egfr receptor is required for differentiation of many cell types during eye development. Mosaic analyses with definitive null mutations were used to analyze the effects of complete absence of Egfr, Ras or Raf proteins during eye development. The Egfr, ras and raf genes are each found to be essential for recruitment of R1-R7 cells. In addition Egfr is autonomously required for MAP kinase activation. Egfr is not essential for R8 cell specification, either alone or redundantly with any other receptor that acts through Ras or Raf, or by activating MAP kinase. As with Egfr, loss of ras or raf perturbs the spacing and arrangement of R8 precursor cells. R8 cell spacing is not affected by loss of argos in posteriorly juxtaposed cells, which rules out a model in which Egfr acts through argos expression to position R8 specification in register between adjacent columns of ommatidia. The R8 spacing role of the Egfr is partially affected by simultaneous deletion of spitz and vein, two ligand genes, but the data suggest that Egfr activation independent of spitz and vein is also involved. The results prove that R8 photoreceptors are specified and positioned by distinct mechanisms from photoreceptors R1-R7 (Yang, 2001).

It is thought that EGFR activity is required for recruiting R1- R7 photoreceptor cells to ommatidia, probably through Ras, Raf and MAPK but the role of this pathway in R8 specification has been less clear. Loss-of-function studies with putative Egfr null clones or temperature sensitivity have suggested that Egfr is dispensable for R8 specification (although involved in R8 spacing); studies with dominant negative approaches have suggested that Egfr is essential for R8 specification. There is also a particular class of Egfr mutants, the Elp alleles, that prevent R8 specification, and there is evidence that R8 specification might depend on Egfr-independent Raf activation. A study of null mutations in the Egfr/Ras/Raf pathway has been undertaken to resolve some of these issues. Two prior studies of Egfr mutant clones used the genetically amorphic point mutations flb1K35 and topCO. For topCO the molecular defect is unknown; flb1K35 corresponds to Gln267 in Ochre, which truncates the Egfr early in the extracellular domain. Although it is a reasonable assumption that these are both null alleles, it is worth noting that another mutation encoding Gln430 in Amber (top38) retains significant function, so the possibility of residual function in topCO or flb1K35 caused by readthrough, translational reinitiation or other mechanisms cannot be completely excluded. However, these possibilities can be excluded for the allele top18A, which deletes all Egfr-coding sequences from the genome. The phenotype of top18A clones is similar to flb1K35 and topCO. ato is expressed in top18A clones. It is concluded that cells completely lacking Egfr-coding capacity can still differentiate R8 photoreceptor cells, although their patterning is abnormal and they later die. Cells that completely lack Egfr are not recruited as any other photoreceptor type (Yang, 2001).

By the late third instar, cells in mutant clones have lacked Egfr gene function for approximately 120 hours. It is possible that cells might have a homeostatic mechanism (such as upregulation of another receptor) that compensates for sustained absence of Egfr function, and that some processes that would be Egfr-dependent in normal eye cells have been rescued in the clones. There is experimental evidence for such homeostasis from studies of the Egfrts2 allele. When Egfr function is interrupted, MAP kinase activation is lost from eye discs within 30 minutes, but levels of activated MAP kinase rebound within a few hours, even in the continued absence of Egfr function. MAP kinase activation was examined within clones of Egfr mutant cells. MAP kinase activation is undetectable. Thus, specification of R8 cells in Egfr mutant clones is not associated with MAP kinase reactivation via an alternative pathway. This finding indicates that the restored dpERK staining seen in the Egfrts2 allele must depend nonautonomously on loss of Egfr function in other cells. For example, loss of Egfr function from the whole animal may lead to changes in humoral signals that nonautonomously affect MAPK by some mechanism (Yang, 2001).

Genetic studies suggest that specification of most ommatidial cells depends on activation of Ras and Raf by Egfr (or by Egfr and Sevenless in the case of R7). R8 cell specification in the absence of Egfr might indicate activation of Ras and Raf by another receptor. Clones of cells null for Ras or Raf have been examined to test this. The null phenotype of Ras closely resembles that of Egfr. Ato expression initiated normally but patterning is affected and more cells than normal retain atonal expression posterior to the furrow. R8 cells are specified and express the R8 protein Senseless. No other Elav-expressing photoreceptor cells are recruited (Yang, 2001).

The phenotype of clones mutant for raf is similar. R8 cell specification begins relatively normally, as indicated by onset of Ato and Senseless expression. R8 cell precursors are improperly spaced, however. More posteriorly, raf mutant R8 cells express the neural protein Elav only transiently. These results also confirm directly that Ras and Raf are required for R1-R7 recruitment, and show that after these clones are induced in the first larval instar, Ras and Raf play no essential roles in the proliferation, survival or maintenance of eye disc identity of most eye disc cells (Yang, 2001).

Since null clones for Egfr, ras, and raf each permit R8 specification, although they affect R8 spacing, it is concluded that R8 specification can occur independently of Egfr, and is also independent of any other receptor that acts through Ras and Raf. Although the requirement for MAP kinase has not been tested directly (since the MAP kinase gene rolled maps proximal to all extant flip recombination target [FRT] sites), it was found that MAP kinase activation is undetectable in Egfr-null clones (Yang, 2001).

For both Egfr and ras, there is a nonautonomous delay of morphogenetic furrow movement and loss of ato, especially in large clones with substantial areas of mutant cells posterior to the furrow. This suggests Egfr and ras are required for expression of factors that push the morphogenetic furrow across the eye disc. Two such factors are Hh and Dpp. Hh is reported to be expressed by photoreceptor cells; therefore, fewer cells are expected to express Hh in ras or Egfr clones. There were some differences between clones mutant for raf and clones mutant for ras or Egfr. Less Elav is detected in raf mutant cells. In Egfr or ras mutant clones, Elav protein is detected in the mutant R8 cells, although at lower levels than in nearby wild type cells. In Egfr mutant clones, normal levels of Elav protein are restored by expression of baculovirus p35, indicating that low Elav levels reflect commitment of Egfr mutant cells to apoptosis. It is possible that Elav is lost more rapidly in raf mutant cells because of more rapid apoptosis than Egfr or ras mutant clones. Delayed furrow progression was not seen in raf mutant clones, but this may be because they were too small (Yang, 2001).

The differences between raf clones and Egfr or ras clones could indicate ras-independent signaling to raf, as has been proposed to occur during the determination of the embryonic termini. Such signaling to permit Elav expression in more R8 precursor cells (or preserve R8 precursor cells from apoptosis for longer) would have to be independent of Egfr as well, whereas all raf activity in the embryonic termini is dependent on torso, the relevant receptor. An alternative explanation is that these apparent differences relate to the much smaller size of raf clones compared with Egfr and ras clones. For the autosomal Egfr and ras mutations, the Minute technique was used to compensate for the growth disadvantage of the homozygous cells. This is not readily possible for the X-linked raf mutation. As a consequence, the raf clones examined were much smaller than the Egfr and ras clones, and grew at a reduced rate relative to neighboring wild-type cells. In the similar situation of Minute heterozygous clones growing slowly in wild-type backgrounds, nonautonomous interactions have been demonstrated, prolonging the cell doubling time of the slow-growing M/+ cells, and accelerating the doubling time of neighboring wild-type cells. If changes in cellular properties are also induced by the differential growth of neighboring homozygous raf mutant and wild-type cells, it is possible that faster loss of Elav might not indicate additional roles for raf in differentiation or survival, but an indirect effect of competition by the nearby wild-type cells on the raf minus cells. At present, experimental evidence to distinguish these models is not available (Yang, 2001).

The common requirements for Egfr, ras and raf in R8 spacing are not shown by null mutations in spi, which codes for an Egfr ligand required for recruitment of R1-R7. It is possible that spi is required redundantly with vn, another ligand with no essential role in ommatidium development. It was found that R8 precursor specification occurs in clones doubly mutant for both spi and vn. R8 spacing occurs almost normally, although there are rare cases of multiple R8 cells like those that occur more frequently in Egfr mutant clones. This raises the possibility that spi and vn do have redundant roles in R8 precursor spacing, but if this is so, there must be another ligand, or ligand-independent process, that is also involved. It has been found that the Drosophila genome sequence predicts another Spi-like protein. Cells doubly mutant for two putative ligand processing molecules encoded by rhomboid and roughoid resemble cells mutant for the Egfr. This suggests that rhomboid and roughoid redundantly process spi and spi-like, which act redundantly on Egfr in R8 spacing. The spi, spi-like double- and spi, spi-like, vn triple-mutant combinations that would directly test the relative contributions of all three ligands have yet to be examined (Yang, 2001).

The inhibitory ligand Argos is also required nonautonomously for R8 spacing. It had been suggested that Argos could diffuse from proneural intermediate groups, where it is expressed in response to Egfr activation, creating an 'exclusion zone' for further Egfr activation that will position future intermediate groups precisely out of phase. It was found, however, that Argos function can be performed by protein secreted several ommatidia away, which questions whether Argos conveys precise spatial information. Crucially, proneural intermediate groups are positioned normally even if immediately posterior regions are null mutant for argos, refuting the 'exclusion zone' model for argos action. Larger argos clones do affect R8 spacing distant from the clone boundary, suggesting that argos may be globally necessary in an unpatterned way to keep Egfr activity in check. An alternative is that argos is required indirectly through its effect on photoreceptor differentiation. Accordingly, ectopic photoreceptor cells in argos mutant territories might alter the expression of furrow progression signals such as Dpp and Hh (Yang, 2001).

The main result of this study is that R8 precursor specification occurs in cells null for Egfr, ras or raf. This is consistent with the proposed Egfr/Ras/Raf pathway of recruitment for photoreceptors R1-R7. These results appear definitively to exclude essential roles for Egfr, ras, raf, spi or vn, in R8 specification (although they support roles in R8 spacing), and show that argos is dispensable for the proposed signaling by each pair of proneural intermediate groups; each pair positions R8 specification in the next most anterior column. It is thought that R8 specification instead relies on autoregulatory transcription of the proneural ato gene promoted by two other DNA-binding proteins, daughterless and senseless that can occur without Egfr signaling. Defects in arrangement of R8 cell precursors show that the Egfr/Ras/Raf pathway nevertheless plays a role in the patterning of R8 cells. The increased number of R8 cells in mutants indicates that Egfr normally activates Ras and Raf to suppress R8 specification in certain locations. The Egfr pathway might modulate Notch. However, the Egfr requirement for R8 spacing was found to be more autonomous than the Egfr requirement for E(spl) expression, raising the possibility of another target. One candidate is the homeobox gene rough (Yang, 2001).

Notch activation of yan expression is antagonized by RTK/Pointed signaling in the Drosophila eye

Receptor tyrosine kinase (RTK) signaling plays an instructive role in cell fate decisions, whereas Notch signaling is often involved in restricting cellular competence for differentiation. Genetic interactions between these two evolutionarily conserved pathways have been extensively documented. The underlying molecular mechanisms, however, are not well understood. Yan, an Ets transcriptional repressor that blocks cellular potential for specification and differentiation, is a target of Notch signaling during Drosophila eye development. The Suppressor of Hairless (Su[H]) protein of the Notch pathway is required for activating yan expression, and Su(H) binds directly to an eye-specific yan enhancer in vitro. In contrast, yan expression is repressed by Pointed (Pnt), which is a key component of the RTK pathway. Pnt binds specifically to the yan enhancer and competes with Su(H) for DNA binding. This competition illustrates a potential mechanism for RTK and Notch signals to oppose each other. Thus, yan serves as a common target of Notch/Su(H) and RTK/Pointed signaling pathways during cell fate specification (Rohrbaugh, 2002).

A role for RTK signaling in regulating yan transcription was investigated. When the RTK pathway is constitutively activated by torD-DER or Ras1V12, the yan enhancer activity is greatly reduced. Thus, RTK signaling appears to negatively regulate yan transcription, in addition to its effect on Yan protein stability. Evidence supports a view that the inhibitory effect of RTK/Ras1 signaling on yan expression is mediated through the pointed (pnt) gene. Taken together, the results demonstrated that Pnt negatively regulates yan expression, and it is likely that Pnt is directly involved in repressing yan transcription. Although a role for Pnt as a transcriptional repressor has not been extensively investigated, pnt has been shown to negatively regulate hid transcription in embryos. Interestingly, a P-DLS motif is present in the Pnt protein (amino acids 356–360 in PntP1), which might mediate interaction with the transcriptional corepressor dCtBP. At this point, the data does not exclude the possibility that Pnt might also activate expression of a repressor, which in turn switches off yan transcription (Rohrbaugh, 2002).

rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

ras1 is a Drosophila homolog of the human ras genes (H-ras, Ki-ras, and N-ras). Ras1 is a GTPase, which functions as the key transducer in several of the receptor tyrosine kinase-activated cellular signal transduction pathways. In the developing eye, ras1 is required for the specification of photoreceptors as well as cone cells. Reduction or loss of Ras1 activity results in the failure of photoreceptor cell determination. A constitutively active form of Ras1 (Rasv12) results in the overrecruitment of retinal cells. The effects were tested of the ras1 mutations on the rg eye phenotype. A 50% reduction in ras1 activity acts as a dominant enhancer of the rg rough eye phenotype. In addition, a single copy of the dominant negative mutant form of RasN17 acts as a strong enhancer of the rg eye phenotype. In these experiments, a single copy of the constitutively active Rasv12 was a weak suppressor of the rough eye phenotype of rg. These data suggest that Ras1 and rugose interact in a dose-dependent manner and may function synergistically in retinal pattern formation (Shamloula, 2002).

Cell cycle withdrawal, progression, and cell survival regulation by EGFR and its effectors in the differentiating Drosophila eye

Receptor tyrosine kinases such as the EGF receptor transduce extracellular signals into multiple cellular responses. In the developing Drosophila eye, Egfr activity triggers cell differentiation. This study focuses on three additional cell autonomous aspects of Egfr function and their coordination with differentiation, namely, withdrawal from the cell cycle, mitosis, and cell survival. Whereas differentiation requires intense signaling, dependent on multiple reinforcing ligands, lesser Egfr activity maintains cell cycle arrest, promotes mitosis, and protects against cell death. Each response requires the same Ras, Raf, MAPK, and Pnt signal transduction pathway. Mitotic and survival responses also involve Pnt-independent branches, perhaps explaining how survival and mitosis can occur independently. These results suggest that, rather than triggering all or none responses, Egfr coordinates partially independent processes as the eye differentiates (Yang, 2003).

Focus was placed on three aspects of Egfr function that occur at two developmental stages. These are the withdrawal from the cell cycle that accompanies the first fate specifications and the mitosis and survival of cells that pass unspecified through a 'second mitotic wave' (SMW) before later recruitment to retinal cell fates. The onset of advancing differentiation is defined by a morphogenetic furrow, which sweeps anteriorly from the posterior part of the eye imaginal disc. Just anterior to the morphogenetic furrow, cells arrest in G1 of the cell cycle. Within the morphogenetic furrow some of the arrested cells are specified as individual R8 photoreceptor cells, the founders of each ommatidium. Each R8 cell then produces ligands that activate Egfr in four neighboring cells. These neighbors are recruited to become the R2, R3, R4, and R5 photoreceptor cells of each ommatidium. While these five cells maintain their G1 arrest and differentiate in response to Egfr activity, the surrounding cells in which Egfr is inactive reenter the cell cycle and begin S phase DNA synthesis. Entry into this SMW occurs around retinal column 1, also the stage at which 'preclusters' of R8, R2, R3, R4, and R5 cells first become morphologically recognizable. The SMW produces unspecified cells that will be recruited to take the remaining 14 retinal cell fates. Egfr is required for survival and G2/M progression of SMW cells as well as later cell fate specification (Yang, 2003).

Because mitosis often occurred earlier than differentiation, whether mitosis also depends on the Ras/Raf/Pnt pathway was investgated. It was critical to distinguish between direct and indirect effects of ras, raf, or pnt mutations. Indirect effects were anticipated because R2-5 cells are a source of Egfr ligands, so mutations that affect R2-5 differentiation will eliminate ligand sources and affect other cells indirectly. If Ras, Raf, or Pnt directly transduces mitotic signaling by Egfr, the respective gene should be required cell autonomously in the dividing cells; effects on ligand production will affect mitosis nonautonomously. An indirect effect on mitosis is illustrated by ru; rho-1 mutant clones, in which most cells remained in G2 arrest posterior to column 3, but both cells in early mitosis with nuclear Cyclin B and postmitotic cells lacking Cyclin B were seen near boundaries with wild-type cells. rho-1 and ru activities are not required autonomously in the mitotic cells themselves, but nonautonomously to activate ligands for mitotic signaling (Yang, 2003).

Cells in ras or raf mutant clones retain premitotic Cyclin B levels, and no mitoses are detected, showing that these mutant cells are cell autonomously arrested in G2 phase. Many cells null for pnt function also retained Cyclin B. In contrast to ras or raf, however, mitotic and postmitotic pnt mutant cells were sometimes observed. Histone H3 phosphorylation was assessed to confirm mitosis of pnt mutant cells. Whereas ras or raf mutant cells never label for phosphorylated H3, labeled mitotic cells are seen in pnt mutant clones at ~40% of the frequency of control, wild-type twin clones. It is concluded that pnt promoted mitosis but is not essential (Yang, 2003).

If the Egfr/Ras/Raf pathway could bypass Pnt to regulate G2/M progression, it would be expected that Ras activated by the Val12 mutation would induce mitosis in clones of cells null for pnt. As predicted, RasV12 expression increases the number of mitotic cells in pnt mutant clones, as expected if pnt-independent mitosis were a target of Egfr signaling. In these experiments mitosis could not be an indirect effect of Ras activation of photoreceptor differentiation because photoreceptor differentiation requires pnt activity (Yang, 2003).

The pathways downstream of activated RasV12 were explored with mutations in the Ras effector loop. Mitosis was also increased by RasV12S35, but not by RasV12G37 or RasV12C40. This was consistent with activation of Raf being the relevant target of Ras, since only RasV12S35 permits full activation of Raf (Yang, 2003).

Ras, Raf, and Pnt should act cell autonomously if they promote survival directly. They could still affect survival nonautonomously if Egfr promotes survival through a distinct transduction pathway, since photoreceptor differentiation would be altered. Autonomy of gene function for survival in clones of mutant cells was assessed using an antibody called CM1, which reacts with the activated caspase ICE from Drosophila and thus labels cells deficient in survival signals. Expression of p35 was used to block apoptosis and thus to preserve the arrangement of cells lacking survival signals. The cell autonomous apoptosis of ras, raf, and pnt mutant cells is seen, indicating that, like Egfr, ras, raf, and pnt are required directly for cell survival, not indirectly through their role in photoreceptor differentiation and ligand production. Caspase activation begins 2-3 hr later in pnt mutant clones than in egfr, ras, or raf clones, suggesting that a pnt-independent pathway downstream of Raf can postpone apoptosis. Consistent with this, pnt mutant clones are often larger than egfr, ras, or raf clones (Yang, 2003).

Why do some cells perform mitosis yet fail to survive? One key question is whether any cell death is unrelated to Egfr. Previous work establishes that lack of the Egfr pathway is the cause of apoptosis in cells mutated for Egfr pathway components. By contrast it is less certain why some wild-type cells die in normal development. Such death may reflect inadequate Egfr activity or loss of another essential pathway (Yang, 2003).

To investigate why cells die in normal development whether activation of the Egfr pathway restored survival was tested. Apoptosis is completely suppressed when activated RasV12 or RasV12S35 is expressed posterior to column 1 with the GMRGal4 driver. This is consistent with low Ras and Raf activity as the cause of normal cell death. Since photoreceptor differentiation is dramatically increased, however, this is equally consistent with release of another signal by photoreceptor cells. Conditions were therefore sought where Egfr signaling was elevated without inducing excess differentiation (Yang, 2003).

