EGF receptor
In Drosophila, the dorsal-ventral polarity of the egg chamber depends on the successful localization of the
oocyte nucleus and Gurken mRNA to the dorsal-anterior corner of the oocyte. Gurken protein
presumably acts as a ligand for the Egfr expressed in the
somatic follicle cells surrounding the oocyte. gurken and Egfr function in an earlier signaling event
that establishes posterior follicle cell fates and specifies the anterior-posterior polarity of the egg
chamber. Mutations in fs(1) K10, squid and orb prevent the formation of a correctly polarized microtubule
cytoskeleton required for proper localization of the anterior and posterior determinants Bicoid and
Oskar, and for the asymmetric positioning of the oocyte nucleus. Such mutation can result in a dorsoventral axis being formed almost parallel to the anterior-posterior axis (Roth, 1995).
The defect in anterior-posterior follicle cell patterning is evident in the morphology of the mutant eggs. The wild-type chorion has distinct anterior and posterior ends: There is a micropyle at the anterior pole and an aeropyle at the posterior end. In gurken, Egfr and cornichon mutant eggs, there is frequently a second micropyle at the posterior pole. This duplication of anterior structures at the posterior is also evident during oogenesis. In wild-type egg chambers, the micropyle is secreted by two anterior follicle cell types: border cells and centripetal follicle cells.
In mutant ovaries, molecular markers specific for these cell types are duplicated at the posterior pole, indicating that in mutants, the presumptive follicle cells take on an anterior fate, resulting in secretion of a second micropyle at the posterior. Also in mutant egg chambers, Bicoid mRNA, which is normally localized in a ring at the anterior cortex, is localized to both anterior and posterior poles, and Oskar mRNA, which is normally localized to the posterior pole, is displaced to the middle. Mislocalization of RNA has its origin in a defect in the cytoskeleton. A second signal back from follicle cells to the oocyte, influencing microtubule polarity, is implied, but its basis is unknown. The second signal appears to involve the cAMP-dependent protein kinase A gene (Ray, 1996).
During oogenesis,
Egfr activation is required for the establishment of the dorsal/ventral axis of the
egg and the embryo. To examine how ectopic Egfr activation affects cell fate
determination, an activated version of the protein was constructed. Expression of this
activated form (lambda top) in the follicle cells of the ovary induces dorsal cell fates in
both the follicular epithelium and the embryo. Different levels of expression result in
different dorsal follicle cell fates. Among the anterior follicle cells, a minimum of three cell fates can be distinguished by their contribution to the final eggshell morphology. The most dorsal cells produce the midline/operculum region; dorsolateral cells secrete the respiratory appendages, and the ventral cells contribute to the main body. The three populations of follicle cells in the anterior of the developing egg chamber express different combinations of downstream genes. The dorsal midline cells express
argos, kekkon1, rhomboid and pointed. The dorsolateral cells express rho and kek1. The ventral follicle cells are distinguished by the expression of CF2 (Queenan, 1997).
Even in cases where all the follicle cells
covering the oocyte express lambda top, dorsal cell fates are expanded in the
anterior, but not the posterior, of the egg. The expression of genes known to respond
to Egfr activation (aos, kek 1 and rho) are also
expanded in the presence of the lambda top construct. When lambda top is
expressed in all the follicle cells covering the oocyte, kek 1 and argos expression are
induced in follicle cells all along the anterior/posterior axis of the egg chamber. In
contrast, rho RNA expression is only activated in the anterior of the egg chamber.
These data indicate that the response to Egfr signaling is regulated by an
anterior/posterior prepattern in the follicle cells. Expression of lambda top in the entire
follicular epithelium results in an embryo dorsalized along the entire anterior/posterior
axis. Expression of lambda top in anterior or posterior subpopulations of follicle cells
results in regionally autonomous dorsalization of the embryos. This result indicates
that subpopulations of follicle cells along the anterior/posterior axis can respond to
Top/Egfr activation independently of one another (Queenan, 1997).
The zinc finger transcription factor CF2 is a mediator of Egfr activated dorsoventral patterning in Drosophila oogenesis. Dorsal ventral polarity is established by a signal from the oocyte to the anterior dorsal follicle cells by the EGF receptor pathway. CF2 is suppressed by Egfr signaling in the anterior dorsal domain of the follicular epithelium where the dorsal signal is received. In turn CF2 depletion expands the expression of dorsal genes such as rhomboid. Since CF2 functionally activates ventral cell fate, it appears that CF2 is a critical target for Egfr signaling in oogenesis (Hsu, 1996).
