Ras oncogene at 85D
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).
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)
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).
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).
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).
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 pupaís 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 ëIPCsí. 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 ëcloverleafí 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).
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).
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).
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).
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 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).
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).
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