Elevated Egfr expression reduces normal apoptosis by 50% without inducing differentiation, supporting the notion that cell death occurs because cells lacked Egfr activity. RasV12 carrying a second G37 mutation rescues all eye disc apoptosis without inducing differentiation. In Drosophila, RasV12G37 has reduced capacity to activate Raf and predominantly activates PI3Kinase. PI3K activity was manipulated directly to distinguish whether PI3K or Raf is relevant to survival. Expression of the catalytic subunit Dp110 does not reduce cell death, as it should have if death were due to inadequate PI3K activity. Expression of a dominant-negative variant of the adaptor protein p60 does not increase cell death, consistent with previous conclusions that the Insulin Receptor/PI3K pathway is dispensable for retinal cell survival. For a more sensitive assay, the same genotypes were examined 2-3 days later, when many cells of the pupal retina undergo apoptosis due to inadequate Egfr activity. Both elevated Egfr expression and RasV12G37 reduces cell death. Ectopic photoreceptor cells were observed, consistent with Raf-mediated differentiation after such sustained expression. Elevated expression of the Drosophila Insulin Receptor also reduces cell death and promotes differentiation in both eye discs and pupal retina. These observations indicate that, when ectopically expressed, both the Insulin Receptor and RasV12G37 promote cell survival as does Egfr, by activating Raf to compensate for inadequate endogenous Egfr activity (Yang, 2003).

Since differentiation, division, and survival affect overlapping, but distinct, sets of cells, the role of Egfr in each response must be distinct in some way. Differentiation requires higher levels of Egfr signaling than G2/M progression. It has been argued that survival requires less Ras activity than does differentiation. Ras acts cell autonomously through Raf and Pnt; a deficit in this pathway causes the cell death that occurs in the wild-type. If survival required less activity than does G2/M progression, this could explain the survival of most cells that remain undivided. It would be necessary to propose declining Egfr activity to account for the death of some cells that have already divided (Yang, 2003).

Survival and G2/M progression were compaired to determine whether they occurred with different thresholds. The expression of Egfr, RasV12G37, or InR reduces apoptosis and also increases the number of SMW mitoses, although not as rapidly as RasV12 expression. It appears that both mitosis and survival are achieved at lower signaling levels than differentiation, but some cell death often remains under conditions when G2/M progression is already stimulated. An Egfr activity level has not been found that promotes survival without mitosis or vice versa, should such a level exist (Yang, 2003).

These data provide some insight into cellular responses to RTK activity. Eye development shares features with the yeast pheromone response, where a MAPK cascade activates transcription of target genes in a graded fashion. Multiple outputs are thought to differ in pheromone dose response because of phosphorylation of multiple substrates by the MAPK Fus3p. By contrast Xenopus oocyte maturation exemplifies an all or none response. In Drosophila, simultaneous differentiation and withdrawal from the cell cycle of R2-5 cells provide an example of tightly coupled responses to Egfr activity, but the mechanism differs from that in Xenopus. Simultaneous differentiation and cell cycle withdrawal depend on enough exposure to ligands Spi and Krn to activate both responses reliably. The responses uncouple if inadequate ligand is present. Other outputs of the Egfr also depend, in part, on different thresholds, and differing dpERK levels exist in individual eye disc cells. Responses might not be separable if the Egfr/Ras/Raf/MAPK pathway provided only all or none output, as when variable progesterone levels are amplified to maximal dpERK levels in Xenopus oocyte maturation (Yang, 2003).

Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6

Egfr signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Evidence is provided for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90° rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90°. rlt is shown to be a rotation-specific allele of the inhibitory Egfr ligand Argos; modulation of Egfr activity shows defects in ommatidial rotation. The data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. Evidence is provided for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway (Gaengel, 2003).

Since ommatidial rotation is a cell motility process requiring cytoskeletal rearrangements, it was of interest to determine if effectors of Egfr other than the Raf/MAPK cascade play a role in this process. The Ras GTPase, the main transducer of Egfr signaling, can utilize distinct effectors in different contexts. In addition to nuclear signaling, mediated by the Raf/MAPK/Pnt cascade, Ras can affect cell growth and cytoskeletal rearrangements via its effectors Rgl/Ral, Phospho-inositol-3-Kinase (PI3K) and Canoe, whose human homolog (AF6) is known as the critical partner of ALL1 in a chimeric protein associated with myeloid leukemia (Gaengel, 2003).

Several point mutations have been identified within the Ras effector loop (amino acids 34-41) that abrogate the binding to and activation by Ras of specific effectors. The specificity of the existing Ras-effector loop mutations has been thoroughly tested in Drosophila imaginal discs (Prober, 2002). RasV12[S35], able to interact with Raf in cell culture, can activate ERK/Rolled (via Raf) and induce Ras/Raf/ERK-specific transcriptional responses in wing and eye imaginal discs (Prober, 2002). In contrast, RasV12[G37], unable to bind Raf in cell culture, cannot activate these responses, but is still capable of activating PI3K-specific read-outs (Prober, 2002). Therefore the Ras effector loop mutations were tested in constitutively activated RasV12 for their effects on ommatidial rotation (Gaengel, 2003).

Expression of RasV12 in developing photoreceptor precursor cells using common eye-specific drivers causes induction of many extra photoreceptors, and thus does not allow the analysis of rotation (the orientation of individual ommatidial clusters cannot be determined unambiguously in the presence of extra photoreceptor cells). To circumvent this problem, RasV12 and its effector loop isoforms were expressed in a limited photoreceptor subset after these had been determined as photoreceptors using the mDelta0.5-Gal4 driver. RasV12 effects on photoreceptor number and fate were thus strongly reduced. To determine the full effect of activated Ras expression under the control mDelta0.5-Gal4, constitutively active RasV12, that activates all known Ras-effectors, was expressed. This gave rise to eyes with some gain and loss of photoreceptors and severe misrotations. The equivalent expression of RasV12[S35], thought to activate mainly Raf, also resulted in rotation abnormalities and occasional gain or loss of photoreceptors, again supporting a requirement of the Raf/MAPK cascade (Gaengel, 2003).

Strikingly, expression of RasV12[G37] and RasV12[C40] also caused misrotations, suggesting an involvement of additional Ras-effectors in this process. In particular, RasV12[G37], in which Raf activation is abolished (or at least strongly reduced), results in severe rotation defects, suggesting that PI3K, Rgl/Ral or Canoe might play a role in this cell motility process. Similarly, RasV12[C40], eliminating Raf activation, but maintaining weaker activation of other effectors, also shows rotation abnormalities, albeit weaker than RasV12[G37]. Moreover, expression of RasV12[C40] under the control of the sevenless (sev) promoter (in R3/R4, R1/R6 and R7) results in strong rotation defects that were comparable to those seen with RasV12[G37] under sev control. Taken together, these data suggested an involvement of PI3K, Rgl/Ral or Canoe in ommatidial rotation (Gaengel, 2003).

To confirm the RasV12[G37] effect and determine which of the three known effectors activated by RasV12[G37] is required in ommatidial rotation, PI3K, Ral and Canoe were analyzed directly. UAS-PI3K expressed under mDelta0.5-Gal4 has no effect on rotation, suggesting that PI3K is not required in this process. This is further supported by the lack of rotation defects in dPI3K mutant clones. In contrast, expression of activated Ral (Sawamoto, 1999) as well as mDelta0.5-Gal4>UAS-Cno exhibits rotation defects (Gaengel, 2003).

Next, a direct canoe (cno) requirement in ommatidial rotation was tested using LOF alleles. First, it was asked whether cno heterozygosity interacts with the Star48-5/+ rotation phenotype. Strikingly, similar to the enhancement observed with Egfr or Ras, the cno2/+ and cno3/+ genotypes enhance the S48-5/+ rotation phenotype. cno is required for cone cell and photoreceptor differentiation and thus clones of null and strong alleles cause a general disorganization of the eye and are difficult to analyze for rotation defects. However, the hypomorphic cnomis1 allele is subviable in trans to the strong alleles cno2 and cno3 with mildly rough eyes, allowing an analysis of ommatidial rotation. Eye sections of such transheterozygous cno flies (e.g. cnomis1/cno2) reveal severe rotation defects. To test whether such defects are already observed at the time when rotation takes place, cno mutant third instar eye discs were analyzed. Strikingly, rotation defects, comparable in strength to the stronger aos alleles, are apparent in cno eye imaginal discs. The discs were counterstained with anti-Elav to ensure that the photoreceptor complement is normal in such cno mutant discs and the observed rotation abnormalities are primary defects, which was indeed confirmed. A similar analysis of Ral/Rgl is precluded by the lack of suitable alleles. In summary, these data indicate that cno plays a critical role in ommatidial rotation and acts as an effector of Egfr/Ras signaling in this context (Gaengel, 2003).

Since ommatidial rotation is a cell biological event, it is probable that among the main read-outs affected are cell-adhesion properties of the precluster cells and effects on cytoskeletal elements. This is further supported by observations that (1) Raf/MAPK-independent and thus transcription-independent Egfr/Ras signaling pathways are important, and (2) that canoe is required in this context. To address this further, two sets of experiments were performed. First, tests were performed for genetic interactions between the dosage-sensitive Star/+ rotation phenotype and selected factors required in cell adhesion and cytoskeletal regulation; and second, whether cell-adhesion components such as cadherins and integrins are normally localized in aosrlt and cnoMis1 mutant backgrounds was directly analyzed (Gaengel, 2003).

To specifically test the involvement of cytoskeletal elements and adhesion as well as junctional components, candidate genes were tested for dominant interaction of the mild Star rotation phenotype. These genetic data argue for an involvement of E-Cadherin/shotgun, the atypical cadherin Flamingo (Fmi), the adherens junction protein canoe, non-muscle myosin II (zipper), the septin peanut, and capulet, a protein with actin and adenylate cyclase-binding ability (Gaengel, 2003).

Next, the expression of Fmi and Shotgun in ommatidial preclusters was examined during rotation. Strong LOF alleles of Egfr and its signaling components also affect cell proliferation, fate specification and survival, making the analysis of cell adhesion and junctional components in the context of rotation rather difficult. Thus localization of the cadherins and Arm/ß-catenin was examined in imaginal discs of the rotation-specific aosrlt allele (Gaengel, 2003).

Although the overall expression and localization of Shotgun and Arm/ß-catenin are largely unaffected, the localization of Fmi is changed in aosrlt discs. In WT, Fmi is initially present apically in all cells of the morphogenetic furrow and subsequently becomes asymmetrically enriched in the R3/R4 precursor pair. In and posterior to column 6, Fmi is expressed at the membrane of R4, and largely depleted from R3 membranes that do not touch R4, forming a horseshoe-like R4-specific pattern. In contrast, in aosrlt discs, Fmi restriction to the R4 precursor is generally delayed, and often not established even in columns 8-12, where high levels of Fmi are still seen around the apical membrane cortex of R3 and R4. Since Fmi is thought to act as a homophyllic cell-adhesion molecule, its increased presence on R3 membranes should have a direct effect on Fmi localization in neighboring cells and thus possibly the adhesive properties of the precluster. It is worth noting that although Fmi is required during PCP establishment and R3/R4 cell-fate specification, the delay in Fmi restriction to R4 has no significant effect on the R3/R4 cell-fate decision. Although Fmi interacts with Fz and Notch in this context, the R4-specific mDelta-lacZ marker does not differ significantly from WT and adult aosrlt eyes also display no defects in R3/R4 specification. Thus, it appears that the delay in Fmi localization specifically affects ommatidial rotation, probably through adhesion, and possibly explains the broad range of rotation angles in aosrlt and other Egfr pathway mutants (Gaengel, 2003).

The Egfr/Ras/Cno link is intriguing for several reasons. The cno gene was originally identified as a mutation affecting the dorsal closure process during embryogenesis. Cno shows a genetic and molecular link to Ras: it contains two Ras-interacting domains and binds both WT Ras and activated Ras-V12. In addition, Cno has been postulated to link cytoskeletal elements to cellular junctions via its ability to bind actin, its interaction with ZO-1/Pyd and its homology with kinesin and myosin-like domains. Thus Cno could directly mediate an Egfr/Ras signal to cytoskeletal and cell architecture elements through its association with adherens junctions and its kinesin and myosin-like domains. Interestingly, Zipper does not only show a similar interaction with Star, like Cno, but it is also required during embryonic dorsal closure, and thus a more general Cno-Zipper link might exist in cell motility contexts (Gaengel, 2003).

A second interesting feature of cno is that it has been genetically linked to sca and Notch signaling. First, the phenotype of the sca1 allele is strongly enhanced by cno/+. Second, cno alleles also display Notch-like phenotypes in the wing and a GOF Notch allele, NotchAbruptex, is suppressed by cno. Although the biochemical role of Sca remains obscure, it has been linked to Notch, possibly as a Notch ligand, in several contexts. Thus, since sca has recently been implicated in ommatidial rotation, the link between Cno and Sca/Notch is intriguing. Taken together, Cno could serve as a factor integrating signaling input from different pathways, e.g., Egfr and Notch in this process, and relaying this to cytoskeletal elements. The Canoe link is also interesting from a disease point of view since its human homolog AF6 is the critical partner of ALL1 in a chimeric protein associated with myeloid leukemia. Thus, taken together, Cno could serve as a factor integrating signaling input from different pathways, e.g., Egfr and Notch in ommatidial rotation, and relaying this to the regulation of cell adhesion and cytoskeletal elements in the context of a developmental patterning process or disease (Gaengel, 2003).

Thus Egfr/Ras signaling plays a general role in the regulation of ommatidial rotation. Canoe has been identified as an effector of Ras in this context. Although much is known about how ommatidial chirality and the associated R3/R4 cell-fate decision are regulated (Fz/PCP-Notch signaling), no clear link between the mechanistic aspects of ommatidial rotation and Fz/PCP signaling previously existed. This is the first link to be demonstrated between Egfr signaling and PCP genes, namely Fmi. A further connection between Egfr signaling and PCP establishment is provided by Zipper, which acts downstream of Fz/Dsh and Rok in wing PCP and modifies the Star rotation phenotype. The identification of the Egfr pathway and its regulation of Fmi/cadherin-mediated cell adhesion will serve as an important entry point to further such studies (Gaengel, 2003).

EGF signaling and ommatidial rotation in the Drosophila eye

The ommatidia of the Drosophila eye initiate development by stepwise recruitment of photoreceptors into symmetric ommatidial clusters. As they mature, the clusters become asymmetric, adopting opposite chirality on either side of the dorsoventral midline and rotating exactly 90°. The choice of chirality is governed by higher activity of the frizzled (fz) gene in one cell of the R3/R4 photoreceptor pair and by Notch-Delta (N-Dl) signaling . The 90° rotation also requires activity of planar polarity genes such as fz as well as the roulette (rlt) locus. Two regulators of EGF signaling, argos and sprouty (sty), and a gain-of-function Ras85D allele, interact genetically with fz in ommatidial polarity. Furthermore, argos is required for ommatidial rotation, but not chirality, and rlt is a novel allele of argos. Evidence is presented that there are two pathways by which EGF signaling affects ommatidial rotation. In the first, typified by the rlt phenotype, there is partial transformation of the 'mystery cells' toward a neuronal fate. Although most of these mystery cells subsequently fail to develop as neurons, their partial transformation results in inappropriate subcellular localization of the Fz receptor, a likely cue for regulating ommatidial rotation. In the second, reducing EGF signaling can specifically affect ommatidial rotation without showing transformation of the mystery cells or defects in polarity protein localization (Strutt, 2003).

Mutations in fz result in defects in planar polarity of the eye, characterized by ommatidia taking on random chirality, or no chirality, and rotating randomly. A hypomorphic combination of fz alleles fz19/fz20 results in a weak eye phenotype in which only 9% of ommatidia show polarity defects. This phenotype is strongly enhanced by removing one copy of the dishevelled (dsh) gene, which acts downstream of Fz in polarity signaling (Strutt, 2003).

In order to identify additional factors involved in regulating ommatidial polarity, a large-scale genetic screen was carried out for loci interacting with fz. Unexpectedly, the principle factors identified were components of the EGF signaling pathway: three complementation groups corresponded to the genes argos, sty, and Ras85D. argos encodes an inhibitory ligand for the Drosophila EGF receptor. The new allele isolated in this screen (argos5F4) and two independent alleles enhanced the fz19/fz20 phenotype, such that about 20% of ommatidia had polarity defects. Similarly, the fz19/fz20 phenotype was also enhanced by two novel alleles and three known alleles of sty, which encodes a cytoplasmic protein that inhibits the Ras signaling pathway. Finally, the 2F4 enhancer mutation had an unusual dominant phenotype, in which a small number of ommatidia had extra R7 cells and very rare defects in specification of outer photoreceptors; also, extra vein tissue was seen in the wing. This phenotype is reminiscent of dominant mutations in the MAPK gene rl (rlSem, and the extra R7 cell phenotype is increased by removing one copy of the negative Ras pathway components sty, Gap1, and yan. Transheterozygotes of 2F4 and loss-of-function Ras85D mutations result in a weak Ras85D phenotype, in which outer photoreceptors were lost from many ommatidia. This phenotype suggests that 2F4 might be a Ras85D allele. This was confirmed by sequencing of the Ras85D gene in 2F4 mutants, which revealed a mutation of Ala59 to Thr. Interestingly, this mutation is a weak activating mutation found in viral oncogenes. Hence, mutations in three EGF pathway components, each of which are predicted to increase levels of pathway activity, are dominant enhancers of an fz ommatidial polarity phenotype (Strutt, 2003).

In wild-type eyes, 1-2 so-called 'mystery cells' are seen associated with the ommatidial cluster at the 5-cell stage of development, but these fail to differentiate as neural cells and are lost from the ommatidium by row 4. In strong argos mutants, most ommatidia in the adult have one or two extra photoreceptor neurons, as a result of mystery cells being transformed into photoreceptors. The phenotype of transheterozygotes of a null argos allele argosΔ7 and argos5F4 (from the screen) was less severe, with only 45% of adult ommatidia having extra photoreceptors. Interestingly, many of the ommatidia with a normal complement of photoreceptor cells had polarity defects; up to 50% of the ommatidia were misrotated, while only about 5% appeared achiral and typically less than 1% had wrong chirality. Therefore, in addition to photoreceptor recruitment defects, argos mutations can be characterized as particularly affecting ommatidial rotation, but not R3/R4 fate (Strutt, 2003).

The phenotype of argos mutations is in fact similar to that of rlt. The most striking defect in rlt mutants is the failure of ommatidia to rotate exactly 90°; however, some ommatidia also have an additional photoreceptor near the R3/R4 pair. rlt maps close to argos, and these loci fail to complement each other. Notably, the phenotypes of argosrlt/argosΔ7 or argosrlt/argos5F4 are identical in strength to that of argosrlt. Sequencing of the argos gene in rlt mutants did not reveal any amino acid changes, suggesting that rlt is a regulatory allele of argos, with only a weak photoreceptor recruitment defect but a strong misrotation phenotype (Strutt, 2003).

sty mutants have a severe rough eye phenotype characterized by transformation of cone cells to R7 photoreceptors and, less frequently, of mystery cells into outer photoreceptors. This phenotype is sufficiently strong that it is not possible to deduce from adult eye sections whether the ommatidia are also misrotated. However, examination of eye imaginal discs from sty homozygotes shows that the developing ommatidial clusters are not uniformly rotated relative to each other (Strutt, 2003).