At stage 10 of oogenesis, mirror is
expressed in anterior-dorsal follicle cells, and this is dependent upon the Gurken signal from the oocyte. The fringe gene is expressed in a complementary
pattern in posterior-ventral follicle cells at the same
stage. Ectopic expression of mirror represses fringe expression,
thus linking the epidermal growth factor receptor
(Egfr) signaling pathway to the Fringe signaling
pathway via Mirror. The Egfr pathway also triggers the
cascade that leads to dorsal-ventral axis determination in
the embryo. twist was used as an embryonic marker for
ventral cells. Ectopic expression of mirror in the follicle
cells during oogenesis ultimately represses twist expression
in the embryo, and leads to phenotypes similar to those that occur due to
the ectopic expression of the activated form of Egfr.
Thus, mirror also controls the Toll signaling pathway, leading to Dorsal nuclear transport. In summary, the Mirror homeodomain protein provides a link that coordinates the Gurken/Egfr signaling pathway (initiated in the oocyte) with the Fringe/Notch/Delta pathway (in follicle cells). This coordination is required for epithelial morphogenesis, and for producing the signal in ventral follicle cells that determines the dorsal/ventral axis of the embryo (Zhao, 2000).
To test whether the complementary expression of mirr
and fng depend upon Egfr signaling at stage 10, their expression patterns were examined in flies expressing activated or dominant negative forms of Egfr.
When the activated form of Egfr (DERAF or lambdatop) is
expressed in the anterior follicle cells, all those cells now
express mirr, but not fng. When the dominant negative
form of Egfr (DER DN) is expressed in those follicle cells surrounding the oocyte, but not the centripetal follicle cells, the
anterior-dorsal follicle cells no longer express mirr. The expression domain of fng expands to include the anterior-dorsal cells in these mutants.
Thus, the expression of both mirr and fng are either positively
or negatively regulated by the activation of Egfr,
and a complementary expression pattern is maintained in
all these experiments. Experiments show that Grk/Egfr signaling
is required to activate the expression of mirr in anterior-dorsal and centripetal follicle cells, which in turn represses
the expression of fng in those cells. As a result,
fng is only expressed in the posterior and ventral follicle cells, where it is required for the normal morphogenesis of the follicle cell layer (Zhao, 2000).
To investigate how the dorsal-ventral polarity
of embryos is affected by ectopic expression of mirr,
expression of the twist (twi) gene was used as a marker
for ventral embryonic cells. After
Egfr activation in anterior dorsal follicle cells, 11 dorsal-group maternal genes are later involved in the establishment
of a gradient of nuclear Dorsal (DL) protein in
the ventral cells of the embryo. This in turn activates
the expression of twi in the whole ventral domain of
the wild-type embryo. In those eggs laid by females
with ectopic expression of mirr driven by the Gal4
line T155, the central expression domain of twi is reduced and in some embryos completely disappears. The terminal domains of twi at both
ends of the embryos are rarely affected. Similar phenotypes
are observed when DERAF is ectopically expressed, but the frequencies of abnormalities are very different. Of the eggs laid, 98% have multiple dorsal appendages, rather than the 2% observed following
ectopic expression of mirr. Also, most eggs lose the
main domain of twi expression following ectopic expression
of DERAF. The terminal regions of twi expression
still remain, using this Gal 4 driver for DERAF (Zhao, 2000).
Further evidence that mirr is responsible for the repression
of ventral genes that direct the dorsal-ventral
polarity of the embryo comes from experiments in which
mirr is ectopically expressed in the posterior of the egg
chamber. Ectopic expression of DERAF in this region
causes the loss of both posterior embryonic segments
and the posterior twi expression domain. When mirr is ectopically expressed in the posterior follicle cells, a similar phenotype is observed,
with both posterior embryonic denticle belts and the twi
expression domain missing. This clearly shows that mirr function is critical for the dorsal-ventral axis of the embryo and that ectopic expression of mirr
can lead to an abnormal localization of the ventral signal, which is required to trigger the initiation of the embryonic ventral pattern (Zhao, 2000).
The ecdysone response
hierarchy mediates egg chamber maturation during
mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific
manner while EcR expression is ubiquitous
throughout oogenesis. Decreasing or increasing the ovarian
ecdysone titer using a temperature-sensitive mutation or
exogenous ecdysone results in corresponding changes in
early gene expression. The stage 10 follicle cell expression
of Ecdysone-induced protein 75B (E75) in wild-type, K10 and EGF receptor (Egfr) mutant
egg chambers reveals regulation of E75 by both the Egfr
and ecdysone signaling pathways. Genetic analysis
indicates a germline requirement for ecdysone-responsive
gene expression. Germline clones of E75 mutations arrest
and degenerate during mid-oogenesis and EcR germline
clones exhibit a similar phenotype, demonstrating a
functional requirement for ecdysone responsiveness during
the vitellogenic phase of oogenesis. Finally, the expression
of Drosophila Adrenodoxin Reductase increases during
mid-oogenesis and clonal analysis confirms that this
steroidogenic enzyme is required in the germline for egg
chamber development. Together these data suggest that the
temporal expression profile of E75, E74 and BR-C may be
a functional reflection of ecdysone levels and that ecdysone
provides temporal signals regulating the progression of
oogenesis and proper specification of dorsal follicle cell
fates (Buszczak, 1999).