Further evidence that EGF signaling was important in regulating rotation came from examination of animals carrying the dominant Ras85D allele, Ras85D2F4. In homozygotes, the extra R7 cell phenotype was not increased above that seen in heterozygotes; however, up to 20% of ommatidia were misrotated, and misrotations were also occasionally seen in heterozygotes (1%-5% of ommatidia). The dominant rotation defects seen in Ras85D2F4 heterozygotes were suppressed when placed in trans to a loss-of-function Ras85D allele; this finding is consistent with the defect being caused by inappropriate activation of Ras85D signaling (Strutt, 2003).

Ommatidial rotation occurs in the eye imaginal disc and begins at the 5-cell cluster stage of development, by row 6. Therefore, the developing ommatidial clusters were examined in argos eye discs by using specific photoreceptor markers. At this stage of development, the seven-up (svp) gene is specifically expressed in the differentiating R3/R4 photoreceptors, and later on in the R1/R6 cells, as they are recruited to the cluster. These cells can therefore be marked by using a svp-lacZ reporter gene. In the intermediate-strength argos allele combination argos5F4/argosΔ7, 65% of clusters had extra svp-lacZ-expressing cells in the R3/R4 position that were first visible in row 4 and were maintained as the clusters matured. Adult eyes of the same genotype contained extra photoreceptors in a position consistent with being R3/R4 type. Thus, the mystery cells that are transformed to a photoreceptor fate in argos mutations take on an R3/R4 fate. Extra R3/R4 cells are never seen in wild-type eye discs (Strutt, 2003).

Interestingly, in argosrlt/argos5F4 eye discs, a large number of immature clusters also have extra svp-lacZ-expressing cells. In particular, 60% of clusters in rows 4-6 have extra cells, a similar proportion to that seen for stronger alleles. However, the number of extra svp-lacZ-expressing cells decreases to about 25% in rows 7 and 8; furthermore, costaining with antibodies against the neuronal antigen Elav reveals that many of the extra cells fail to take on a neuronal fate. This is consistent with the adult phenotype in which only 15% of ommatidia have extra R3/R4 cells. Therefore, in argosrlt mutants, mystery cells are partially transformed into R3/R4 cells; but, most of them ultimately fail to develop into neurons (Strutt, 2003).

In addition, expression of mδ0.5-lacZ, a marker for high N activity and thus R4 fate, was examined. In wild-type eye discs, mδ0.5-lacZ is initially expressed at a low level in both R3 and R4, but the pattern is rapidly resolved to high-level expression in just R4. The expression of mδ0.5-lacZ is largely unperturbed in argosrlt/argos5F4, and expression fails to be resolved to a single cell in only occasional clusters. Therefore, the presence of transient, extra R3/R4 cells in the cluster does not affect signaling between the R3 and R4 cells to define high N activity in R4, as expected from the lack of chirality defects in argosrlt adults (Strutt, 2003).

Whether the transient presence of extra R3/R4 cells in argosrlt had any effect on the subcellular localization of the Fz receptor was examined. In the early stages of photoreceptor recruitment and rotation, Fz exhibits a dynamic localization pattern; in particular, it localizes differentially in the R3 and R4 photoreceptors. The planar polarity protein Flamingo (Fmi) also colocalizes with Fz in the R3 and R4 cells. In the absence of Fz activity, or its correct localization in R3/R4, ommatidial chirality and rotation is disrupted, suggesting that Fz localization in R3/R4 may provide a subcellular cue that controls both ommatidial chirality and rotation (Strutt, 2003).

Since the mystery cells are partially transformed into photoreceptors of the R3/R4 type in argosrlt, it was predicted that this might lead to aberrant Fz localization in the early ommatidium. In row 4 of wild-type eye discs, Fz-GFP is localized to the apicolateral membranes of the R3 and R4 cells, except where they contact R2/R5, and to the posterior side of R8. By row 6, Fz-GFP in the R3 cell is localized specifically at the R3/R4 boundary, whereas in the R4 cell, it is excluded from the R3/R4 boundary and the boundary with R5 but remains enriched on other apical membranes. In argosrlt mutants, a dramatically altered localization pattern is observed. In row 4 of most clusters, Fz-GFP is enriched on the apical membranes of several cells, which from their position correspond to the R3/R4 cells and a variable number of partially transformed mystery cells. By row 6, Fz-GFP is still apically localized in these additional cells in most clusters, rather than specifically in the R3/R4 pair. As expected, Fmi is also mislocalized in an identical pattern in argosrlt eye discs. Therefore, at the time when ommatidia begin to rotate, Fz-GFP distribution is abnormal, and it is asymmetrically distributed in multiple cells that are partially transformed to the R3/R4 fate (Strutt, 2003).

Extra R3/R4 cells and corresponding mislocalization of Fz-GFP are also seen in sty mutants, and these extra cells may be an underlying cause of the ommatidial rotation defect observed. Nevertheless, evidence was also sought of EGF signaling affecting rotation independently of the induction of extra photoreceptors (Strutt, 2003).

The phenotype caused by overexpression of a second Ras homolog in flies, Ras64B, was examined. Overexpression of activated Ras64BV14 under control of the sevenless enhancer or heat shock promoter causes rough eyes, in which ommatidia are improperly oriented. A similar phenotype is seen if Ras64BV14 is expressed by using the actin promoter; the predominant defect is misrotations, with occasional loss of pigment cells and fusion of ommatidia. A role for Ras64B in eye development has not yet been determined, since no mutants have been identified. However, the actin-Ras64BV14 misrotation phenotype can be suppressed by removing one copy of argos or sty, or in Ras85D2F4 heterozygotes; this finding is consistent with Ras acting in the EGF signaling pathway, but, in this context, as a negative regulator (Strutt, 2003).

Since actin-Ras64BV14 appears to act by lowering EGF signaling, it is unlikely that its rotation phenotype is due to extra photoreceptor cells or mislocalization of polarity proteins. Indeed, no extra photoreceptor cells were visible in the adult, and staining of imaginal discs from actin-Ras64BV14 males also failed to show any extra R3/R4 photoreceptor recruitment. Furthermore, Fz-GFP and Fmi localization was normal in these eye discs. Therefore, it is believed that alteration in EGF pathway activity by expression of Ras64BV14 causes misrotations without resulting in defects in cell fate determination or polarity protein localization (Strutt, 2003).

Since the exact role of Ras64B in EGF signaling is unclear, the effect on ommatidial rotation was examined of lowering the amount of a known component of the pathway, the EGF receptor, by using a temperature-sensitive allele, Egfrts1a. To generate a very weak phenotype, flies were raised at just above the restrictive temperature, at 18°C-19°C: examination of adult eyes revealed that, in addition to loss of photoreceptors in some clusters, occasional ommatidia were misrotated. While the number of misrotations was low (1-2 per eye section), the degree of misrotation was generally at least 45°, supporting a role for EGF signaling in this process (Strutt, 2003).

It is concluded that EGF signaling is required for correct ommatidial rotation. A fz ommatidial polarity phenotype is dominantly enhanced by argos, Ras85D2F4, and sty, all of which result in excess EGF pathway activation. Additionally, ommatidial rotation defects are seen in conditions in which EGF pathway activity is either increased or decreased (Strutt, 2003).

It is proposed that there are two mechanisms by which EGF signaling affects ommatidial rotation. The first is that this is a result of mystery cells inappropriately taking on an R3/R4 fate. In argosrlt, most ommatidia show partial transformation of mystery cells into R3/R4 photoreceptors. Although most of these extra R3/R4 cells do not ultimately differentiate into neurons, Fz-GFP is mislocalized in them at the time of ommatidial rotation. Since fz is required in the R3/R4 photoreceptor pair for correct ommatidial chirality and rotation, the presence of extra cells containing localized Fz-GFP could be providing the ommatidium with conflicting cues that disrupt normal rotation (Strutt, 2003).

The largely normal expression of mδ0.5-lacZ and the lack of chirality defects in argosrlt suggest that the presence of Fz-GFP in extra cells does not affect N-Dl signaling between the cells that finally take on the R3 and R4 fates. It is supposed that only cells that eventually take on neural fate are competent to participate in the N-Dl signaling event (Strutt, 2003).

Evidence was also found for a second mechanism whereby EGF signaling affects ommatidial rotation, without induction of extra R3/R4 cells. Lowering EGF signaling by using a temperature-sensitive allele of Egfr results in misrotations, even though this would be predicted to cause loss rather than gain of photoreceptors. In addition, expression of activated Ras64B causes rotation defects without inducing extra photoreceptors. While the role of Ras64B in EGF signaling has not been fully characterized, its rotation phenotype is dominantly suppressed by argos, sty, and Ras2F4, suggesting that it is acting as a negative regulator of the EGF pathway in this context. One possibility is that it acts by competing with Ras85D for binding to exchange factors or downstream effectors, thus reducing Ras85D activity. These observations support an additional, more direct role for the EGF pathway in control of ommatidial rotation, downstream of Fz localization (Strutt, 2003).

Finally, it is noted that the RhoA locus, which also controls ommatidial rotation, interacts with neither fz19/fz20 nor actin-Ras64BV14. Hence, RhoA may be required for another aspect of ommatidial rotation, perhaps via regulation of dynamic changes in actin structure needed for cell movement (Strutt, 2003).

Autophagy regulates tissue overgrowth in a context-dependent manner

Autophagy is a catabolic process that has been implicated both as a tumor suppressor and in tumor progression. This study investigated this dichotomy in cancer biology by studying the influence of altered autophagy in Drosophila models of tissue overgrowth. The impact of altered autophagy was found to depend on both genotype and cell type. As previously observed in mammals, decreased autophagy suppresses Ras-induced eye epithelial overgrowth. In contrast, autophagy restricts epithelial overgrowth in a Notch-dependent eye model. Even though decreased autophagy did not influence Hippo pathway-triggered overgrowth, activation of autophagy strongly suppresses this eye epithelial overgrowth. Surprisingly, activation of autophagy enhances Hippo pathway-driven overgrowth in glia cells. These results indicate that autophagy has different influences on tissue growth in distinct contexts, and highlight the importance of understanding the influence of autophagy on growth to augment a rationale therapeutic strategy (Perez, 2014).

Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth

Oncogenic mutations in Ras deregulate cell death and proliferation to cause cancer in a significant number of patients. Although normal Ras signaling during development has been well elucidated in multiple organisms, it is less clear how oncogenic Ras exerts its effects. Furthermore, cancers with oncogenic Ras mutations are aggressive and generally resistant to targeted therapies or chemotherapy. This study identified the exocytosis component Sec15 as a synthetic suppressor of oncogenic Ras in an in vivo Drosophila mosaic screen. Oncogenic Ras elevates exocytosis and promotes the export of the pro-apoptotic ligand Eiger (Drosophila TNF). This blocks tumor cell death and stimulates overgrowth by activating the JNK-JAK-STAT non-autonomous proliferation signal from the neighboring wild-type cells. Inhibition of Eiger/TNF exocytosis or interfering with the JNK-JAK-STAT non-autonomous proliferation signaling at various steps suppresses oncogenic Ras-mediated overgrowth. These findings highlight important cell-intrinsic and cell-extrinsic roles of exocytosis during oncogenic growth and provide a new class of synthetic suppressors for targeted therapy approaches (Chabu, 2014).

The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, this study identified grindelwald (grnd; CG10176), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. This study shows that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib-/- tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth (Andersen, 2015).

A genome-wide screen was carried to identify molecules that are required for neoplastic growth. The condition used for this screen was the disc-specific knockdown of avalanche, also known as syntaxin 7), a gene encoding a syntaxin that functions in the early step of endocytosis2. avl-RNAi results in ectopic Wingless (Wg) expression, neoplastic disc overgrowth, and a 2-day delay in larva-to-pupa transition. A collection of 10,100 transgenic RNA interference (RNAi) lines were screened for their ability to rescue the pupariation delay, and 121 candidate genes were identified. Interestingly, only eight candidate genes also rescued ectopic Wg expression and neoplastic overgrowth. These included five lines targeting core components of the JNK pathway (Bendless, Tab2, Tak1, Hemipterous and Basket. Using a puckered enhancer trap (puc-lacZ) as a readout for JNK activity, it was confirmed that JNK signalling is highly upregulated in avl-RNAi discs. One of the remaining lines targets CG10176, a gene encoding a transmembrane protein. Reducing expression of CG10176 by using two different RNAi lines was as efficient as tak1 silencing to restore normal Wg pattern and suppresses JNK signalling and neoplastic growth in the avl-RNAi background. Sequence analysis of GC10176 identified a cysteine-rich domain (CRD) in the extracellular part with homology to vertebrate TNFRs harbouring a glycosphingolipid-binding motif (GBM) characteristic of many TNFRs including Fas. CG10176 was named grindelwald (grnd) , after a village at the foot of Eiger, a Swiss mountain that lent its name to the unique Drosophila TNF, Egr. Immunostaining and subcellular fractionation of disc extracts confirmed that Grnd localizes to the membrane. Moreover, co-immunoprecipitation experiments showed that both Grnd full-length and Grnd-intra, a form lacking its extracellular domain, directly associate with Traf2, the most upstream component of the JNK pathway. This interaction is disrupted by a single amino acid substitution within a conserved Traf6-binding motif (human TRAF6 is the closest homologue to Traf2. Overexpression of Grnd-intra, but not full-length Grnd, is sufficient to induce JNK signalling, ectopic Wg expression and apoptosis, and Grnd-intra-induced apoptosis is efficiently suppressed in a hep (JNKK) mutant background, confirming that Grnd acts upstream of the JNK signalling cascade (Andersen, 2015).

The Drosophila TNF Egr activates JNK signalling and triggers cell death or proliferation, depending on the cellular context. Therefore tests were performed to see whether Grnd is required for the small-eye phenotype generated by Egr-induced apoptosis in the retinal epithelium (via Egr overexpression). Inhibition of JNK signalling by reducing tak1 or traf2 expression, or by overexpressing puckered, blocks Egr-induced apoptosis and rescues the small-eye phenotype. In contrast to a previous report, RNAi silencing of wengen (wgn) , a gene encoding a presumptive receptor for Egr, does not rescue the small-eye phenotype. Furthermore, the small-eye phenotype is not modified in a wgn-null mutant background, confirming that Wgn is not required for Egr-induced apoptosis in the eye. By contrast, reducing grnd levels partially rescues the Egr-induced small-eye phenotype, producing a 'hanging-eye' phenotype that is not further rescued in a wgn-knockout mutant background. A similar phenotype was previously reported as a result of non-autonomous cell death induced by a diffusible form of Egr. This suggests that Grnd prevents Egr from diffusing outside of its expression domain. Co-immunoprecipitation experiments show that both full-length Grnd and Grnd-extra, a truncated form of Grnd lacking the cytoplasmic domain, associate with Egr through its TNF-homology domain. Although Grnd-extra can bind Egr, it cannot activate JNK signalling. Therefore, it was reasoned that Grnd-extra expression might prevent both cell-autonomous and non-autonomous apoptosis by trapping Egr and preventing its diffusion and binding to endogenous Grnd. Indeed, GMR-Gal4-mediated expression of grnd-extra fully rescues the Egr small-eye phenotype. To confirm that the removal of Grnd induces Egr-mediated non-autonomous cell death, wing disc clones were generated expressing egr alone, egr + tak1 RNAi, or egr + grnd RNAi. As expected, reducing tak1 levels in egr-expressing clones prevents their elimination by apoptosis. Similarly, reducing grnd levels prevents autonomous cell death, but also induces non-autonomous apoptosis. This suggests that Egr, like its mammalian counterpart TNF-α, can be processed into a diffusible form in vivo whose interaction with Grnd limits the potential to act at a distance. Flies carrying homozygous (grndMinos/Minos) or transheterozygous (grndMinos/Df) combinations of a transposon inserted in the grnd locus express no detectable levels of Grnd protein and are equally resistant to Egr-induced cell death. In addition, grndMinos/Minos mutant flies are viable and display no obvious phenotype, suggesting that Grnd, like Egr, participates in a stress response to limit organismal damage. Collectively, these data demonstrate that Grnd is a new Drosophila TNF receptor that mediates most, if not all, Egr-induced apoptosis (Andersen, 2015).

TNFs probably represent a danger signal produced in response to tissue damage to rid the organism of premalignant tissue or to facilitate wound healing. Disc clones mutant for the polarity gene scribbled (scrib) induce an Egr-dependent response resulting in the elimination of scrib mutant cells by JNK-mediated apoptosis. To test the requirement for Grnd in this process, scrib-RNAi and scrib-RNAi + grnd-RNAi clones obtained 72 h after heat shock induction were compared. As expected, scrib-RNAi cells undergo apoptosis and detach from the epithelium. By contrast, scrib-RNAi clones with reduced grnd expression survive, indicating that Grnd is required for Egr-dependent elimination of scrib-RNAi cells. Similar results were obtained by generating scrib mutant clones in the eye disc (Andersen, 2015).

In both mammals and flies, TNFs are double-edged swords that also have the capacity to promote tumorigenesis in specific cellular contexts. Indeed, scrib minus eye disc cells expressing an activated form of Ras (RasV12) exhibit a dramatic tumour-like overgrowth and metastatic behaviour, a process that critically relies on Egr. RasV12/scrib-/- metastatic cells show a strong accumulation of Grnd and Mmp1, and invade the ventral nerve cord. Primary tumour cells reach peripheral tissues such as the fat body and the gut, where they form micro-metastases expressing high levels of Grnd. Reducing grnd levels in RasV12/scrib-/- clones is sufficient to restore normal levels of Mmp1 and abolish invasiveness in a way similar to that observed in an egr mutant background. Therefore, Grnd is required for the Egr-induced metastatic behaviour of RasV12/scrib-/- tumorous cells. Similarly, reducing grnd, but not wgn levels, strongly suppresses Mmp1 expression in RasV12/dlg-RNAi cells and limits tumour invasion, indicating that Wgn does not have a major role in the progression of these tumours (Andersen, 2015).

Perturbation of cell polarity is an early hallmark of tumour progression in epithelial cells. In contrast to small patches of polarity-deficient cells, for example, scrib mutant clones, organ compartments or animals fully composed of polarity-deficient cells become refractory to Egr-induced cell death and develop epithelial tumours. The formation of these tumours requires JNK/MAPK signalling, but not Egr, suggesting Egr-independent coupling between loss of polarity and JNK/MAPK-dependent tumour growth. In line with these observations, it was noticed that, in contrast to Grnd, Egr is not required to drive neoplastic growth in avl-RNAi conditions. This suggests that, in addition to its role in promoting Egr-dependent functions, Grnd couples loss of polarity with JNK-dependent growth independently of Egr. Disc immunostainings revealed that Grnd co-localizes with the apical determinant Crb in the marginal zone, apical to the adherens junction protein E-cadherin (E-cad) and the atypical protein kinase C (aPKC). In avl-RNAi discs, Grnd and Crb accumulate in a wider apical domain. Apical accumulation of Crb is proposed to be partly responsible for the neoplastic growth induced by avl knockdown, since overexpression of Crb or a membrane-bound cytoplasmic tail of Crb (Crb-intra) mimics the avl-RNAi phenotype. Therefore whether Grnd might couple the activity of the Crb complex with JNK-mediated neoplastic growth was examined. Indeed, reducing grnd levels, but not wgn, in ectopic crb-intra discs suppresses neoplastic growth as efficiently as inhibiting the activity of the JNK pathway. Notably, Yki activation is not rescued in these conditions, illustrating the ability of Crb-intra to promote growth independently of Grnd by inhibiting Hippo signalling through its FERM-binding motif (FBM). Indeed, neoplastic growth and polarity defects induced by a form of Crb-intra lacking its FBM (CrbΔFBM-intra) are both rescued by Grnd silencing. As expected, the size of ectopic crbΔFBM-intra;grnd-RNAi discs is reduced compared to the size of ectopic crb-intra; grnd-RNAi discs (Andersen, 2015).

Crb, Stardust (Sdt; PALS1 in humans), and Pals1-associated tight junction protein (Patj) make up the core Crb complex, which recruits the adaptor protein Veli (MALS1-3 in humans). In agreement with previous yeast two-hybrid data, this study found that Grnd binds directly and specifically to the PDZ domain of Veli through a membrane-proximal stretch of 28 amino acids in its intracellular domain. Grnd localization is unaffected in crb and veli RNAi mutant clones. However, reducing veli expression rescues the patterning defects and disc morphology of ectopic crb-intra mutant cells, suggesting that Grnd couples Crb activity with JNK signalling through its interaction with Veli. Interestingly, aPKC-dependent activation of JNK signalling also depends on Grnd. aPKC is capable of directly binding and phosphorylating Crb, which is important for Crb function. This suggests that aPKC, either directly or through Crb phosphorylation, activates Grnd-dependent JNK signalling in response to perturbation of apico-basal polarity (Andersen, 2015).

These data are consistent with a model whereby Grnd integrates signals from Egr, the unique fly TNF, and apical polarity determinants to induce JNK-dependent neoplastic growth or apoptosis in a context-dependent manner. Recent work reveals a correlation between mammalian Crb3 expression and tumorigenic potential in mouse kidney epithelial cells. The conserved nature of the Grnd receptor suggests that specific TNFRs might carry out similar functions in vertebrates, in which the link between apical cell polarity and tumour progression remains elusive (Andersen, 2015).

Antagonistic regulation of Yan nuclear export by Mae and Crm1 may increase the stringency of the Ras response

Phosphorylation of Yan, a major target of Ras signaling, leads to Crm1-dependent Yan nuclear export, a response that is regulated by Yan polymerization. Yan SAM (sterile {alpha} motif) domain mutations preventing polymerization result in Ras-independent, but Crm1-dependent Yan nuclear export, suggesting that polymerization prevents Yan export. Mae, which depolymerizes Yan, competes with Crm1 for binding to Yan. Phosphorylation of Yan favors Crm1 in this competition and counteracts inhibition of nuclear export by Mae. These findings suggest that, prior to Ras activation, the Mae/Yan interaction blocks premature nuclear export of Yan monomers. After activation, transcriptional up-regulation of Mae apparently leads to complete depolymerization and export of Yan (Song, 2005).

Yan polymerization is mediated by two hydrophobic SAM domain interfaces termed the mid-loop (ML) and end-helix (EH) surfaces, which bind one another to form a head-to-tail polymer. Mutagenesis of key residues in these surfaces (e.g., Ala86 on the ML surface or Val105 on the EH surface) converts Yan from a polymer into a monomer (Song, 2005).

In unstimulated Drosophila S2 cells, wild-type Yan remains in the nucleus, while introduction of constitutively active Ras (RasV12) results in Yan export. In contrast, the two monomeric mutants, YanA86D and YanV105R, are both exported from the nucleus even in the absence of Ras signaling. Nuclear localization was quantified by categorizing cells according to whether they displayed predominant nuclear localization, predominant cytoplasmic localization, or localization in both the nucleus and cytoplasm. Approximately 90% of cells expressing wild-type Yan displayed predominant nuclear localization. In contrast, only ~20% of cells expressing monomer Yan displayed predominant nuclear localization, with ~50% displaying localization in both the nucleus and cytoplasm, and ~30% displaying predominant cytoplasmic localization. Export of monomeric Yan mutants is fully Crm1-dependent, since cotransfection of dsRNA against Crm1 results in predominant nuclear localization of monomeric Yan in 90% of the cells. Conversely, overexpression of Crm1 enhances monomeric Yan export; >90% of YanA86D-expressing cells display predominant cytoplasmic localization of monomeric Yan (Song, 2005).

Since unstimulated S2 cells may nonetheless contain low levels of Ras signaling, it was determined whether or not monomer export requires the critical phosphoacceptor Ser127 residue in Yan. Monomeric Yan harboring the S127A mutation is still exported to the cytoplasm, although to a lesser extent than is monomeric Yan without the S127A mutation. Once again, the export was completely dependent upon Crm1 as demonstrated by Crm1 RNA interference (RNAi). In conclusion, the export of monomeric Yan is dependent on Crm1. Ras signaling stimulates but is not strictly required for export of the monomeric protein (Song, 2005).

The Mae SAM domain contains a functional ML surface that binds the Yan SAM EH surface, blocking Yan polymerization. Mae lacks a functional EH surface and so cannot bind to the Yan ML surface. Because the intrinsic affinity of Yan EH for Mae ML is three orders of magnitude greater than the intrinsic affinity of Yan EH for Yan ML, complete depolymerization of Yan occurs whenever Mae is present in stoichiometric excess over Yan. Under these conditions, all the Yan is driven into Yan:Mae heterodimers (Song, 2005).

If nuclear export of the Yan monomer is due to the exposure of Crm1-binding site(s) in the monomer that are buried in the Yan polymer, the Yan:Mae interaction might also mask the Crm1-binding site(s), resulting in the nuclear retention of the monomer. In accord with this idea, cotransfection of Mae prevents the nuclear export of YanA86D, a monomeric Yan mutant that retains the ability to bind Mae. The nuclear retention of the Yan monomer is completely dependent upon the interaction between Yan and Mae, since a Mae mutant with a defective ML surface, which is unable to bind to Yan, fails to interfere with the export of YanA86D. In addition, the localization of YanV105R, a monomeric Yan mutant that cannot bind Mae, is not altered by cotransfection of wild-type Mae (Song, 2005).

If Mae masks the Crm1-binding site(s) on Yan, then Crm1 might compete with Mae for binding to Yan. In this case, Crm1 overexpression would be expected to antagonize nuclear retention of Yan by Mae. In addition, if Yan phosphorylation facilitates Yan export by helping Crm1 to compete with Mae, cotransfection of RasV12 should also antagonize Mae-mediated Yan nuclear retention. To test these possibilities YanA86D and Mae were coexpressed in the presence of RasV12, Crm1, or both. Overexpression of Mae results in predominant nuclear localization of monomeric Yan in ~85%-95% of the cells. Coexpression of Crm1 reduces this proportion to ~20%-30%, dependent on the dose of transfected Mae. Coexpression of RasV12 alone has a much smaller effect on Mae-mediated Yan nuclear retention -- the proportion of cells with predominant nuclear localization was ~65%-85%. The level of RasV12 expression used in these experiments is sufficient to drive essentially all Yan from the nucleus in the absence of cotransfected Mae. Coexpression of RasV12 and Crm1 results in additive or slightly greater than additive effects on monomeric Yan nuclear retention, supporting the idea that Ras signaling facilitates Crm1-mediated Yan export (Song, 2005).

To test directly for competition between Crm1 and Mae, coprecipitation assays were employed. Because the interaction between Crm1 and its targets is affected by multiple factors (e.g., it is stabilized by RanGTP in the nucleus and destabilized by RanGDP in the cytoplasm, and may depend upon additional adaptors) S2 cell nuclear extracts were employed as a source of Crm1. Transiently expressed Flag-tagged Yan was purified on anti-Flag agarose beads, and then incubated with S2 nuclear extract. Crm1 precipitates with the Flag-Yan-bound beads. The bound Crm1 is displaced from the beads upon addition of the wild-type MBP-MaeSAM domain, but not upon addition of MBP-MaeSAMA141D, in which the SAM domain contains a point mutation that disrupts the ML surface (Song, 2005).

Next it was determined whether phosphorylation of Yan enhances the coprecipitation of Crm1. Flag-tagged wild-type Yan was transiently expressed in S2 cells in the absence or presence of RasV12 and then tested in coprecipitation assays for binding to Crm1 in S2 cell nuclear extracts. Like untagged Yan, Flag-tagged Yan localizes to the nucleus in the absence of Ras signaling and is exported to the cytoplasm in the presence of Ras signaling. The amount of RasV12 used in these experiments was sufficient to drive nearly all the Yan protein into the cytoplasm, indicating that Yan phosphorylation is likely quantitative. This was further verified by anti-Flag immunoblots showing a quantitative mobility shift of Flag-Yan upon coexpression with RasV12. The Crm1-Yan interaction was more stable when the Yan was expressed with the activated form of Ras, as shown by the coprecipitation and Mae competition assays. This enhanced interaction was likely dependent on phosphorylation of Yan on Ser127; coexpression with activated Ras has no effect on the ability of Flag-tagged YanS127A to bind Crm1 (Song, 2005).

In contrast to the results in which a Crm1-containing nuclear extract was imployed, purified Crm1 does not show significant affinity for Yan, indicating that the nuclear extract provides additional components that stabilize the interaction. This is consistent with studies on exportins showing that they often require adaptor molecules for high-affinity binding to their targets. In addition, the GTP-bound form of Ran is required for the stable binding of Crm1 to its targets, and therefore GMP-PNP was included as a standard component in all the Crm1 binding assays (Song, 2005).

A model is proposed in which Mae plays multiple roles in regulating the Crm1:Yan interaction. In cells that have not received the RTK signal, polymeric Yan is in equilibrium with depolymerized Yan. The formation of the depolymerized Yan is likely favored by the presence of a small amount of Mae in the nucleus of unstimulated cells, which binds and blocks one of the Yan polymerization interfaces (the Yan EH surface). It is suspected that by promoting limited Yan depolymerization, Mae ensures that unstimulated cells will be poised to receive the RTK signal. However, at this stage Mae also appears to block access of Crm1 to depolymerized Yan. This is important since if depolymerized Yan was accessible to Crm1, the resulting export of Yan would pull the equilibrium away from the polymer, leading to further Yan export and ultimately to premature expression of Yan target genes (Song, 2005).

It is essential to maintain Mae at low levels in undifferentiated cells, because Mae-mediated Yan depolymerization is sufficient to result in derepression, even in the absence of Ras signaling. Maintenance of low levels of Mae prior to differentiation appears to be at least partially ensured by the Yan-dependent repression of Mae. Upon activation of Ras, Ser127 in the Yan-Mae dimer is phosphorylated by the activated Rolled MAPK, favoring Crm1 in the competition with Mae for binding to Yan. The resulting increased Yan export is expected to relieve the transcriptional repression of Mae, and the accumulation of additional Mae is then expected to result in further Yan depolymerization, phosphorylation, and nuclear export. This feedback loop ensures that while Yan targets will be stably repressed prior to differentiation, they will be completely derepressed upon reception of the RTK signal. Furthermore, this feedback loop could serve to amplify a small change in the level of the RTK ligand, thereby allowing for sharp threshold responses to graded extracellular ligands (Song, 2005).

Capicua regulates cell proliferation downstream of the receptor tyrosine kinase/ras signaling pathway

Signaling via the receptor tyrosine kinase (RTK)/Ras pathway promotes tissue growth during organismal development and is increased in many cancers. It is still not understood precisely how this pathway promotes cell growth (mass accumulation). In addition, the RTK/Ras pathway also functions in cell survival, cell-fate specification, terminal differentiation, and progression through mitosis. An important question is how the same canonical pathway can elicit strikingly different responses in different cell types. This study shows that the HMG-box protein Capicua (Cic) restricts cell growth in Drosophila imaginal discs, and its levels are, in turn, downregulated by Ras signaling. Moreover, unlike normal cells, the growth of cic mutant cells is undiminished in the complete absence of a Ras signal. In addition to a general role in growth regulation, the importance of cic in regulating cell-fate determination downstream of Ras appears to vary from tissue to tissue. In the developing eye, the analysis of cic mutants shows that the functions of Ras in regulating growth and cell-fate determination are separable. Thus, the DNA-binding protein Cic is a key downstream component in the pathway by which Ras regulates growth in imaginal discs (Tseng, 2007).

A genetic screen was performed, by using mitotic recombination in the developing eye, for mutations that allow homozygous mutant cells to outgrow their wild-type neighbors. In addition to mutations in genes, such as Tsc1, Tsc2, Pten, salvador, warts and hippo, that encode negative regulators of growth and result in grossly enlarged eyes, mutations were identified where the only observable abnormality was an overrepresentation of mutant over wild-type tissue. Four such mutations belonged to a single lethal complementation group. Eyes containing mutant clones showed an increased relative representation of mutant tissue over wild-type tissue. Eyes containing mutant clones also consistently contained more ommatidia (mean = 763 ommatidia) and were thus slightly larger than eyes containing clones that were homozygous for the parent chromosome (mean = 703 ommatidia). Otherwise, the eyes were normal in appearance (Tseng, 2007).

All four alleles failed to complement the lethality of cicfetU6 and cicfetE11, which are alleles of capicua (cic). Mutations in the cic locus (also known as fettucine and bullwinkle) have been isolated in screens for mutations that disrupt either embryonic patterning or patterning of the eggshell, but the role of cic as a negative regulator of growth has not been described previously. cic encodes a protein with a single high-mobility group (HMG)-box that localizes to the nucleus and that is likely to bind DNA via its HMG-box motif. Each of the four mutant chromosomes isolated in the screen has a mutation in the coding region of the cic gene (Tseng, 2007).

An antibody that recognizes the C-terminal portion of Cic stains nuclei throughout the eye imaginal disc. There is a stripe of increased expression immediately anterior to the morphogenetic furrow and reduced expression in the morphogenetic furrow itself. Staining is not detected in clones of cicQ474X cells, thus confirming that the antibody recognizes the C-terminal portion of the Cic protein (Tseng, 2007).

In the eye imaginal disc, loss-of-function mutations in cic appear to increase tissue growth but do not seem to perturb cell-fate specification or differentiation. cic mutant ommatidia were indistinguishable from wild-type ommatidia in terms of the size, number, and arrangement of photoreceptor cells in the adult retina and appear to develop normally at earlier stages. Discs containing cic clones also showed normal patterns of BrdU incorporation throughout the eye imaginal disc. However, cic clones anterior to the morphogenetic furrow contained a 2- to 3-fold higher density of cyclin-E-positive cells per unit of pixel area than wild-type clones, consistent with the increased rate of cell proliferation in mutant clones. As in wild-type discs, no BrdU incorporation was observed in cic mutant discs posterior to the second mitotic wave, and ectopic cyclin E protein was not observed in cic clones posterior to the second mitotic wave. The patterns of mitosis as assessed by staining with anti-phospho-histone H3 were also unchanged. Thus, cic cells maintain a relatively normal pattern of S phases and mitoses in the eye disc and are still able to exit from the cell cycle in a timely manner. In mature pupal eye discs, occasional extra interommatidial cells are observed in mutant clones, suggesting that cic cells may have a subtle defect in developmental apoptosis (Tseng, 2007).

To examine the growth characteristics of cic cells at greater resolution, cells from the eye and wing discs of early third instar larvae (120 hr AED) were dissociated and analyzed by flow cytometry. The distribution of mutant cells in the different phases of the cell cycle as assessed by their DNA content was very similar to that of wild-type cells, as was cell size as assessed by forward scatter in cells of the eye disc or the wing disc. As in the adult eye and the eye imaginal disc, the area occupied by mutant clones in the wing disc was larger than the corresponding wild-type twin spots, suggesting that the mutant cells collectively grow (accumulate mass) more quickly than their wild-type neighbors. Also, mutant clones typically contained more cells than their wild-type twin spots. The inferred population doubling time calculated from the median clone size was 10.3 hr in mutant clones compared to 12.3 hr in the wild-type twin spots. The simplest interpretation of all of these observations is that cic cells have an increased rate of growth (mass accumulation) compared to wild-type cells but maintain a normal size because of a commensurate acceleration of the cell cycle. These findings indicate that a normal function of cic is to restrict cell growth in both the eye and wing imaginal discs (Tseng, 2007).

Previous work has shown that the levels of Cic protein are responsive to the level of signaling via RTKs and Ras. In the embryo, the level of Cic protein in the terminal regions is decreased upon signaling via the Tor RTK. Activation of Ras in the cells of the wing imaginal disc also reduces Cic levels in those cells. In eye discs, loss-of-function clones of Egfr or Ras, although small, had clearly elevated levels of Cic protein. Conversely, clones of cells expressing the activated form of Ras, Ras (Val12), had reduced levels of Cic. Thus, as in other tissues, increased signaling via the Egfr/Ras pathway reduces Cic protein levels in the eye disc. Furthermore, studies with mutations in the effector domain of Ras suggest that Ras regulates Cic primarily via the Raf/MAPK pathway. This is consistent with a recent study that has shown a direct interaction between Cic and MAPK (Tseng, 2007).

In the eye imaginal disc, clones of RasΔC40b, a null allele of Ras, were much smaller than their wild-type twin spots. Strikingly, clones of cells that were mutant for both cic and RasΔC40b were indistinguishable from cic clones in that they were typically larger than their twin spots. Thus, the loss of cic function completely bypasses the requirement for Ras in promoting cell growth. In contrast to the result obtained with cic, clones that were doubly mutant for Ras as well as a different negative regulator of growth, Tsc1, were no larger than Ras clones. Hence, the ability of cic to suppress the growth defect of Ras clones is specific and not a general property of negative regulators of growth. Also, cic mutations did not suppress the growth defect resulting from mutations in the Insulin Receptor (InR), Akt, and Rheb. Thus, cic mutations appear capable of rendering cell growth independent of Ras-mediated signaling but not independent of InR/PI3K- or Tor-mediated signaling. Taken together, these findings support the notion that the ability of Cic to restrict cell growth is specific to its function as a downstream component of the Ras pathway (Tseng, 2007).

In addition to promoting tissue growth, the recruitment of photoreceptor cell precursors to the developing ommatidia occurs via reiterated use of the EGFR/Ras pathway. Clones of cells that are mutant for RasΔC40b do not contain clusters of cells expressing the neural marker Elav, and instead they contain only the regularly spaced single Elav-positive nuclei that belong to the R8 photoreceptor cells. Although clones doubly mutant for cic and RasΔC40b are of normal size, they, like Ras clones, contain single nuclei that stain with anti-Elav and express the R8-specific marker Senseless. Thus, loss of cic function does not bypass the requirement for Ras function in the specification of photoreceptor cells R1-R7. Mutations in Tsc1 suppress the requirement for Ras neither in growth nor in photoreceptor differentiation. Thus, adult eyes containing clones doubly mutant for cic and RasΔC40b have large patches of tissue lacking any recognizable ommatidia. In retinal sections, there are no photoreceptor cells in the cic Ras double-mutant clones, and all the photoreceptor cells at the borders of the clone are wild-type for Ras. Thus, although they exhibit impaired photoreceptor differentiation, cic Ras double-mutant clones are not impaired in their growth and, unlike Ras clones, are not outcompeted by neighboring cells. Indeed, the phenotype of cells doubly mutant for Ras and cic is extremely similar to that of large Ras clones that are generated in a Minute background, suggesting that cic mutations primarily rescue the growth disadvantage of Ras clones (Tseng, 2007).

Thus, in the eye disc, there may be a branching of the Egfr/Ras pathway. One branch, functioning via Cic, appears important for growth regulation, whereas the other branch, acting via Pnt, appears important for photoreceptor cell-fate specification. In contrast to Ras clones, clones of pnt in the eye imaginal disc do not show a marked growth defect, suggesting that pnt has a minor role in regulating tissue growth in the eye disc (Tseng, 2007).