During stage 10, the follicle cell expression of E75 becomes
enriched in the dorsal anterior cells. This suggested
that inputs in addition to ecdysone are needed to refine E75
expression. Previous work has shown that follicle cell polarity
is established during mid- to late-oogenesis and depends on the
interaction between Gurken and the Drosophila
homolog of the mammalian EGF receptor (Egfr). To determine whether E75 expression is
under control of the dorsoventral signaling pathway, ovarian E75 mRNA distribution was examined in dorsalized and
ventralized mutant backgrounds. In fs(1)K10 mutants,
mislocalization of Grk protein results in activation of Egfr
in all anterior follicle cells surrounding the oocyte. In
fs(1)K10 mutant egg chambers, E75 expression expands to a
ring of anterior follicle cells surrounding the oocyte.
Mutations in Egfr prevent signal transduction by the receptor
and lead to the ventralization of the eggshell and embryo. In situ analysis indicates that stage 10
follicle cells overlying the oocyte in Egfr mutants no longer
express E75. However, E75 expression in the nurse
cells is unaffected. These experiments show that
the Egfr signaling pathway regulates E75 expression in the
dorsal follicle cells but not in the germline (Buszczak, 1999).
The Drosophila BMP homolog DPP can function as a
morphogen, inducing multiple cell fates across a
developmental field. However, it is unknown how graded
levels of extracellular DPP are interpreted to organize a
sharp boundary between different fates. Opposing DPP and EGF signals are shown to set the boundary for an
ovarian follicle cell (FC) fate. First, DPP regulates gene
expression in the follicle cells that will create the operculum
of the eggshell. Global increase in DPP levels, using
heat-shock-GAL4 to drive UAS-dpp expression throughout all
FCs gives rise to eggs that show expanded opercula
and reduced dorsal appendages. In other respects,
the eggshells are normal. At the extreme anterior, normal
micropyles were formed. The mutant opercula generally have a
normal organization of large cell imprints surrounded by a
raised structure, the collar. Significantly, expansion
of the operculum always occurs over the dorsal side of the
egg, indicating that dorsal-ventral patterning is unperturbed. DPP induces expression of the enhancer
trap reporter A359 and represses expression of bunched,
which encodes a protein similar to the mammalian
transcription factor TSC-22. Second, DPP signaling
indirectly regulates A359 expression in these cells by
downregulating expression of bunched. Reduced bunched
function restores A359 expression in cells that lack the
Smad protein Mad; ectopic expression of Bunched
suppresses A359 expression in this region. Importantly,
reduction of bunched function leads to an expansion of the
operculum and loss of the collar at its boundary. Third,
EGF signaling upregulates expression of bunched. The bunched expression
pattern requires the EGF receptor ligand Gurken. Activated EGF receptor is sufficient to induce
ectopic bunched expression. Thus, the balance of DPP and
EGF signals sets the boundary of bunched expression. It is proposed that the juxtaposition of cells with high and low
Bunched activity organizes a sharp boundary for the
operculum fate (Dobens, 2000).
Gurken signaling through the
Egfr is necessary for normal bunched-lacZ expression in the
dorsal anterior FC. Ectopic expression of activated Egfr is sufficient to
induce ectopic bunched-lacZ in the centripetal migrating FCs.
Conversely, Dpp signaling is both necessary and sufficient to
repress bunched-lacZ in columnar FCs. Thus the dorsal anterior
boundary of bunched-lacZ expression is set by a balance of
positive EGF and negative Dpp signals. Dpp also sets the
anterior boundary for Broad-Complex expression; however, the
regulation of this gene by EGF signaling is more complex.
In summary, a model is proposed where the
boundary for the operculum is set by the boundary of
Bunched activity, which is positioned by opposing activity
of Dpp and EGF signals in the dorsal FCs. Dorsal anterior FC
are exposed to high levels of EGF ligands Grk, Spitz and Vein, and thus have elevated bunched expression.
High anterior Dpp signaling represses bunched expression.
The close apposition of these signals in the dorsal anterior FCs
creates a sharp boundary of bunched expression. BUN-1
functions to repress A359 and define the boundary to
centripetal migrating FC fates, including the operculum. These
data indicate that the ventral operculum boundary is also set
by bunched; however, another signal appears to promote
ventral bunched expression at late stages. The normal operculum border is
defined by the eggshell collar. This structure is lost as bunched
activity is lowered, suggesting that the boundary of bunched
expression may serve to further organize cell fates at the
operculum boundary (Dobens, 2000).