In mammalian cells, several extracellular growth factors that act via RTKs increase the activity of cyclin D/Cdk4 or cyclin D/Cdk6 complexes that can phosphorylate and inactivate the retinoblastoma protein (pRb) and thus promote S phase entry. However, it is still unclear how inactivation of pRb can cause cell growth (mass accumulation). At least in Drosophila, the role of Cic appears distinct from cyclin D because neither are cyclin D protein levels elevated in cic clones nor is the growth advantage of cic cells over wild-type cells compromised in flies that completely lack Cdk4/6 function. Other studies suggest that Ras can promote cell growth by stabilizing Myc protein via MAPK-mediated phosphorylation. This mode of Ras function also appears to be dispensable under conditions where cic function is inactivated but may still be relevant at physiological levels of Ras signaling (Tseng, 2007).

Notably, these data also show that Cic also functions as a negative regulator of tissue growth in the wing disc. However, in this tissue, Cic has a role in specifying cell fates as well because others have shown that cic mutations result in the formation of ectopic vein tissue. Thus, although the role of Cic as a regulator of growth in imaginal discs appears to be general, the importance of Cic in pathways that regulate cell-fate determination may vary from one tissue to another (Tseng, 2007).

The human and mouse genome each appear to have a single cic ortholog whose function in the regulation of growth has not been addressed to date. However, a recent study that determined the DNA sequence of 13,023 genes from 11 breast and 11 colorectal cancers found missense mutations in the human cic ortholog in three of the breast cancers. Although the functional consequences of these mutations have not been evaluated, these data suggest that Cic may indeed function in restricting cell growth in human cells (Tseng, 2007).

A MAPK docking site is critical for downregulation of Capicua by Torso and EGFR RTK signaling

Early Drosophila development requires two receptor tyrosine kinase (RTK) pathways: the Torso and the Epidermal growth factor receptor (EGFR) pathways, which regulate terminal and dorsal-ventral patterning, respectively. Previous studies have shown that these pathways, either directly or indirectly, lead to post-transcriptional downregulation of the Capicua repressor in the early embryo and in the ovary. This study shows that both regulatory effects are direct and depend on a MAPK docking site in Capicua that physically interacts with the MAPK Rolled. Capicua derivatives lacking this docking site cause dominant phenotypes similar to those resulting from loss of Torso and EGFR activities. Such phenotypes arise from inappropriate repression of genes normally expressed in response to Torso and EGFR signaling. These results are consistent with a model whereby Capicua is the main nuclear effector of the Torso pathway, but only one of different effectors responding to EGFR signaling. Finally, differences in the modes of Capicua downregulation by Torso and EGFR signaling are described, raising the possibility that such differences contribute to the tissue specificity of both signals (Astigarraga, 2007).

Ras and bract development

A long-standing mystery in Drosophila has been how certain bristles induce adjacent cells to make bracts (a type of thick hair) on their proximal side. The apparent answer, based on loss- and gain-of-function studies, is that these bristles emit a signal that neighbors then transduce via the epidermal growth factor receptor pathway. Suppressing this pathway removes bracts, while hyperactivating it evokes bracts indiscriminately on distal leg segments. Misexpression of the diffusible ligand Spitz (but not its membrane-bound precursor) elicits extra bracts at normal sites. What remains unclear is how a secreted signal can have effects in one specific direction (Held, 2002).

The EGFR pathway is involved in the development of ommatidia of the fly eye via dosage-sensitive interactions between loss-of-function (LOF) alleles of Star and Ras1. If the EGFR pathway were instrumental in bract development, then those same alleles might be expected to also manifest dosage effects on the frequencies of bracts. Indeed, they do. Star5671/+ heterozygotes have a missing-bract phenotype (31%Ti and 89%Ba), which is aggravated slightly in deficiency heterozygotes such as Df(2L)ast4/+ (17%Ti and 78%Ba). In contrast, Ras1e1B/+ heterozygotes look nearly wild-type (96%Ti and 100%Ba). The double heterozygote shows synergistic effects: Star5671/+; Ras1e1B/+ flies have fewer bracts than either heterozygote alone (2%Ti and 60%Ba). In each of the above genotypes, the Ti was more strongly affected than the Ba. This disparity was seen in other contexts as well. Another trend in differential sensitivity was found among the basitarsal bristle rows: dorsal bristles tend to lose bracts more readily than ventral ones (Held, 2002).

If all epidermal cells are competent to make bracts in response to EGFR stimulation, then it should be possible to fool them into 'thinking' that they have 'heard' a signal (when in fact they have not) by activating the pathway downstream of the receptor. For this purpose, a constitutively active Ras1 transgene was used under the control of a heat shock promoter. When hs-Ras1*M11.2 males are heat-shocked at any time from 5 to 27 h AP, their legs acquire extra bracts. On the Ti, these excess bracts are patchily distributed. On the Ba, the bracts are also patchy (mainly found near bristles) for shocks between 11 and 27 h AP, but earlier shocks (5-10 h AP) typically yield a confluent lawn of unpigmented bracts (Held, 2002).

Since Star acts upstream of the EGF receptor, the missing-bract defect of Star5671/+; Ras1e1B/+ heterozygotes should be rescueable by hyperactivating Ras1. When the hs-Ras1*M11.2 transgene is introduced and the resulting Star5671/hs-Ras1*M11.2; Ras1e1B/+ pupae are heat-shocked during the extra-bract sensitive period (24 h AP), a partial rescue is indeed observed. The shocked flies have significantly more bracts (30%Ti and 59%Ba) than their unshocked control siblings (0%Ti and 40%Ba) (Held, 2002).

Ras pathway and oogenesis

During Drosophila oogenesis, spatially restricted activity of the Epidermal growth factor receptor tyrosine kinase first recruits follicle cells adjacent to the oocyte to a posterior cell fate and subsequently, in a later function, specifies dorsal follicle cell fate. Gurken is known to act as the ligand stimulating Egf-r in both instances. Another receptor tyrosine kinase, Breathless, stimulates migration of the anterior follicle cells known as border cells. Since Ras is known to mediate many receptor tyrosine kinase effects, the role of Ras was investigated in follicle cell fate determination, differentiation, and migration throughout oogenesis. Early ectopic Ras activity induces transient expression of posterior follicle cell markers in anterior follicle cells, but does not inhibit anterior differentiation. Among the posterior follicle cell markers is the gene pointed expressed in anterior follicle cells during stages 8 and 9 after being subjected to ectopic Ras during stage 2. Later ectopic Ras activity inhibits anterior follicle cell differentiation but does not induce posterior marker expression. Complete transformation of anterior follicle cells to posterior follicle cells required early ectopic Ras activity in egg chambers where terminal differentiation of anterior cells is inhibited. Although Ras alone is insufficient to completely transform anterior cells into posterior cells, complete transformation of anterior cells to posterior cells does occur when transient Ras activity is induce in a slow border cells (slbo) mutant. Thus Ras and C/EBP play antagonistic roles in the terminal differentiation of border cells (T. Lee, 1997).

slbo targets breathless and is essential for terminal differentiation and migration of the anterior follicle cells known as border cells. Ectopic Ras activity prior to stage 6 of oogenesis impairs border cell migration. An additional copy of slbo is able to rescue the border cell migration defect in egg chambers with elevated ras, indicating that increased C/EBP expression counteracts increased Ras activity. It is concluded that Ras and C/EBP appear to antagonize each other in the terminal differentiation of border cells, and that it does not appear that low Ras activity per se is required for initiation of border cell migration (Lee, 1997).

Two other cell populations were studied: dorsal follicle cells and outer follicle cells. It was found that activated Ras is sufficient to specify dorsal follicle cell fate. In addition, a surprising role for border cells was found in the establishment of outer follicle cell fate. During stage 9, follicle cells covering the nurse cells gradually flatten, and, simultaneously, most follicle cells undergo movement from anterior to posterior and finally form a columnar epithelium in contact with the oocyte. These columnar epithelial cells constitute the outer follicle cells of the oocyte. Interestingly, outer follicle cell rearrangement is impaired when border cell fate is suppressed by activated Ras. These results suggest that, in vivo as in vitro, Ras can have diverse effects on different cells, however Ras activity can also have different effects on the same cells at different stages in their development (Lee, 1997).

The synthesis of dorsal eggshell structures in Drosophila requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. Advantage of this process was taken to identify genes that link patterning and morphogenesis; lethal mutations on the second chromosome were screened for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. These genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61ß, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway: Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a 'neurospecific' receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype (Schnorr, 2001).

Ras1 signaling downstream of the Egfr, Torso, and Sev RTKs has been studied extensively in the fly. Consequently, the recovery of mutations in second chromosomal genes known to function in Ras1 signal transduction was expected, as well as genes necessary for the D/V patterning of the eggshell. Indeed, l(2)k05115, an allele of Egfr was recovered. Another Ras1 pathway member identified is Star, a member of the Spitz group of genes that functions during Egfr-mediated formation of the embryonic ventral midline. Star mutations appear repeatedly in screens for RTK-related eye phenotypes and these Star alleles suppress gain-of-function mutations affecting Egfr and sev signaling pathways. In addition, a dominant female sterile allele, StarKojak, produces phenotypes that suggest a dual function for Star in A/P and D/V patterning in oogenesis. Finally, Star encodes a single-pass transmembrane protein; recent evidence supports the hypothesis that Star is involved in processing the Egfr ligands Spitz and Gurken. Both the recovery of Star and epistasis tests with lambdatop (which place Star upstream of or in parallel with Egfr) support these previous findings (Schnorr, 2001).

Another known Ras1 pathway member identified was blistered (bs), which encodes the Drosophila homolog of the human SRF. SRF is a MADS domain-containing transcription factor that binds the serum response element, an enhancer sequence named for its presence upstream of genes that respond to growth factor stimulation. Although the functional requirements of bs in dorsal appendage formation has not been tested, it is likely that SRF acts in follicle cell nuclei (Schnorr, 2001).

Genetic observations in wing and tracheal development reveal a role for SRF in processes regulated by Egfr and FGF-R signaling pathways. In Drosophila wing imaginal discs, bs is expressed in the future intervein tissue in a pattern complementary to that of Rhomboid, an Egfr accessory protein that facilitates presentation of ligand. Loss-of-function mutations in bs interact strongly with Egfr and other Ras1 signaling components in the wing and suppress the effects of disruptions in that pathway. In contrast, bs mutations enhance Ras1 defects in the egg, revealing important differences in the regulation of these two processes (Schnorr, 2001).

In tracheal development, bs functions in the terminal branching process that results from activity of breathless (FGF-R), an RTK that can employ the Ras signaling cascade. Lack of bs or breathless function eliminates cellular outgrowths and terminates tracheal branching prematurely. Thus, SRF and FGF-R act in concert to regulate cellular morphogenesis and in these ways resemble SRF and Ras1 function in dorsal appendage formation (Schnorr, 2001).

Finally, five alleles of the DSec61ß gene, which encodes a subunit of the DSec61 protein translocation channel, were identified. DSec61ß is required for embryonic development; mutants die with poorly developed, transparent cuticles due to defective cuticle secretion. DSec61ß has also been implicated in D/V patterning of the egg. Loss of DSec61ß from the germ line results in variable dorsal appendage phenotypes ranging from forked to fused single dorsal appendages. It has been proposed that DSec61ß is involved in Grk secretion and that the eggshell phenotypes result from secretion of lower levels of Grk ligand from the oocyte to the overlying follicle cells (Schnorr, 2001).

Identification of five alleles of DSec61ß, some with distinct phenotypes, is consistent with two potential roles for this protein during oogenesis. Four of the five alleles produce some level of dominant eggshell phenotypes characterized by translucent and flaccid eggs with shortened dorsal appendages. This phenotype was observed in a Ras+ background and may be due to a defect in chorion secretion. Nevertheless, these DSec61ß alleles also enhance the Ras1 dorsal appendage phenotype, increasing the number of fused and forked appendages observed in Ras1ix12a homozygotes. This enhancement may be an additive effect between two mutations affecting coupled biological processes, patterning and secretion. One DSec61ß allele, however, l(2)k07819b, enhances the Ras1 eggshell phenotype but does not produce the dominant chorion defects observed with the other four alleles. This latter observation suggests that the DSec61ß enhancement is specific and supports the hypothesis that the DSec61 channel participates in the secretion of Grk ligand. For these reasons, DSec61ß is likely to function in the germ line (Schnorr, 2001).

A surprising and rewarding feature of these results is the identification of signaling and cytoskeletal components that had not been linked previously to the Ras1 pathway. Molecules such as Dock, Tec29, POSH, and potentially Nrk or TppII function in signal transduction pathways or share homology with known signaling proteins but are new to Egfr-regulated D/V patterning processes. Molecules such as Profilin (chic), Peanut, and Smt3 may not be directly involved in signaling, but all are associated with cytoskeletal processes in some way. These proteins may organize the signal transduction machinery or effect the reorganization of the cytoskeleton in response to signaling (Schnorr, 2001).

dock is the Drosophila homolog of the mammalian oncogene Nck and belongs to the SH2/SH3 adapter protein family. dock has an essential role during axon guidance in the development of the eye, functioning in the growth cones of photoreceptor axons. Although the mammalian Nck protein interacts with the guanine nucleotide releasing protein, Sos, no previous evidence links Drosophila dock to Sos or the Ras1 signaling cascade. Recent proposals for Dock function in the nervous system are based on its interactions with Misshapen, Ste20-like kinases that may regulate growth cone changes through phosphorylation of the cytoskeleton. Epistasis tests place dock downstream of Egfr, potentially interacting with Sos and/or the Drosophila homologs of the Ste-20-like kinases (Schnorr, 2001).

Tec29 is a non-RTK molecule that was originally identified based on its homology to Src kinases. Mutations in Tec29 are embryonic lethal but clonal analyses reveal a role for Tec29 in ring canal formation during oogenesis. Egg chambers lacking Tec29 protein in the germ cells contain ring canals that are reduced in size and lack phosphoproteins as well as Tec29 itself. In addition, the nurse cells fail to transfer their cytoplasmic contents into the oocyte, producing small eggs. In spite of these defects, developing embryos deficient for maternally provided Tec29 exhibit no D/V patterning defects; indeed, 60% of embryos hatch. This result suggests that Tec29 interacts with Ras1 through a function in the follicle cells (Schnorr, 2001).

Drosophila POSH is homologous to a mouse protein identified in a two-hybrid screen with RAC, a Ras-like GTPase that regulates cytoskeletal function. Data from cultured cells support the hypothesis that mouse POSH mediates RAC signaling by activating the JNK cascade and inducing subsequent transcriptional changes, rather than affecting cytoskeletal structure or activity directly. This study is the first to demonstrate a function for this gene in Drosophila. Although POSH function has not been tested through mosaic analysis, its homology to other known signaling components suggests that POSH acts in the follicle cells (Schnorr, 2001).

l(2)k14301 is inserted at 49F7 and potentially affects two genes, Nrk and TppII. Nrk encodes an RTK of the Ror family. These proteins contain a cytoplasmic tyrosine kinase domain structurally related to the Trk family of receptors but differ in that their extracellular regions contain a 'kringle' domain thought to mediate protein-protein interactions. To date, the ligand for this family of receptors is unknown. One other Drosophila gene shows homology to the Ror family of RTKs; both genes were thought to be expressed exclusively in the nervous system. These results reveal unexpected expression of Nrk in the follicle cells and suggest that additional signaling events modulate follicle cell activity during eggshell morphogenesis. Moreover, the existence of a second RTK in this process could explain some aspects of the differential phenotypes produced by Ras1 compared to grk and Egfr mutations. It is possible, for example, that Nrk interacts with Ras, Tec29, POSH, and Dock to modulate cytoskeletal structure or function. Alternatively, l(2)k14301 could be affecting TppII, a serine protease with demonstrated activity in degrading neuropeptide signals. In mammalian systems, TppII homologs can compensate for loss of ubiquitin-mediated degradation pathways. If TppII acts in oogenesis, it may interact with Smt3 in a novel pathway to modulate signaling or cytoskeletal functions in response to Ras. Thus, the recovery of a Nrk/TppII mutation in this screen is an exciting result that will facilitate further analyses of these patterning and morphogenetic events (Schnorr, 2001).

The second main class of Ras1 Enhancers consists of mutations that disrupt bona fide cytoskeletal regulatory genes. These Enhancers may interact in the pathway in a variety of ways (Schnorr, 2001). chickadee (chic) is the Drosophila homolog of Profilin, a conserved actin binding protein. The chic gene produces both constitutive and ovary-specific transcripts through alternative promoters. Mutations that disrupt the ovary-specific transcript result in sterile females that produce dumpless eggs; at a low frequency, these eggs exhibit fused dorsal appendages. In these mutants, cytoplasmic actin cables in the nurse cells are missing. As a result, the rapid transfer of cytoplasm that occurs in late-stage 10 and 11 is disrupted when the nurse cell nuclei drift into the ring canals and impede further cytoplasmic flow (Schnorr, 2001).

The cause of the D/V patterning defect exemplified by the fused dorsal appendages is more obscure. The female sterile alleles do not disrupt follicle cell expression of chic, suggesting the defect is linked to chic function in the germ line. In situ hybridization studies, however, do not reveal a significant change in the level or degree of localization of grk mRNA, which encodes the TGFalpha-like D/V morphogen. These results suggest that the defect is more subtle than a gross perturbation of the cytoskeleton. Mutations disrupting either the ovary transcript or both the ovary and constitutive transcript of chic enhance a weak Ras1 eggshell phenotype. One explanation may involve the interaction of Chic, the actin cytoskeleton, and the microtubule-based cytoplasmic streaming thought to facilitate the uniform distribution of molecules in the oocyte during nurse cell cytoplasmic transfer. Premature streaming may inhibit the localization or function of molecules involved in translation or secretion of the Grk morphogen. Alternatively, Chic might be necessary to form an actin-based anchoring network critical for tethering Grk protein (Schnorr, 2001).

Although the data suggest that Chic must have a germ-line function, these data do not rule out an additional function in the follicle cells. During egg formation, follicle cells undergo shape changes and migrations while secreting the eggshell. These functions require the reorganization and directed movement of the follicle cell cytoskeleton, possibly effected in part by the Chic protein (Schnorr, 2001).

Likewise, the current evidence concerning the function of Peanut and Smt3 links these proteins to signaling and the cytoskeleton. peanut (pnut) is one of five Drosophila septin genes and was originally identified as a genetic enhancer of a weak allele of seven in absentia (sina). pnut is required for cytokinesis and is localized to the cleavage furrow of dividing cells. How does heterozygous Pnut enhance a weak Ras1 phenotype? Both Pnut and Ras1 have been implicated in cell division and a reduction in these proteins may inhibit cell division early in oogenesis resulting in later effects on egg morphogenesis. This possibility seems remote, however, since no obvious defects are observed in the follicular layer (Schnorr, 2001).

Alternatively, septins may be critical for the organization of the cytoskeleton and/or the localization of signal transduction components in the follicle cells. Septins are found in the cytoplasmic bridges during spermatogenesis, but their distribution in oogenesis differs. In nurse cells, septins are not part of the ring canals or intracellular bridges but are found in the cytoplasm, while in follicle cells, septins are specifically localized to baso-lateral surfaces. Septins also localize to areas of cortical reorganization and thus may be important for follicle cell shape changes and migrations. In yeast, septins function to localize signaling molecules to the future bud site. Thus, peanut may be critical for the correct localization, anchoring, and stability of the Ras1 signal transduction machinery in the follicle cells (Schnorr, 2001).

Interestingly, the ubiquitin-like gene smt3 was also identified as a Ras1 Enhancer. The smt3 gene product is a member of a new family of proteins that share many functional similarities with ubiquitin. Like ubiquitin, Smt3 proteins are post-translationally conjugated to target proteins, but the functional significance of this tagging is still under investigation. In human cells, cytosolic RanGAP1 is targeted to the nuclear pore after receiving an SMT3C (SUMO-1) protein tag. In contrast, SMT3C conjugation to IkappaBalpha renders the inhibitory factor resistant to ubiquitination and results in the retention of NFkappaB in the cytoplasm. In Drosophila, the NFkappaB homolog Dorsal and the transcriptional repressor Tramtrack69 (TTk69) are tagged by Smt3. Although the function of the Smt3 tag on TTk69 is not clear, Dorsal/Smt3 conjugates appear to migrate preferentially from the cytoplasm into the nucleus (Schnorr, 2001).

These observations suggest that Ras1 signaling may be affected by Smt3 conjugation to transcription factors. The ability of the smt3 mutant to suppress the activated Egfr eggshell phenotype suggests that Smt3 functions downstream of the Egfr receptor in the follicle cells. Thus, Smt3 might be involved in the modulation of transcription factors in the follicle cells. One candidate molecule is CF2, a zinc finger transcription factor negatively regulated by Egfr signaling. CF2 is retained in the cytoplasm of cells experiencing high levels of Ras1 signaling activity. Smt3 may function together with MAP kinase to alter the conformation of CF2 and prevent import into the nucleus. Alternatively, Smt3 may be conjugated to transcription factors to facilitate their nuclear import. For example, Smt3 is known to show punctate nuclear staining in some tissues of the fly and interact with TTk69. Thus, Ras1 activation may lead to the conjugation of Smt3 to transcription factors, leading to nuclear importation and/or modification of their binding activities (Schnorr, 2001).

Recently, the conjugation of Smt3 protein to yeast septins has been observed. Genetic studies in yeast suggest that Smt3p may be important for the disassembly of septin rings during cytokinesis. Considering these yeast interaction data, the identification of pnut and smt3 as independent modifiers of Ras1 in this screen suggest that the smt3/Ras1 interaction may be dependent on pnut. Thus, rather than modifying a transcription factor, the Smt3 protein may affect Ras1 signaling by regulating septin dynamics and the cytoskeleton (Schnorr, 2001).

Together, these results suggest that effective Ras1 signaling during eggshell morphogenesis depends on molecules that control the dynamic cytoskeleton. As described above, molecules such as Chic, Tec29, Pnut, Smt3, and Dock are involved in cytoskeletal reorganization. The Ras1 pathway may require a properly assembled cytoskeletal scaffold to achieve adequate signaling levels to correctly pattern the egg. Alternatively, the Ras1 signal may induce reorganization of the follicle cell cytoskeleton during the later cell migrations and subsequent secretion of the eggshell. Such large-scale reorganization may depend on a number of these Ras1 Enhancers (Schnorr, 2001).

Juxtaposition between two cell types is necessary for dorsal appendage tube formation

The Drosophila egg chamber provides an excellent model for studying the link between patterning and morphogenesis. Late in oogenesis, a portion of the flat follicular epithelium remodels to form two tubes; secretion of eggshell proteins into the tube lumens creates the dorsal appendages. Two distinct cell types contribute to dorsal appendage formation: cells expressing the rhomboid-lacZ (rho-lacZ) marker form the ventral floor of the tube and cells expressing high levels of the transcription factor Broad form a roof over the rho-lacZ cells. In mutants that produce defective dorsal appendages (K10, Ras and ectopic decapentaplegic) both cell types are specified and reorganize to occupy their stereotypical locations within the otherwise defective tubes. Although the rho-lacZ and Broad cells rearrange to form a tube in wild type and mutant egg chambers, they never intermingle, suggesting that a boundary exists that prevents mixing between these two cell types. Consistent with this hypothesis, the Broad and rho-lacZ cells express different levels of the homophilic adhesion molecule Fasciclin 3. Furthermore, in the anterior of the egg, ectopic rhomboid is sufficient to induce both cell types, which reorganize appropriately to form an ectopic tube. It is proposed that signaling across a boundary separating the rho-lacZ and Broad cells choreographs the cell shape-changes and rearrangements necessary to transform an initially flat epithelium into a tube (Ward, 2005).

Each dorsal appendage is made from a population of cells that reorganizes from an initially flat epithelium into a tube. This process most closely resembles wrapping, one of a variety of mechanisms that produce epithelial tubes. Dorsal appendage tube formation exhibits all three characteristics of the wrapping mechanism. (1) The dorsal appendage cells maintain epithelial contacts during tube formation. (2) The Broad cells constrict apically, causing the flat epithelium to curve. (3) The dorsal appendage tubes are parallel to the follicular epithelium, rather than perpendicular as observed in budding tubes. These features also characterize vertebrate neural tube and Drosophila ventral furrow formation (Ward, 2005).

Dorsal appendage tube formation differs from neural tube/ventral furrow formation in one key respect. The neural tubes and ventral furrow are each made by a symmetric fold in the epithelium. In contrast, asymmetric shape-changes and movements produce each dorsal appendage tube. Prior to tube formation, the rho-lacZ rows are perpendicular to one another in a pattern resembling an open hinge. Then, during tube formation the anterior row of rho-lacZ cells moves posterior, thereby closing off the ventral midline of the dorsal appendage tube. Cells in the medial (dorsal) row elongate concomitant with the apical constriction of the roof cells, but these floor cells do not swing anteriorly. Thus, during dorsal appendage formation the perpendicular rows of rho-lacZ cells do not move equivalent distances to seal the tube. Evidently, this process is both robust and malleable, since normal tubes can still form in patterning mutants with altered primordia (Ward, 2005).

What mechanism ensures proper tube closure? Recall, during tube formation the Broad pattern simultaneously shortens and lengthens along two perpendicular axes via likely convergent-extension rearrangements. Since the rho-lacZ and Broad cells maintain epithelial contacts with one another during tube formation, it is proposed that the rearrangements among the Broad cells contribute to a reorganization of the adjacent, underlying rho-lacZ cells. Thus, the anterior-medial movement of the Broad cells simultaneously lengthens the tube and draws the anterior row of rho-lacZ cells posteriorly. This process allows the rho-lacZ cells on either side of the hinge to associate with one another in a pair-wise fashion to close off the ventral midline of the tube. Similarly, convergent extension during neural tube development narrows the distance between the neural folds, allowing them to meet and fuse (Ward, 2005).

Additional insight into patterning and morphogenesis is provided by analysis of loss of function (Ras1 and K10) and gain of function (UAS-dpp and UAS-rho) mutants. In each mutant, the rho-lacZ and Broad cell types are specified and occupy their stereotypical locations within the otherwise defective tubes. Four features of these aberrant tubes are noteworthy. (1) The number of cells contributing to each primordium can vary widely, from a few cells in the UAS-rho clones to as many as hundreds in K10 egg chambers. Nevertheless, the rho-lacZ and Broad cells coordinate their movements to form a tube. (2) The position of the primordium within the egg chamber is not restricted to the dorsal anterior. Dorsal appendages shift posteriorly when dpp expression is greatly expanded, and ventral/lateral tubes may form when UAS-rho is expressed in the collar. Apparently, as long as both cell types form, other factors are not limiting in tube formation. (3) The posterior and ventral limits of both rho-lacZ and Broad expression precisely mirror one another. Even when ectopic rhomboid enlarges the normal domain of the dorsal appendage primordium, Broad and rho-lacZ expression expand coordinately. These results suggest that the patterning of these two cell types is a linked process. (4) In some K10 egg chambers and in the Ras1 hypomorphs and UAS-rho mutants, the rho-lacZ cells flank only the anterior margin of each Broad domain. Although the rho-lacZ cells are not arranged in a hinge pattern, the dorsal appendage cells reorganize appropriately to form a tube. Thus, the hinge pattern of rho-lacZ cells is not essential for tube formation (Ward, 2005).

These results indicate that the juxtaposition of rho-lacZ and Broad cells is necessary for tube formation and suggests that communication between the two cell types promotes the cell shape changes and rearrangements necessary to make a tube from an initially flat epithelium (Ward, 2005).

How do the dorsal appendage cells reorganize in a coordinated manner to form a tube? It is proposed that the rho-lacZ and Broad cells are separated by a 'boundary' and that signaling across this boundary choreographs the cell shape-changes and rearrangements necessary to make a tube from an initially flat epithelium. Boundaries between two different cell types occur frequently in developing tissues. Importantly, cells on one side of a boundary are free to mix with one another, but do not mix with cells on the other side of the boundary. This fence-like property of boundaries may be maintained by differences in cell adhesion. Although this hypothesis provides a satisfying explanation for cell behaviors, the adhesive mechanisms that prevent intermingling between different cell types are not understood. Finally, a boundary can function as an ‘organizer’ to instruct cells about their position and fate within a developing tissue (Ward, 2005).

Boundaries are of two types: lineage restricted (compartment) and non-lineage restricted. Since the patterning processes that define the dorsal appendage primorida occur after the cessation of cell division, a boundary between the rho-lacZ and Broad cells would be of the non-lineage-restricted type. Previous researchers have proposed that another boundary exists in the dorsal anterior follicle cells. This boundary is established by differential Bunched activity and lies between operculum/non-operculum cells. The boundary described in this paper is between the rho-lacZ and Broad cells (Ward, 2005).

What is the evidence that a boundary separates the rho-lacZ and Broad cells? (1) The roof and floor cells express unique cell-fate markers, display differential levels of cell-adhesion proteins, and exhibit distinct behaviors such as directed elongation and convergence/extension. Clearly they are different cell types. (2) Throughout the elaborate cell shape-changes and rearrangements of dorsal-appendage morphogenesis, the rho-lacZ and Broad cells coordinate their behaviors and never intermingle, even when patterning goes awry. Finally, the membrane(s) between the rho-lacZ and Broad cells accumulates high levels of phosphorylated proteins, consistent with signaling between the two cell types (Ward, 2005).

Signaling via an organizer established at the boundary may direct the cell shape-changes and rearrangements necessary to make a tube, perhaps by instructing the rho-lacZ cells to elongate and the Broad cells to constrict apically. The boundary could also direct the convergent-extension rearrangements of the Broad cells. Consistent with an organizer acting at the boundary between the rho-lacZ and Broad cells, ectopic expression of rhomboid in the anterior of the egg chamber produces an ectopic boundary capable of reorganizing the rho-lacZ and Broad cells into a tube. Domains of high-Broad-expressing cells merely produced warts, whereas clusters of cells containing both the Broad and rho-lacZ cell types reorganized properly and synthesized dorsal appendage tubes (Ward, 2005).

Altogether, these wild type, mutant, and ectopic rhomboid studies indicate that the juxtaposition of rho-lacZ and Broad cells is necessary to make a dorsal appendage tube. These two sub-populations of cells express many different cell-fate and adhesion markers, exhibit distinct behaviors, and never intermingle. It is hypothesized that a boundary exists between these two cell types and that signaling across the boundary coordinates the cell shape-changes and rearrangements that form the tube. These studies offer insight into the processes that regulate tubulogenesis, reveal mechanistic links between patterning and morphogenesis, and provide a foundation for inquiry in other systems (Ward, 2005).

Ras and apoptosis

The strong eye ablation phenotype in Drosophila, caused by expressing head involution defective under the control of an eye-specific promoter, was used to perform a genetic screen aimed at identifying components that regulate and mediate Hid activity. Mutations in genes that regulate the EGF receptor (EGFR)/Ras1 (Ras oncogene at 85D) pathway were recovered as strong suppressors of Hid-induced apoptosis. The survival effect of the EGFR/Ras1 pathway is specific for Hid-induced apoptosis, since neither Reaper- nor Grim-induced apoptosis is affected by the EGFR/Ras1 pathway. The Ras1 pathway has been shown to inhibit Hid activity apparently by the direct phosphorylation of Hid by MAPK (Rolled). Alteration of the MAPK phosphorylation sites within the HID sequence blocks the survival signals generated by constitutively activate Ras1 and constitutively active MAPK. It is concluded that the hid gene in Drosophila provides a mechanistic link between the survival activity of Ras1 and the apoptotic machinery. Post-translational modification of Hid is a survival signal regulating Hid activity (Bergmann, 1998).

A gain-of-function germline mutation in Drosophila ras1 affects apoptosis and cell fate during development

The RAS/MAPK signal transduction pathway is an intracellular signaling cascade that transmits environmental signals from activated receptor tyrosine kinases (RTKs) on the cell surface and other endomembranes to transcription factors in the nucleus, thereby linking extracellular stimuli to changes in gene expression. Largely as a consequence of its role in oncogenesis, RAS signaling has been the subject of intense research efforts for many years. More recently, it has been shown that milder perturbations in Ras signaling during embryogenesis also contribute to the etiology of a group of human diseases. This study reports the identification and characterization of the first gain-of-function germline mutation in Drosophila ras1 (ras85D), the Drosophila homolog of human K-ras, N-ras and H-ras. A single amino acid substitution (R68Q) in the highly conserved switch II region of Ras causes a defective protein with reduced intrinsic GTPase activity, but with normal sensitivity to GAP stimulation. The ras1R68Q mutant is homozygous viable but causes various developmental defects associated with elevated Ras signaling, including cell fate changes and ectopic survival of cells in the nervous system. These biochemical and functional properties are reminiscent of germline Ras mutants found in patients afflicted with Noonan, Costello or cardio-facio-cutaneous syndromes. Finally, ras1R68Q was used to identify novel genes that interact with Ras and suppress cell death (Gafuik, 2011).

Genetic screens were conducted for dominant modifiers of cell death induced by the Drosophila IAP-antagonists, hid and rpr. From over 150 mutants initially isolated, secondary screens allowed identification of 58 cell death specific modifiers. Of these, 40 alleles were placed into six complementation groups that define both known and unknown genes. These include Star, gap and sprouty involved in EGFR/MAPK signaling, the known cell death regulator diap1, the very large BIR and UBC containing dbruce, and an unknown gene, Su(GMRhid)2A that remains unidentified. This study focused on a previously uncharacterized cell death suppressor originally termed Su(21-3s). Using a combination of meiotic and P-element induced male recombination, genetic reversion, biochemistry and in vivo analysis, this mutant was demonstrated to be a gain-of-function mutation in ras1 (ras85D), the Drosophila homolog of human K-ras, N-ras and H-ras. This allele affects cell fate decisions and the pattern of normal, developmental apoptosis in paradigms known to depend on Ras-signaling (Gafuik, 2011).

One important role of Ras signaling during development is the transmission of an anti-apoptotic signal. The pro-apoptotic protein Hid contains 5 potential MAPK phosphorylation sites that are essential for its sensitivity to Ras-mediated inhibition. A Hid protein with either 3/5 or 5/5 mutant MAPK sites (HidAla3 and HidAla5, respectively) was refractory to suppression by the gain-of-function MAPK allele rlSem (a very mild suppression by rlSem is due to phosphorylation of the endogenous wildype Hid protein). In contrast, there was still some suppression of HidAla3 and HidAla5 by RasV12. It was postulated that this might be due to the ability of Ras, unlike MAPK, to exert additional anti-apoptotic effects through activation of the PI3-K/Akt-kinase effector branch. The current study found that rasR68Q was able to partially suppress HidAla3 but not HidAla5. Because HidAla3 retains two phosphorylation sites, it appears that partial phosphorylation of Hid is sufficient for a mild inhibitory effect, and that all five phospho-acceptor sites need to be eliminated in order for Hid to become refractory to inhibition by MAPK. Furthermore, it appears that RasR68Q, unlike RasV12, is unable to exert an additional suppressive effect via PI3-K/Akt-kinase. Perhaps the enhanced signaling activity of RasR68Q is able to activate the MAPK effector branch, but does not reach a required threshold to engage the PI3-K/Akt-kinase pathway. This may also help to explain the organismal viability of RasR68Q as compared to RasV12. Along the same line, RasR68Q was able to suppress Hid-induced cell death of lymphocytes within the protective environs of the lymph gland but not of those that were circulating. In sharp contrast, over-expression of Rasv12 in hemocytes not only leads to survival of circulating hemocytes but in fact results in a massive overproliferation of hemocytes. These results serve to highlight the exquisite sensitivity of biological systems to the degree of Ras signaling and suggest that between the extremes of wildtype Ras and constitutively active RasV12 lies a large spectrum of biological responsiveness (Gafuik, 2011).

Ras is highly conserved among metazoans and a number of Ras structures have been published that make it possible predict how mutations in specific regions might affect function. In the case of RasR68Q, it was considered that this change may affect the transition state of Ras. According to the 'arginine-finger hypothesis' GTPase-activating-proteins (GAPs) dramatically accelerate the GTPase reaction of Ras by supplying an arginine side chain (arginine-789 in the case of GAP-334) into the active site of Ras to neutralize developing charges in the transition state. A detailed analysis of the interactions between Ras and GAP-334 showed no role for R68 of Ras, explaining why RasR68Q can be stimulated by GAP. However, a close inspection of the Ras catalytic site shows that R68 extends its side chain towards the catalytic center. Mutating R68 to glutamine removes a stabilizing positive charge from the transition state and, according to the arginine-finger hypothesis, would be expected to result in less efficient hydrolysis of GTP. This prediction was tested biochemically and indeed it was found that RasR68Q hydrolyzes GTP intrinsically at a reduced rate, approximately 30% of that of wild type GTP (Gafuik, 2011).

Oncogenic mutations in Ras occur most frequently at codons 12,13 or 61 and result in an enzyme with deficient GTPase activity. This renders Ras inactive because Ras is 'on' when bound to GTP and switches 'off' by hydrolyzing bound GTP to GDP. Inhibition of Ras GTPase activity therefore stabilizes Ras in its active conformation, prolonging its recruitment and activation of downstream signaling components. The reduced GTPase activity of RasR68Q means that it would remain in its active GTP-bound conformation for longer periods of time allowing for enhanced signaling to downstream effector pathways. As noted above, however, RasR68Q may not remain in an active state sufficiently long to engage the catalytic p110 subunit of PI3K. An interesting alternative possibility however may be that R68 is directly involved in an interaction with PI3K and a mutation in R68 negatively affects this interaction. This raises the intriguing possibility that some of the phenotypes described for RasR68Q may actually be due to a loss, rather than a gain of PI3K activity (Gafuik, 2011).

During the initial mapping and characterization of ras1R68Q, a reversion screen was conducted in order to provide genetic evidence for the hypothesis that a rare gain-of-function allele in ras85D was identified. While searching for revertants, several mutants were recovered that were strong suppressors of GMR-hid. Recognizing that these mutants might be synergizing with ras1R68Q to produce such a strong suppression, 14 of these suppressors were successfully recovered and mapped. Most were mapped to a single candidate gene. Since these mutants were essentially derived from a dominant modifier screen for suppression of GMR-hid induced cell death, but within a sensitized ras1R68Q background, the mutational spectrum was expected to be overlapping, yet distinct from that of previous GMR-hid or UAS-RasV12 based screens. Indeed several suppressors turned out to overlap with ones identified previous screens. However, two novel interactors were also isolated: one allele of notum and four alleles of Su(Tpl). This demonstrates the utility of ras1R68Q to identify novel genetic interactions. While notum affects the Wnt/Wingless signaling pathway, Su(Tpl) is thought to function in the regulation of transcription in response to stress (Gafuik, 2011).