Although the data suggest that EGF signals antagonize
operculum patterning, EGF signaling is essential for
operculum formation. (1) grk and Egfr mutant eggs have
no opercula. (2) Overexpression of
activated Egfr can result in operculum expansion, although
interpretation of the specific phenotype is not straightforward. Thus, it is expected that Dpp does not
prevent all Egfr-induced events in the operculum-forming
FCs. It is likely that EGF signaling is active in cells that lack
Bunched activity, and that Dpp inactivation of Bunched
modifies the response of these cells to EGF signals. In cultured
mammalian cells, RTK signaling can directly antagonize BMP
signaling by preventing nuclear accumulation of Smad protein,
offering a possible molecular mechanism for these interactions (Dobens, 2000).
Invasive cell migration in both normal development and metastatic cancer is
regulated by various signaling pathways, transcription factors and
cell-adhesion molecules. The coordination between these activities in the
context of cell migration is poorly understood. During Drosophila oogenesis, a
small group of cells called border cells (BCs) exit the follicular epithelium to
perform a stereotypic, invasive migration. The ETS transcription
factor Yan is required for border cell migration and Yan expression is
spatiotemporally regulated as border cells migrate from the anterior pole of the egg chamber towards the nurse cell-oocyte boundary. Yan expression is dependent on inputs from the JAK/STAT, Notch and Receptor tyrosine kinase pathways (Egfr and Pvr) in border cells. Mechanistically, Yan functions to modulate the turnover of DE-Cadherin-dependent adhesive complexes to facilitate border cell migration. These results suggest that Yan acts as a pivotal link between signal transduction, cell adhesion and invasive cell migration in Drosophila border cells (Schober, 2005).
Interestingly, Yan expression levels gradually decrease as BCs
move along an increasing PVR/EGFR activity gradient. Yan
has been shown to be phosphorylated by the EGFR-MAPK pathway, which triggers
its nuclear export and protein degradation.
Consistent with these previous studies, expression of dominant-active PVR and
EGFR in BCs blocks BC migration and abrogates Yan protein expression, whereas
yan transcript or enhancer trap expression is still detectable.
Expression of activated Ras and Raf similarly induced Yan downregulation,
consistent with an involvement of the canonical Ras/MAPK pathway in mediating
PVR/EGFR signaling. It is noted, however, that although BC migration is
significantly delayed upon ectopic expression of activated Ras, activated Raf
hardly affects their ability to migrate. The basis of this difference, which
might be due to complex feedback loops between the implicated signaling
pathways, is unclear at the present time and will need to be investigated
further (Schober, 2005).
Anterior-posterior axis formation in the Drosophila oocyte requires activation of the EGF receptor (EGFR) pathway in the posterior follicle cells (PFC), where it also redirects them from the default anterior to the posterior cell fate. The relationship between EGFR activity in the PFC and oocyte polarity is unclear, because no EGFR-induced changes in the PFC have been observed that subsequently affect oocyte polarity. This study shows that an extracellular matrix receptor, Dystroglycan, is down-regulated in the PFC by EGFR signaling, and this down-regulation is necessary for proper localization of posterior polarity determinants in the oocyte. Failure to down-regulate Dystroglycan disrupts apicobasal polarity in the PFC, which includes mislocalization of the extracellular matrix component Laminin. These data indicate that Dystroglycan links EGFR-induced repression of the anterior follicle cell fate and anterior-posterior polarity formation in the oocyte (Poulton, 2006; full text of article).
This study has identified DG as a gene whose expression pattern is both regulated by EGFR signaling in the PFC and necessary for oocyte polarity. These findings provide a mechanistic link between EGFR activity in the PFC and polarization of the oocyte. Furthermore, it was discovered that defects in apicobasal polarity caused by ectopic DG also are present in the PFC where EGFR signaling is disrupted, possibly due to the misexpression of DG in these cells. In addition, the findings that ectopic DG leads to mislocalizations of Lan at the apical surface of the PFC indicates a process of cell–cell communication in which EGFR-regulated DG expression in the PFC controls Lan organization in the ECM that in turn may affect localization of posterior determinants in the oocyte (Poulton, 2006).