Much of the understanding of Ras-mediated signaling is derived from a combination of biochemical experiments conducted in mammalian tissue culture, and genetic studies in model organisms. For example, Ras-mediated signaling regulates the specification and differentiation of R7 photoreceptors in the Drosophila eye. However, until now, studies on the physiological consequences of elevated Ras in Drosophila have relied on overexpression of the activated ras1v12 allele. The viable hypermorphic ras1 allele described in this study, ras1R68Q, represents the first endogenous gain-of-function mutation in Drosophila Ras and hence offers a new tool for the analysis of Ras biology in situ. In particular, certain aspects of Ras biology have remained largely inaccessible to the use of constitutively active versions of this protein. This is because mutants, such as ras1v12, do not cycle normally between off and on states, are insensitive to regulatory circuits and are generally not compatible with organismal development. As a consequence, in certain paradigms and contexts, ras1v12 actually behaves as a loss-of-function mutant rather than a hypermorph, occluding the biological interpretation of Ras function in vivo. Therefore, the use of milder, viable hypermorphs of Ras, such as ras1R68Q, offers the potential for a refined understanding of the normal physiological roles of this important protein. Significantly, the ras1R68Q allele described in this study shares overall biochemical properties with recently discovered mutations in k-ras and h-ras that underlie human developmental disorders, such as Noonan, Costello and CFC syndromes (Gafuik, 2011).

Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila; JNK and Ras signaling cooperate in promoting tumor growth

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic RasV12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).

Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (RasV12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of RasV12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of RasV12 in these mutant cells prevents this cell death (Igaki, 2006).

To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).

In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of RasV12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).

To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (BskDN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with RasV12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by BskDN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of RasV12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by BskDN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).

Next, whether JNK activation is sufficient to trigger metastatic behavior in RasV12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (HepCA) can also activate JNK signaling. However, the eye phenotype caused by HepCA overexpression was only slightly suppressed by Bsk-IR, suggesting that HepCA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in RasV12-induced benign tumors, and it was found that the RasV12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of RasV12/scrib−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both RasV12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in RasV12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of RasV12+Eiger/shg−/− tumors was completely blocked by coexpression of BskDN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in RasV12-induced benign tumors. In contrast, overexpression of HepCA in RasV12/shg−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the RasV12+Eiger/shg−/− does not completely phenocopy the effect of RasV12/scrib−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in RasV12-induced benign tumors (Igaki, 2006).

Aside from its evolutionarily conserved role in cell migration and invasion, JNK signaling is also a potent activator of cell death in Drosophila and mammals. Although RasV12-expressing tissues showed a weak and restricted activation of JNK at later stages of development, mutation of cell polarity genes in combination with RasV12 expression constitutively activated JNK signaling. Striking acceleration of tumor growth occurred during days 5 and 6, and these tumors outcompeted surrounding wild-type tissues, resulting in a loss of the unlabeled wild-type cells and a dramatic increase in the GFP-expressing mutant tissue. The activated JNK was correlated with this accelerated tumor growth, suggesting that JNK signaling may play a role in tumor growth. Indeed, in addition to blocking metastatic behavior, inactivation of JNK pathway components strongly suppressed the accelerated tumor growth caused by cell polarity disruption. These results reveal that JNK signaling activated by loss of cell polarity also stimulates tumor growth (Igaki, 2006).

Since JNK signaling is required for both tumor growth and invasion, it was next asked whether these two phenotypes are separable processes. To address this, different types of tumors caused by alterations in genes involved in cell proliferation, growth, and cell polarity were analyzed. Day 6 RasV12/scrib−/− tumors showed moderate tumor growth and VNC invasion phenotypes. Loss of the Akt gene, a component of insulin growth signaling, considerably reduced the tumor load of RasV12/scrib−/− animals but did not impair metastatic behavior. In contrast, overexpression of Akt, combined with mutations in both the scrib gene and the lats gene, a potent tumor suppressor, did not cause metastatic behavior despite accelerated tumor growth comparable to RasV12/scrib−/−. In addition, although RasV12/Tsc1−/− mutant cells resulted in extremely large tumors, these tumor cells never exhibited metastatic behavior. These data indicate that tumor growth and invasion are separable processes in this model system (Igaki, 2006).

It was found that JNK signaling is indeed activated in polarity-deficient cells, and acridine orange staining revealed that most of these cells die. Interestingly, ectopic cell death was mostly blocked within clones of polarity-deficient cells also expressing RasV12, despite strong JNK activation. In addition, coexpression of RasV12 and Eiger, a potent inducer of cell death, resulted in accelerated tumor growth, although neither RasV12 alone nor Eiger alone caused dramatic overgrowth. This massive overgrowth was completely blocked by coexpression of BskDN. Moreover, stimulation of JNK signaling by expressing Eiger dramatically enhanced tumor growth of RasV12/shg−/− tissues, although Eiger/shg−/− clones were very small, probably because of cell death of these mutant clones. The accelerated growth of the RasV12+Eiger/shg−/− tumors was again completely blocked by BskDN. Together, these data indicate that, in the context of oncogenic Ras, JNK activation is the primary mediator of tumor growth downstream of cell polarity disruption. These observations suggest that JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras, and that the dramatic tumor growth is caused by cooperation between oncogenic Ras and JNK signaling (Igaki, 2006).

This study provides a molecular link between loss of cell polarity and tumor malignancy, namely activation of JNK signaling and inactivation of the E-cadherin/catenin complex in the context of oncogenic Ras activation. Although RasV12 alone only induces noninvasive overgrowth, and loss of cell polarity alone results in JNK-mediated cell death, the combination of these two alterations promotes both tumor growth and invasion through oncogenic cooperation. Thus, the tumor-promoting alterations caused by loss of cell polarity do not function alone and rather act as oncogenic Ras modifiers or “oncomodifiers” (Igaki, 2006).

The JNK signaling is essential for a variety of biological processes such as morphogenesis, cell proliferation, migration, invasion, and cell death. Genetic studies in Drosophila have demonstrated that JNK signaling is essential for epithelial cell movements and invasive behavior during normal development. A genetic study in mice revealed that TNF-triggered JNK signaling stimulates epidermal proliferation. These studies suggest that JNK may play an important role in tumorigenesis, tumor growth, and metastasis. Indeed, a substantial body of evidence indicates that JNK activation and c-Jun phosphorylation play important roles in cancer development. In mammalian cell culture systems, Ras acts cooperatively with JNK or c-Jun to enhance cellular transformation. Furthermore, knockin mice expressing a mutant form of c-Jun (JunS63A,S73A) suppress development of skin tumors in response to Ras activation and also block development of intestinal epithelial cancers caused by APC mutation. Moreover, liver-specific inactivation of c-Jun impairs development of chemically induced hepatocellular carcinomas. Furthermore, JNK signaling is activated in many tumor types. On the contrary, however, it has been also shown that JNK functions as a negative regulator for tumor development in Ras/p53-transformed fibroblasts. Thus, the role of JNK signaling seems to be highly dependent on cellular context, and, this study provides the first evidence for a cell-autonomous oncogenic cooperation between JNK and Ras signaling that promotes tumor growth and malignancy (Igaki, 2006).

How is JNK signaling activated? Loss of cell polarity may directly influence activity of a JNK pathway component. Alternatively, cell polarity defects may activate a cell surface receptor that triggers JNK signaling. The genetic analysis of multiple JNK pathway components suggests that the pathway is activated through a cell surface receptor, Wengen. It would be interesting to further investigate whether mislocalization or disregulation of Wengen, which should be normally tightly regulated in polarized epithelial cells, results in stimulation of JNK pathway signaling (Igaki, 2006).

The discovery that metastasis-promoting alterations (i.e., JNK activation) also increase tumor growth may explain why tumor cells acquire such mutations; that is, they primarily provide a selective advantage in tumor growth. Given that cell polarity defects are frequently associated with human tumor malignancy, and that the pathways identified in Drosophila are evolutionarily conserved, similar molecular mechanisms could be involved in human tumor progression. It would be particularly interesting to study these processes in human tumors with high frequencies of Ras mutations. If such processes prove conserved, components of these pathways, especially JNK signaling, could serve as potential therapeutic targets against such cancers (Igaki, 2006).

Identification of novel Ras-cooperating oncogenes in Drosophila melanogaster: a RhoGEF/Rho-family/JNK pathway is a central driver of tumorigenesis

Nutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (RasACT) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with RasACT in tumorigenesis, a genome-wide screen was carried out for genes that when overexpressed throughout the developing Drosophila eye enhance RasACT-driven hyperplasia. RasACT-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1ACT), RhoGEF2, and pbl cooperated with RasACT, resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or >Rac1 with RasACT upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system, upregulation of JNK alone was not sufficient for cooperation with RasACT, while in the clonal setting, JNK upregulation was sufficient for RasACT-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of RasV12-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2+ human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg and aPKC, with RasACT in the whole-tissue context. Collectively this analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with RasACT (Brumby, 2011).

Conserved mechanisms of tumorigenesis in the Drosophila adult midgut

Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophila's intestines share many similarities, this study explored the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Compound Apc-Ras clones, but not clones bearing the individual mutations, were shown to expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, these results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression (Martorell, 2014).

Reduced growth of Drosophila neurofibromatosis 1 mutants reflects a non-cell-autonomous requirement for GTPase-Activating Protein activity in larval neurons

Neurofibromatosis type 1 (NF1) is among the most common genetic disorders of humans and is caused by loss of neurofibromin, a large and highly conserved protein whose only known function is to serve as a GTPase-Activating Protein (GAP) for Ras. However, most Drosophila NF1 mutant phenotypes, including an overall growth deficiency, are not readily modified by manipulating Ras signaling strength, but are rescued by increasing signaling through the cAMP-dependent protein kinase A pathway. This has led to suggestions that NF1 has distinct Ras- and cAMP-related functions. This study reports that the Drosophila NF1 growth defect reflects a non-cell-autonomous requirement for NF1 in larval neurons that express the R-Ras ortholog Ras2, that NF1 is a GAP for Ras1 and Ras2, and that a functional NF1-GAP catalytic domain is both necessary and sufficient for rescue. Moreover, a Drosophila p120RasGAP ortholog, when expressed in the appropriate cells, can substitute for NF1 in growth regulation. These results show that loss of NF1 can give rise to non-cell-autonomous developmental defects, implicate aberrant Ras-mediated signaling in larval neurons as the primary cause of the NF1 growth deficiency, and argue against the notion that neurofibromin has separable Ras- and cAMP-related functions (Walker, 2006).

Enhancing the GTPase activity of Ras family members is the only known biochemical activity of neurofibromin, the protein defective in patients with NF1. This has focused much attention on manipulating Ras signaling as a way to correct the diverse symptoms of NF1. However, most Drosophila NF1 phenotypes lack dosage-sensitive genetic interactions with mutants that affect signaling by Ras1, the single fly ortholog of mammalian H-Ras, K-Ras, and N-Ras. Rather, an NF1 mutant growth deficiency, an electrophysiological defect, and a defect in olfactory learning are rescued by manipulations that increase signaling through the cAMP/PKA pathway. These findings have led to suggestions that neurofibromin may affect cAMP/PKA signaling in a Ras-independent manner, a hypothesis supported by a recent report that human NF1 suppresses Drosophila NF1 mutant size independent of GAP activity. In contrast, the current experiments using Drosophila NF1 transgenes suggest that loss of RasGAP activity is inseparable from the NF1 size defect. The reason for this discrepancy remains unclear, but may reflect inappropriate interactions between human neurofibromin and Drosophila GTPases or other proteins involved in growth regulation (Walker, 2006).

The current results show that the impaired growth of Drosophila mutants reflects a non-cell-autonomous role for NF1 in larval neurons. While runting is relatively common in mutant mice, it has been noted that mice engineered to specifically lack neuronal Nf1 expression are small. Growth in Drosophila proceeds during three larval instars that culminate in pupariation, pupation, and adult eclosion. As in other animals, growth is affected by feeding, which in Drosophila occurs during the first two and most of the third larval instar. Early in the third instar, larvae reach what is known as critical weight, a point at which holometabolous insects commit to metamorphosis and can develop without further feeding. Two neuroendocrine pathways have been implicated in coordinating feeding with Drosophila development and overall growth, but the results argue against obvious roles for NF1 in either one. Perhaps the best-understood growth-related pathway involves Drosophila insulin-like proteins (dILPs), three of which are produced -- two in a nutrient-dependent manner -- by bilateral symmetric groups of seven neurosecretory cells in the pars intercerebralis of the larval CNS. Ablating these cells causes a severe growth defect that is rescued by expression of a dILP2 transgene. In peripheral tissues, dILPs activate the insulin receptor, leading to the phosphorylation of Chico and the recruitment of a class I PI3 kinase, consisting of Dp110 catalytic and p60 regulatory subunits. Genetic manipulations that increase signaling through this pathway increase the size of peripheral tissues in a cell-autonomous manner, whereas loss-of-function mutations have the opposite effect. Recently, insulin was found to control developmental timing, but not body or organ size, during the period before Drosophila achieves critical weight, whereas after reaching this set point insulin no longer affected developmental timing, but only body and organ size. Analysis of mutant development and behavior found no differences in feeding or developmental timing between NF1 mutants and isogenic controls. Moreover, the lack of dosage-sensitive genetic interactions between NF1 and PI3 kinase p60 or Tor mutants, and the observation that dILP2-GAL4-driven UAS-NF1 expression in insulin-producing neuroendocrine cells does not modify NF1 size, all argue that insulin deficiency is not likely to be a major contributor to the NF1 size defect (Walker, 2006).

Drosophila growth and development are also coordinated by a hormonal cascade involving juvenile hormone (JH), prothoracicotrophic hormone (PTTH), and ecdysone. JH and ecdysone are produced by the corpora allata and the thoracic gland, respectively, which together with the corpora cardiaca form the neuroendocrine ring gland. PTTH stimulates ecdysone release and is made by neurons that innervate the thoracic gland in response to a developmentally controlled reduction in JH titer. JH production, in turn, is controlled by insulin, explaining the developmental delay and increased longevity of some hypomorphic insulin pathway mutants. It has recently been reported that increasing the size of the prothoracic gland by manipulations that activate Ras1 or its Dp110 PI3 kinase effector impairs Drosophila growth, possibly through ecdysone-mediated attenuation of insulin signaling in peripheral tissues. Again, the inability to modify NF1 size by expressing UAS-NF1 in the prothoracic gland, in other parts of the ring gland, or in neurons that innervate the ring gland suggests that excess Ras activity resulting from a loss of NF1 in these cells or tissues does not provide an easy explanation for the impaired growth of NF1 mutants. Further arguing against such a role, no obvious NF1 expression was detected in the ring gland (Walker, 2006).

Ras2-GAL4 is among the most restricted drivers that rescue NF1 size when driving UAS-NF1. This fact, combined with the observation that neuronal but not glial drivers similarly rescue, suggests that Ras2-GAL4-expressing cells are neuronal. It remains unclear in what proportion of these cells NF1 is required to restore growth, but costaining experiments revealed substantial overlap between endogenous NF1 and Ras2-GAL4-driven UAS-GFP expression. Moreover, Ras2-GAL4-driven UAS-NF1 expression strongly suppressed the larval CNS p-ERK phenotype. Several other findings support the conclusion that a Ras signaling defect in Ras2-GAL4-expressing cells is the primary cause of the NF1 size defect. (1) Ras2-GAL4-driven expression of a functional NF1 GAP-related domain (GRD) is necessary and sufficient for rescue. (2) Ras2-GAL4-driven expression of activated Ras1 or Ras2 phenocopied the NF1 size defect. (3) Ras2-GAL4-driven expression of a Drosophila p120RasGAP ortholog also rescued, arguing that the ability to rescue reflects a property shared between NF1 and RasGAP. Interestingly, expression of a third Drosophila RasGAP, Gap1, did not rescue either size or p-ERK phenotypes. Whether the inability of Gap1 to substitute for NF1 reflects an inappropriate expression level or some other factor (such as different regulation, localization, or GTPase substrate specificity) remains to be determined (Walker, 2006).

Initial reports that increasing cAMP/PKA activity rescues Drosophila NF1 phenotypes has generated much interest, in part because cAMP plays a prominent role in learning, which is impaired in many children with NF1. However, subsequent studies showed that genetic or pharmacologic manipulations that attenuate Ras signaling restored learning in heterozygous Nf1 mutant mice. Altered Ras signaling in the CNS appears capable of regulating the growth of the larval epidermis and imaginal discs. This could occur by modulating the levels of diffusible growth factors or growth inhibitors. Conceivably, cAMP/PKA signaling could be of importance at a more downstream component of this pathway, such as the release of, or response to, such diffusible factors (Walker, 2006).

The current results also demonstrate that heterozygous loss of individual genes encoding canonical Ras pathway components is insufficient to restore p-ERK activity in homozygous null or hypomorphic NF1 mutants. Interestingly, combined loss of Raf and rl, Ras1 and Raf, and Ras2 and Raf fully rescued the larval p-ERK defect, while the former two double mutants partially restored pupal size. Thus, Ras1 and Ras2 may jointly contribute to ERK activation in NF1-deficient CNS. Whether Ras effectors other than Raf/ERK contribute to the NF1 size defect, and how enhanced PKA activity rescues NF1 phenotypes remain to be determined (Walker, 2006).

Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling

Cancer cells demand excessive nutrients to support their proliferation but how cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar was previously demonstrated to not only direct metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2015).

The prevalence of obesity is increasing globally. Obesity impacts whole-body homeostasis and is a risk factor for severe health complications including type 2 diabetes and cardiovascular disease. Accumulating epidemiological evidence indicates that obesity also leads to elevated risk of developing several types of cancers. However, the mechanisms that link obesity and cancer remain incompletely understood. Using Drosophila, a whole-animal model system has been developed to study the link between diet-induced obesity and cancer: this model has provided a potential explanation for how obese and insulin resistant animals are at increased risk for tumor progression (Hirabayashi, 2015).

Drosophila fed a diet containing high levels of sucrose (high dietary sucrose or ‘HDS') developed sugar-dependent metabolic defects including accumulation of fat (obesity), organismal insulin resistance, hyperglycemia, hyperinsulinemia, heart defects and liver (fat body) dysfunctions. Inducing activation of oncogenic Ras and Src together in the Drosophila eye epithelia led to development of small benign tumors within the eye epithelia. Feeding animals HDS transformed Ras/Src-activated cells from benign tumor growths to aggressive tumor overgrowth with tumors spread into other regions of the body (Hirabayashi, 2013). While most tissues of animals fed HDS displayed insulin resistance, Ras/Src-activated tumors retained insulin pathway sensitivity and exhibited an increased ability to import glucose. This is reflected by increased expression of the Insulin Receptor (InR), which was activated through an increase in canonical Wingless (Wg)/dWnt signaling that resulted in evasion of diet-mediated insulin resistance in Ras/Src-activated cells. Conversely, expressing a constitutively active isoform of the Insulin Receptor in Ras/Src-activated cells (InR/Ras/Src) was sufficient to elevate Wg signaling, promoting tumor overgrowth in animals fed a control diet. These results revealed a circuit with a feed-forward mechanism that directs elevated Wg signaling and InR expression specifically in Ras/Src-activated cells. Through this circuit, mitogenic effects of insulin are not only preserved but are enhanced in Ras/Src-activated cells in the presence of organismal insulin resistance (Hirabayashi, 2015).

These studies provide an outline for a new mechanism by which tumors evade insulin resistance, but several questions remain: (1) how Ras/Src-activated cells sense the organism's increased insulin levels, (2) how nutrient availability is converted into growth signals, and (3) the trigger for increased Wg protein levels, a key mediator that promotes evasion of insulin resistance and enhanced Ras/Src-tumorigenesis consequent to HDS. This study identifies the Hippo pathway effector Yorkie (Yki) as a primary source of increased Wg expression in diet-enhanced Ras/Src-tumors. Ras/Src-activated cells are sensitized to Hippo signaling, and even a mild perturbation in upstream Hippo pathway is sufficient to dominantly promote Ras/Src-tumor growth. Functional evidence is provided that increased insulin signaling promotes Salt-inducible kinases (SIKs) activity in Ras/Src-activated cells, revealing a SIKs-Yki-Wg axis as a key mediator of diet-enhanced Ras/Src-tumorigenesis. Through this pathway, Hippo-sensitized Ras/Src-activated cells are positioned to efficiently respond to insulin signals and promote tumor overgrowth. These mechanisms act as a feed-forward cassette that promotes tumor progression in dietary rich conditions, evading an otherwise insulin resistant state (Hirabayashi, 2015).

Previously work has demonstrated that Ras/Src-activated cells preserve mitogenic effects of insulin under the systemic insulin resistance induced by HDS-feeding of Drosophila (Hirabayashi, 2013). Evasion of insulin resistance in Ras/Src-activated cells is a consequence of a Wg-dependent increase in InR gene expression (Hirabayashi, 2013). This study identified the Hippo pathway effector Yki as a primary source of the Wnt ortholog Wg in diet-enhanced Ras/Src-tumors. Mechanistically, functional evidence is provided that activation of SIKs promotes Yki-dependent Wg-activation and reveal a SIK-Yki-Wg-InR axis as a key feed-forward signaling pathway that underlies evasion of insulin resistance and promotion of tumor growth in diet-enhanced Ras/Src-tumors (Hirabayashi, 2015).

In animals fed a control diet, at most a mild increase was observed in Yki reporter activity within ras1G12V;csk-/- cells. A previous report indicates that activation of oncogenic Ras (ras1G12V) led to slight activation of Yki in eye tissue. Activation of Src through over-expression of the Drosophila Src ortholog Src64B has been shown to induce autonomous and non-autonomous activation of Yki. In contrast, inducing activation of Src through loss of csk (csk-/-) failed to elevate diap1 expression. The results indicate that activation of Yki is an emergent property of activating Ras plus Src (ras1G12V;csk-/-). However, this level of Yki-activation was not sufficient to promote stable tumor growth of Ras/Src-activated cells in the context of a control diet: Ras/Src-activated cells were progressively eliminated from the eye tissue (Hirabayashi, 2013). It was, however, sufficient to sensitize Ras/Src-activated cells to upstream Hippo pathway signals: loss of a genetic copy of ex-which was not sufficient to promote growth by itself-dominantly promoted tumor growth of Ras/Src-activated cells even in animals fed a control diet. These data provide compelling evidence that Ras/Src-transformed cells are sensitive to upstream Hippo signals (Hirabayashi, 2015).

SIK was recently demonstrated to phosphorylate Sav at Serine-413, resulting in dissociation of the Hippo complex and activation of Yki (Wehr, 2013). SIKs are required for diet-enhanced Ras/Src-tumor growth in HDS. Conversely, expression of a constitutively activated isoform of SIK was sufficient to promote Ras/Src-tumor overgrowth even in a control diet. Mammalian SIKs are regulated by glucose and by insulin signaling. However, a recent report indicated that glucagon but not insulin regulates SIK2 activity in the liver. The current data demonstrate that increased insulin signaling is sufficient to promote SIK activity through Akt in Ras/Src-activated cells. It is concluded that SIKs couple nutrient (insulin) availability to Yki-mediated evasion of insulin resistance and tumor growth, ensuring Ras/Src-tumor growth under nutrient favorable conditions (Hirabayashi, 2015).

The results place SIKs as key sensors of nutrient and energy availability in Ras/Src-tumors through increased insulin signaling and, hence, increased glucose availability. SIK activity promotes Ras/Src-activated cells to efficiently respond to upstream Hippo signals, ensuring tumor overgrowth in organisms that are otherwise insulin resistant. One interesting question is whether this mechanism is relevant beyond the context of an obesity-cancer connection: both Ras and Src have pleiotropic effects on developmental processes including survival, proliferation, morphogenesis, differentiation, and invasion, and these mechanisms may facilitate these processes under nutrient favorable conditions. From a treatment perspective the current data highlight SIKs as potential therapeutic targets. Limiting SIK activity through compounds such as HG-9-91-01 may break the connection between oncogenes and diet, targeting key aspects of tumor progression that are enhanced in obese individuals (Hirabayashi, 2015).

Overexpression screen in Drosophila identifies neuronal roles of GSK-3 beta/shaggy as a regulator of AP-1-dependent developmental plasticity

AP-1, an immediate-early transcription factor comprising heterodimers of the Fos and Jun proteins, has been shown in several animal models, including Drosophila, to control neuronal development and plasticity. In spite of this important role, very little is known about additional proteins that regulate, cooperate with, or are downstream targets of AP-1 in neurons. This paper outlines results from an overexpression/misexpression screen in Drosophila to identify potential regulators of AP-1 function at third instar larval neuromuscular junction (NMJ) synapses. First, >4000 enhancer and promoter (EP) and EPgy2 lines were used to screen a large subset of Drosophila genes for their ability to modify an AP-1-dependent eye-growth phenotype. Of 303 initially identified genes, a set of selection criteria were used to arrive at 25 prioritized genes from the resulting collection of putative interactors. Of these, perturbations in 13 genes result in synaptic phenotypes. Finally, one candidate, the GSK-3α-kinase homolog, shaggy, negatively influences AP-1-dependent synaptic growth, by modulating the Jun-N-terminal kinase pathway, and also regulates presynaptic neurotransmitter release at the larval neuromuscular junction. Other candidates identified in this screen provide a useful starting point to investigate genes that interact with AP-1 in vivo to regulate neuronal development and plasticity (Franciscovich, 2008).

The transcription factor AP-1 is a key regulator of neuronal growth, development, and plasticity, and in addition to cAMP response element binding (CREB) protein, it controls transcriptional responses in neurons during plasticity. Acute inhibition of Fos attenuates learning in mice and in invertebrate models such as Drosophila; AP-1 positively regulates developmental plasticity of motor neurons. Essential to the understanding of AP-1 activity in neurons is the knowledge of other proteins that influence AP-1 function or are downstream transcriptional targets. This study describes a forward genetic screen for modifiers of AP-1 in Drosophila (Franciscovich, 2008).

Using a conveniently scored AP-1-dependent adult-eye phenotype, 4307 EP and EPgy2 lines were screened for genes that modified this phenotype. Several advantages of this screen include: (1) the ease and rapidity of screening as compared to the neuromuscular junction, (2) immediate gene identification, (3) the potential to analyze in vivo phenotypes that arise from overexpression/misexpression, and finally (4) the scope for rapidly generating loss-of-function mutations through imprecise excision of the same P-element. A total of 249 known genes were isolated of which 73 can be directly implicated in eye development. The selection was prioritized using several criteria, to derive a short list of 13 final candidates that were then tested at the NMJ. Future work will focus on other predicted but as yet unstudied genes that are likely to have important functions at the NMJ (Franciscovich, 2008).

The prescreening strategy using the adult eye was successful because (1) almost all the genes selected did not result in eye phenotypes when expressed on their own, but selectively modified a Fbz dependent phenotype (Fbz is a dominant-negative transgenic construct that expresses the Bzip domain of Drosophila Fos); (2) several genes were identified that are known to interact with AP-1 in regulating synaptic phenotypes (these include ras and bsk); (3) multiple alleles of some genes were recovered confirming the sensitivity of the screening technique; (4) several genes involved in eye development were isolated (including cyclinB, which has been shown to be a downstream target of Fos in the regulation of G2/M transition in the developing eye); (5) a large number of putative interactors have connections with neural physiology and/or AP-1 function in other cell types; (6) some candidates with strong phenotypes have previously been shown to play important roles in motor neurons; and finally (7) the majority of candidates (but not all) isolated as enhancers or suppressors of Fbz in the eye exerted a similar effect on AP-1 at the synapse (Franciscovich, 2008).

Although the relative success and merits of a functional screen are considerable, there are a few disadvantages. First, the use of P-element transposons naturally excludes a large fraction of genes that are refractory to P-element transposition events. Second, insertions of EP elements within or in inverse orientation to the gene make it difficult to assign phenotypes to specific genes. Even in instances where overexpression was predicted, it has to be verified that this is indeed the case and also the phenotypes derive from hypomorphic mutations that result from the insertion of the P-element close to the target gene have to be tested. Third, although recover genes that play conserved roles in AP-1 biology is to be expected, those genes that specifically affect synaptic physiology and play no role in the eye will be excluded by this scheme. Finally, this screen will not discriminate between genes that function upstream or downstream of AP-1 in neurons. In spite of these deficiencies, it is believed that candidates identified in this screen provide strong impetus for the investigation of additional factors that are involved in the regulation of synaptic plasticity and development by AP-1 (Franciscovich, 2008).

Following their identification, it was found that several candidates had synaptic functions since several of these genes resulted in significant differences in synaptic size when compared to appropriate controls. This provided the first confirmation of the screening strategy. Next, experiments to determine genetic interaction with AP-1 showed that expression of four genes (pigeon, lbm, Cnx99A, and sty) suppressed the Fbz-dependent small synapse phenotype. Of these, sty had been isolated as an enhancer while the other three similarly suppressed the Fbz-derived eye phenotype, suggesting potentially conserved functions of these genes in the two tissues (Franciscovich, 2008).

Four genes isolated as enhancers, similarly enhanced an Fbz-mediated small synapse (cnk, pde8, fkbp13, and sgg). Notably, expression of these genes also suppressed an AP-1-dependent synapse expansion at the NMJ. These two lines of evidence indicate that these genes are negative regulators of AP-1 function in these neurons. Together with the fact that all four have previously described functions in the nervous system, these observations confirm the validity of the screen and highlight the utility of genetic screens to uncover novel molecular interactions. Further studies will provide a more comprehensive understanding of the interplay between these genes and AP-1 in the regulation of neuronal development and plasticity. For instance, more careful analysis needs to be carried out to discern whether synaptic phenotypes in each of these cases are due to overexpression or potential insertional mutagenesis of specific genes (Franciscovich, 2008).

Although GSK-3β-signaling has been implicated in several neurological disorders such as Alzheimer's disease, it is only recently that neuronal roles for this important kinase have come to light. For instance, several studies have demonstrated the role of GSK-3β in the regulation of long-term potentiation (LTP) in vertebrate hippocampal synapses (Hooper, 2007; Peineau, 2007; Zhu, 2007). In particular, these reports highlight the negative regulatory role of GSK-3β in the induction of LTP or in one case, the switching of long-term depression (LTD) into LTP. Interestingly, LTP induction leads to GSK-3β-inhibition thus precluding LTD induction in the same neurons. In flies, sgg mutations have defects in olfactory habituation, circadian rhythms and synaptic growth. These observations point to a conserved and central role for GSK-3β in neuronal physiology (Franciscovich, 2008).

GSK-3β-dependent modulation of transcriptional responses is widely acknowledged. Among several transcription factors that are known to be regulated by this kinase, are AP-1, CREB, NFAT, c/EBP, and NF-kappaB. In the context of neuronal function, for instance, RNA interference-based experiments in cultured rat cortical neurons have shown that GSK-3β-activity influences CREB and NF-kappaB-dependent transcription. Additionally, two other transcription factors, early growth response 1 and Smad3/4 have been identified in DNA profiling experiments in the same study. Significantly, GSK-3β is also a primary target of lithium, a drug used extensively to treat mood disorders. Lithium treatment has been reported to result in an upregulation of AP-1-dependent transcription, though a role for GSK-3β in this phenomenon has not been tested directly (Franciscovich, 2008).

In Drosophila, recent experiments have described the negative regulation of synaptic growth by the GSK3β-homolog shaggy (Franco, 2004). These studies demonstrate that sgg controls synaptic growth through the phosphorylation of the Drosophila MAP1B homolog futsch. The current studies suggest that Sgg-dependent regulation of synapse size occurs through the immediate-early transcription factor AP-1. GSK-3β is believed to inhibit transcriptional activity of AP-1 in cultured cells by direct inhibitory phosphorylation of c-Jun. Circumstantial evidence also suggests that GSK-3β provides an inhibitory input into AP-1 function in neurons (Franciscovich, 2008).

It was intriguing to find that Sgg inhibition leads to an expanded synapse with reduced presynaptic transmitter release, similar to highwire mutants. Given that in several instances, Sgg-dependent phosphorylation targets a protein for ubiquitination, and that Highwire encodes an E3 ubiquitin ligase, it is conceivable that sgg and hiw function in the same signaling pathway. Consistent with this hypothesis, both hiw and sgg function at the synapse seem to impinge on AP-1-dependent transcription through modulation of the JNK signaling pathway. Considering previous reports of GSK-3β-involvement in multiple signaling cascades, it will be interesting to study how sgg controls multiple aspects of cellular physiology to regulate neural development and plasticity, particularly in the context of brain function and action of widely used drugs such as lithium (Franciscovich, 2008).

Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe

A large number of neural and glial cell cell types differentiate from neuronal precursor cells during nervous system development. Two types of Drosophila optic lobe neurons, lamina and medulla neurons, are derived from the neuroepithelial (NE) cells of the outer optic anlagen. During larval development, epidermal growth factor receptor (EGFR)/Ras signaling sweeps the NE field from the medial edge and drives medulla neuroblast (NB) formation. This signal drives the transient expression of a proneural gene, lethal of scute, and its signal array is referred to as the 'proneural wave', since it is the marker of the EGFR/Ras signaling front. This study shows that the atypical cadherin Fat and the downstream Hippo pathways regulate the transduction of EGFR/Ras signaling along the NE field and, thus, ensure the progress of NB differentiation. Fat/Hippo pathway mutation also disrupts the pattern formation of the medulla structure, which is associated with the regulation of neurogenesis. A candidate for the Fat ligand, Dachsous is expressed in the posterior optic lobe, and its mutation was observed to cause a similar phenotype as fat mutation, although in a regionally restricted manner. It was also shown that Dachsous functions as the ligand in this pathway and genetically interacts with Fat in the optic lobe. These findings provide new insights into the function of the Fat/Hippo pathway, which regulates the ordered progression of neurogenesis in the complex nervous system (Kawamori, 2011).

The Fat/Hippo pathway has been known as a tumor suppressor pathway. This study and in the report of Reddy (2010), it was shown that the loss of Fat/Hippo signaling causes a delay of NB differentiation in the optic lobe. In contrast, dachs;ft double mutation, which is expected to stabilize the Fat/Hippo pathway, causes an advance of NB differentiation. This led to the question of how the Fat/Hippo pathway controls NB differentiation (Kawamori, 2011).

It has been reported that EGFR/Ras signaling is necessary and sufficient for NB induction, and its transduction is the driving force of the progress of the proneural wave. EGFR/Ras signaling sweeps the NE field through the gradual activation of Ras and its downstream EGF secretion by Rho. It was reasoned that this signal transduction is the target of the Fat/Hippo pathway in the control of NB differentiation. Indeed, ectopic expression of the EGFR/Ras signaling components RasV12 and rho was sufficient to induce NBs in the ft mutant background (Kawamori, 2011).

Which step of this cycling process does Fat/Hippo pathway mutation affect? Based on ectopic expression experiments, the Fat/Hippo pathway lies upstream of Ras and Rho, and it is expected to control the process from EGF transmission to Ras activation. The phenotypic difference produced by RasV12 and rho overexpression should be noted. When rho was expressed in the ft mutant background, several NE clones with abnormal morphology remained. This phenotype was not observed when RasV12 was expressed. In this model, RasV12 drives NB differentiation in RasV12-expressing cells in a cell-autonomous manner. In contrast, Rho activates Ras signaling in neighboring cells through the secretion of an EGF ligand. Based on these phenotypic differences, the Fat/Hippo pathway is expected to control the cell-to-cell EGF transmission, including its secretion, distribution or reception at the cell surface. This hypothesis is supported by the fact that several signaling components of the EGFR/Ras pathway, including Rho and EGFR, are localized to the apical side of epithelial tissues, and it is thought that this signal is transmitted along the apical side in epithelial tissues. It has also been reported that Fat/Hippo pathway mutations enhance the expression level of several apically localized molecules, such as aPKC, PatJ, Crumbs and E-cadherin. Thus, Fat/Hippo signaling targets could include unknown apical components that are involved in EGF transmission and this could account for the incomplete NB induction by rho overexpression. rho-expressing cells secret the EGF ligand, which diffuses in the NE surface, but Fat/Hippo pathway mutation would prevent its cell-to-cell transmission and subsequent EGFR/Ras pathway activation in the receiving cells. In this hypothesis, EGF transmission would be disturbed in the NE mutant for the Fat/Hippo pathway, causing the delay of proneural wave progress (Kawamori, 2011).

As an alternative hypothesis, the Fat/Hippo pathway could regulate signal transduction from the EGFR to Ras activation. If this is the case, the Fat/Hippo pathway regulates the intracellular signal transduction of the EGFR pathway. Many of the known targets of the Fat/Hippo pathway are components of growth regulatory, cell survival and cell adhesion molecules. There could be unknown targets that modulate other signaling pathways, including the EGFR pathway, and the NB differentiation defect would thus be caused by a failure in the activation of differentiation signals in the absence of Fat/Hippo signaling (Kawamori, 2011).

This study shows that the Fat/Hippo pathway mutation also affected the morphological character of the NE. Fat/Hippo pathway mutant clones were induced, and they often included NE tissue with a folded morphology and disrupted the medulla structure. The results showed that the Fat/Hippo pathway functions in the regulation of NB differentiation and in NE morphology are distinct, but the two functions could affect each other. The morphological defect of the NE could affect EGFR/Ras signal transduction. The possibility is discussed that the EGF ligand could be distributed along the apical membrane of the NE. The invagination of the apical membrane of the folded NE into the inner region could prevent EGF ligand signaling. There were clones with a normal NE morphology in which NB differentiation was delayed and, thus, morphological defects are not determinate, but they could promote the delay of NB induction (Kawamori, 2011).

How is the activity of the Fat/Hippo pathway regulated throughout the development of the optic lobe? Ft is a member of the cadherin family, and an extracellular molecule is expected to regulate its activity. Ds is a candidate for the Ft ligand that regulates planar cell polarity and Fat/Hippo signaling activity in other epithelial tissues. The expression of ds with a posterior-specific pattern in the developing optic lobe (Reddy, 2010) was confirmed. In the rescue experiments for the ds mutation, the expression of either ds lacking its intracellular domain (dsΔICD) or ft lacking its extracellular domain (ftΔECD) was sufficient to compensate for ds function, suggesting that Ds functions as a ligand and that Ft lies downstream of Ds in this context (Kawamori, 2011).

The phenotypes of ds and ft mutants were compared to assess whether the mutation of ds by itself accounts for the phenotype of the ft mutants. In contrast to the ft mutants that exhibited altered NB differentiation in the entire outer optic anlagen, the ds mutant phenotype was regionally specific; NB differentiation was severely delayed in the posterior region, and the development of the anterior region was not significantly affected. These differences suggest that there might be some regulatory mechanisms that control Ft activity independently of Ds in the anterior region of the optic lobe (Kawamori, 2011).

The Fat/Hippo pathway is known as a tumor suppressor pathway, and many studies related to this pathway have focused on tissue growth or cell survival. This study has reported a new function of the Fat/Hippo pathway in the regulation of neural differentiation. The Fat/Hippo pathway regulates the progress of neural differentiation signaling, and the EGFR/Ras pathway is a candidate target of this pathway. The data suggest that the Fat/Hippo pathway includes unknown targets involved in EGFR/Ras signal transduction. Further studies are required to identify the targets of the Fat/Hippo pathway and determine the interplay between Fat/Hippo and EGFR/Ras pathways, specifically in NB differentiation (Kawamori, 2011).

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Ras85D: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | References

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