It was reported that loss of LanA in the PFC disrupts oocyte polarity, which seems to be in conflict with the suggestion that high levels of apical Lan in the PFC perturbs oocyte polarity. However, a model in which Lan is required in early oogenesis, but must be localized basally after EGFR activation and DG down-regulation, reconciles these findings. In the previous research on loss-of-function lanA mosaic egg chambers, oocyte polarity defects observed at stage 9/10 could be generated only by larger lanA PFC clones. Because follicle cells are only mitotically active until stage 6/7 of oogenesis, these large PFC clones present at stage 9/10 would have represented sizeable lanA clones in prestage-6 follicle cells. Because Lan is present on the apical surface of these pre-PFCs, the polarity defects observed at stage 9/10 may have resulted from perturbation of some earlier Lan-dependent processes, such as organizing receptors on the facing surfaces of the oocyte or follicle cells. Consistent with this model, the addition of Lan to myotubes in culture is sufficient to organize the receptors integrin and DG, as well as their respective cytoplasmic counterparts, vinculin and dystrophin. Alternatively, it could be that the role of Lan in mediating the relationship between the PFC and oocyte is sensitive to any disruption of the ECM stemming from either the loss or misexpression of Lan, which then is sufficient to negatively affect oocyte polarity. Either of these models demonstrates the importance of the ECM in this process and ultimately may lead to a mechanistic understanding of the oocyte polarity defects caused by mutation in the putative Lan receptor Dlar (Poulton, 2006).
Precisely how ectopic DG on the surface of the PFC translates to mislocalizations of posterior polarity markers in the adjacent oocyte remains to be determined, however, several different explanations for this process can be considered. (1) DG down-regulation in the PFC may be necessary to allow the actin-based cortical anchoring of the posterior determinants in the oocyte. (2) The down-regulation of DG after EGFR activation might serve as a cue to the oocyte, which leads directly to MT reorganization and AP axis formation. In this analysis, however, DG overexpression did not result in defects in global microtubule organization or mislocalization of anterior oocyte polarity markers, phenotypes that have been reported in grk and top mutant egg chambers. Furthermore, simply reducing DG levels in non-PFCs by RNAi was not sufficient to mislocalize Stau to nonposterior regions of the oocyte. Therefore, DG down-regulation alone probably cannot serve as the signal to repolarize the microtubule network and, thus, establish oocyte polarity, but it is possible that changes in cell adhesion mediated through the DG/Lan complex could be part of a complex signal involving additional ECM receptors or even other signaling mechanisms that have yet to be identified. A similar model has been proposed for this signal in which changes in cell adhesion between the oocyte and PFCs serve as a nontraditional signal initiating AP axis formation. Alternatively, EGFR-mediated changes in DG/Lan patterns could regulate a novel mechanism that is required specifically for localization of posterior determinants at the oocyte cortex but is independent of the signal provided by the PFC to repolarize the oocyte microtubule cytoskeleton (Poulton, 2006).
(3) The apicobasal defects caused by up-regulation of DG may have led to the loss of apical targeting of the polarizing signal from the PFC, as has been proposed for oocyte polarity defects caused by Merlin mutation. This explanation does not seem likely, however, given the ability of DG RNAi to rescue the CAM phenotype even though the Ras clones still should be unable to produce the signal, because they do not take the PFC fate. Instead, a model is favored in which the apicobasal defects caused by ectopic DG results in apical accumulations of Lan, thereby modifying the ECM between the clones and oocyte so as to preclude diffusion of a secreted signal from the adjacent wild-type cells. Therefore, in the Ras rescue experiment, down-regulation of DG allows the basal restriction of Lan, facilitating diffusion of the polarizing signal from the remaining wild-type cells. The fact that the rescue of the CAM phenotype by DG RNAi in Ras clones was not complete (34% of these egg chambers continued to show some defect in Stau localization) may support this model, because the diffusion of a signal from the neighboring cells probably would not be expected to replace fully the endogenous signal absent from the clone cells in every case. Whether mutations in other genes required for both apicobasal polarity and oocyte polarity also disrupt the ECM will be interesting to discover (Poulton, 2006).
The study of axis formation in the Drosophila oocyte has demonstrated the importance of cell–cell communication in the tightly regulated patterning of the follicle cells, which ultimately leads to the establishment of those axes. The key findings presented here suggest a multifaceted role for EGFR signaling in PFC differentiation and oocyte polarization, highlighting the need for further study of EGFR activity, differentiation of the PFC, and formation of the AP axis (Poulton, 2006).
α-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between α-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific α-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, α-actinin is ubiquitously expressed. The non-muscle α-actinin mutant ActnΔ233, which is viable and fertile, lacks α-actinin expression in ovarian germline cells, while somatic follicle cells express α-actinin at late oogenesis. This latter population of α-actinin, termed FC-α-actinin, is shown to be absent from the dorsoanterior follicle cells, and evidence is presented that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increases the F-actin bundling activity of ectopically expressed muscle-specific α-actinin. A novel morphogenetic event in the follicle cells is described that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior β-integrin-dependent adhesion site accumulating α-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although α-actinin is not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling is perturbed, and Enabled does not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that α-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila (Wahlström, 2006).
The dorsoanterior follicle cells express only NC-α-actinin (α-Actinin produced from an mRNA that is transcribed from the upstream promoter), whereas the main body follicle cells also express FC-α-actinin. Furthermore, these two cell populations responded differently to overexpression of the adult muscle-specific α-actinin isoform. Prominent actin spikes were induced in the dorsal appendage cells, while the main body follicle cells remained unaffected. This effect might be specific for the adult muscle-specific isoform. Alternatively, the dorsal appendage cells might be sensitive to an overload of any isoform of α-actinin. In either case, it provides a plausible explanation as to why FC-α-actinin needs to be downregulated in these cells. The appearance of spikes roughly coincided with the start of tube elongation, suggesting a connection with events that regulate dorsal appendage migration. At stages 9 and 10A, also stages at which the follicle cells undergo migration, a thickening of the basal actin fibres was observed. Since the dorsal appendage cells are characterised by their maintained EGFR signalling, whether EGFR signalling directly increased α-actinin's bundling activity was tested. Indeed, co-expression of λtop and muscle-specific α-actinin at stages 9 and 10A resulted in extensive bundling of the basal actin fibres, an effect that was not observed when either protein was expressed alone. No effect was seen prior to stage 9, suggesting that the factor needed for α-actinin’s bundling activity was induced independent of EGFR signalling at stage 9, but subsequent to the induction, its activity was modulated by EGFR signalling (Wahlström, 2006).
Regardless of whether or not the effect is specific for a certain α-actinin isoform, it is concluded that EGFR signalling modulates α-actinin's bundling activity. A possible mediator could be, for example, an enzyme that regulates the phosphoinositide levels in the cell. The F-actin crosslinking activity of vertebrate α-actinin is regulated by phosphoinositide binding, although some controversy exists as to whether phosphoinositides increase or decrease the crosslinking activity. Interestingly, mammalian cells transfected with a mutant form of α-actinin-1 with a reduced affinity for phosphoinositides displayed excessive bundling of actin filaments in a manner somewhat similar to what was observed in stage 9 follicle cells overexpressing both λtop and muscle-specific α-actinin. The phosphoinositide binding site in vertebrate α-actinin has been mapped to the N-terminal actin-binding domain. All residues shown to be important for phosphoinositide binding are conserved in Drosophila α-actinin, indicating that the same regulatory mechanism probably exists in Drosophila as well (Wahlström, 2006).
Neurofibromatosis type I (NFI) is a common genetic disorder that causes nervous system tumors, and learning and memory defects in human and in other animal models. A novel growth factor stimulated adenylyl cyclase (AC) pathway has been identified in the Drosophila brain, which is disrupted by mutations in the epidermal growth factor receptor (EGFR), neurofibromin (NF1) and Ras, but not Galphas. This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galphas, can activate AC. This study also shows that Galphas is the major Galpha isoform in fly brains, and a second AC pathway is defined stimulated by serotonin and histamine requiring NF1 and Galphas. A third, classical Galphas-dependent AC pathway, is stimulated by Phe-Met-Arg-Phe-amide (FMRFamide) and dopamine. Using mutations and deletions of the human NF1 protein (hNF1) expressed in Nf1 mutant flies, it is shown that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, it is demonstrated that sequences in the C-terminal region of hNF1 are sufficient for NF1/Galphas-dependent neurotransmitter stimulated AC activity, and for rescue of body size defects in Nf1 mutant flies (Hannan, 2006).
This study defines three separate pathways for AC activation: (1) a novel pathway for AC activation, downstream of growth factor stimulation of EGFR that requires both Ras and NF1, but not Galphas; (2) an NF1/Galphas-dependent AC pathway operating through the Rutabaga-AC (Rut-AC) and stimulated by serotonin and histamine, as observed in the larval brain; (3) a classical G-protein coupled receptor-stimulated AC pathway operating through Galphas alone. The Rut-AC pathway may also be stimulated by PACAP38 at the larval neuromuscular junction and in adult heads as shown in previous studies. The AC activated by NF1/Ras (AC-X), or Galphas (AC-Y), has not yet been identified (Hannan, 2006).
This study shows for the first time that Ras can stimulate AC in an NF1-dependent manner in higher organisms, via an RTK-coupled pathway that is independent of the Galphas G-protein. The functionality of human NF1 in the fly system, and the high degree of identity between human and fly NF1 (60%), suggests that similar pathways for AC activation may also operate in mammals. Previous studies failed to detect stimulation of AC by Ras in cultured vertebrate cell lines, and in Xenopus oocytes, however, these cell types may not contain sufficient NF1 to support NF1/Ras-dependent AC activation. This is consistent with the observation that levels of both Ras and NF1 are critical for stimulation of AC activity in adult head membranes. The reported EGF activation of AC in cardiac myocytes and other tissues requires both Galphas, and the juxtamembrane domain of the EGFR, which is not present in the Drosophila EGFR (Hannan, 2006).
Experiments with human NF1 mutants show that the GRD domain and the RasGAP activity of NF1 are both necessary and sufficient for growth factor-stimulated NF1/Ras-dependent AC activity. It is also concluded that C-terminal residues downstream of the GRD are critical for both body size regulation and neurotransmitter-stimulated NF1/Galphas-dependent AC activity, thus defining for the first time a region outside the GRD that contributes to this pathway. Interestingly, expression of a human NF1 GRD fragment in Nf1-/- astrocytes results in only partial restoration of NF1-mediated increases in cAMP levels in response to PACAP. Thus, regions outside the GRD also seem to be necessary for activation of AC in these mammalian cells (Hannan, 2006).
Thus, NF1, while being a negative regulator of Ras, is also actively involved in stimulation of AC activity. Moreover, it regulates AC activity through at least two different mechanisms, one of which depends on the RasGAP activity of NF1. The multifunctional nature of the NF1 protein illuminates its importance in nervous system development, tumor formation and behavioral plasticity, and may also explain the wide range of clinical manifestations in neurofibromatosis type I (Hannan, 2006).
Epidermal growth factor receptor (EGFR) controls a wide range of developmental events, from body axes specification in insects to cardiac development in humans. During Drosophila oogenesis, a gradient of EGFR activation patterns the follicular epithelium. Multiple transcriptional targets of EGFR in this tissue have been identified, but their regulatory elements are essentially unknown. This study reports the regulatory elements of broad (br) and pipe (pip), two important targets of EGFR signaling in Drosophila oogenesis. br is expressed in a complex pattern that prefigures the formation of respiratory eggshell appendages. This pattern is generated by dynamic activities of two regulatory elements, which display different responses to Pointed, Capicua, and Mirror, transcription factors involved in the EGFR-mediated gene expression. One of these elements is active in a pattern similar to pip, a gene repressed by EGFR and essential for establishing the dorsoventral polarity of the embryo. This similarity of expression depends on a common sequence motif that binds Mirror in vitro and is essential for transcriptional repression in vivo (Fuchs, 2012).
Current models of pattern formation in Drosophila oogenesis
involve multiple components, signaling pathways, and network motifs. Critical tests of these models require direct analysis of the cis-regulatory sequences of genes comprising the network. As a first step in this direction, this study identified the regulatory elements of br, a gene that plays a key role in eggshell patterning and morphogenesis. The dynamic pattern of br was found to be generated by superposition of the activities of two distinct regulatory regions, which drive br expression in nonoverlapping regions of space and display differential sensitivity to three transcription factors that act downstream of EGFR (Fuchs, 2012).
It was shown that loss of Mirr induces ectopic br expression in
the dorsal midline follicle cells, but leads to a complete loss of br
in the lateral cells, which form dorsal appendages. This region-specific effect can be now explained, and is fully consistent with our finding that Mirr represses the brE and activates brL regions, respectively. Previous studies suggest that Mirr functions as a dedicated repressor. Based on this
theory, it is speculated that the activating effect of Mirr on the
expression of the brL region is indirect and involves intermediate
factors. In contrast, these results strongly suggest that
Mirr represses the brE region directly (Fuchs, 2012).
In contrast to the brL region, which generates br expression in
a two-domain pattern that is necessary for the formation of two
eggshell appendages, the function of the brE region is unclear. At
the same time, this regulatory region was instrumental in identification of a critical cis-element that controls the expression of pip, a gene which must be repressed in the dorsal follicle cells
for proper induction of the DV polarity of the embryo. The
regulatory regions of both br and pip contain a sequence essential
for their transcriptional restriction to the ventral follicle cells. Moreover, the data suggest that the identified sequence is
a direct sensor of Mirr, which is derepressed by EGFR.
Thus, thus this study has upheld an earlier proposal that Mirr connects the
EGFR-mediated patterning of the follicle cells to the DV patterning of the embryo. In the emerging transcriptional cascade, EGFR signaling down-regulates CIC, which derepresses Mirr, which in turn represses pip (Fuchs, 2012).
Previous studies have demonstrated that Mirr
can repress pip, but suggested that this effect requires a relay
mechanism. The current results, based on marked mirr overexpression
clones, demonstrate that the effect is cell-autonomous. Other studies argue against Mirr-dependent pip repression, based on the fact that mirr mutant clones did not induce ectopic expression of pip. These results may be
because of the fact that the mirr allele that was used is not
a complete null and has residual activity sufficient for pip repression.
It is argued that the current data, demonstrating pip derepression
by deletion of a sequence that binds Mirr, provide a strong
support for Mirr-dependent repression of pip. Thus, these findings
close a long-standing gap in the chain of events that convert
EGFR signaling to pipe repression, a key step in transmitting the
DV polarity from the egg to the embryo (Fuchs, 2012).
EGFR-dependent patterning of the follicle cells and the
resulting effects for patterning of the embryo represent canonical
examples of inductive effects in development. Indeed, genetic
connection between EGFR signaling and pipe repression are
found in essentially all textbooks of development. However, as
discussed above, the identity of transcription factors involved in
pipe regulation remained controversial and the cis-regulatory
sequences responsible for pipe repression were unknown. The current
results, which established Mirr as a direct repressor or pipe and
identified the regulatory element responding to Mirr, clearly
change this status. Thus, the results provide a significant addition
to a very important model of inductive signaling. The
regulatory element of pipe was discovered using an approach that harnesses both conventional and modern techniques of gene regulation research
and can be extended to other transcriptional targets of EGFR
pathway in the follicle cells. Finally, it is noted that most of the
available information on the transcriptional effects of EGFR
signaling is related to gene activation (mediated by Pnt) or
derepression (mediated by Cic). The current work reveals a mechanism
for EGFR-dependent gene repression, mediated by Mirr. Given
the central role played by the EGFR signaling in development,
the identified regulatory sequences can shed light on other
EGFR-dependent pattern formation events (Fuchs, 2012).
The Drosophila embryonic CNS develops from the ventrolateral
region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To
gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, loss of function was combined with ectopic activation of Egfr activity. Ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. This work has identified the genetic criteria required for the development of the medial and intermediate column cell fates. ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. This study also demonstrates that Egfr and the genes of the achaete-scute complex
act in parallel to regulate the individual fate of neural stem cells (Zhao, 2007).
The Dorsal gradient initiates patterning of the CNS via the
transcriptional regulation of the expression vnd, rhomboid and
zen. Dorsal-mediated activation of rhomboid, the rate-limiting factor in Egfr-signaling and vnd establishes the initial expression domains of two of the
earliest positive activators of CNS patterning along the DV axis.
Similarly, Dorsal-mediated repression in the ventral and ventrolateral
ectoderm limits the expression of zen and decapentaplegic
(dpp) to the dorsal ectoderm. Dpp functions as a morphogen and defines via a repressive mechanism the lateral limit of the developing CNS (Zhao, 2007).
Within the CNS, vnd and rhomboid exhibit differential sensitivity
to the dorsal gradient with vnd being activated solely
within the medial column and rhomboid in both the intermediate
and medial columns. Since rhomboid is the limiting factor in Egfr signaling, its
presence activates Egfr-signaling activity in the medial and
intermediate columns. In wild-type embryos, Egfr activity maintains vnd expression in the medial column and is necessary to promote ind
expression in the intermediate column. The ability of vnd to repress ind expression explains the restriction of ind expression to the intermediate
column. vnd expression persists throughout most of the
medial column until the end of embryogenesis; in contrast, ind
expression is extinguished in the intermediate column neuroectoderm
by stage 10 after the first two (of five) waves of NB segregation (Zhao, 2007).
This work adds a new regulatory relationship into the genetic
regulation of CNS patterning, since it was found that ind helps establish
the lateral limit of vnd expression. ind could perform this
function via the direct repression of vnd, a possibility supported
by gain-of-function and loss-of-function experiments. If this
model is correct, the mutual repression of vnd and ind would
bear striking similarity to the reciprocal repressive interactions
observed for the class I and class II homeodomain proteins that
pattern the DV axis of the vertebrate CNS. In this context, it is important to note that the vertebrate ortholog of vnd, Nkx2.2., is a class II protein that plays a key role in patterning some of the ventral-most regions of the vertebrate
CNS. Alternatively or additionally, vnd and ind could establish
their mutual sharp boundary indirectly via the regulation of
other factors. For example, differential regulation of homophilic
cell-adhesion molecules could account for the observed phenotype.
Differential expression of cell-adhesion molecules on
medial versus intermediate column cells would cause these
cells to associate preferentially with cells from the same column
and result in a sharp boundary between the two cell populations
that minimized interaction. Loss of such differences would
reduce the requirement to minimize interactions and likely
result in a jagged boundary. Additional work is necessary to
identify the precise mechanism through which ind helps establish
the lateral limit of vnd expression. Previous work has shown
that misexpression of ind along the anterior-posterior axis
using the Kruppel enhancer failed to repress vnd expression in
the medial column. However, this is not contradictory to the current findings of this study. This work suggests that ind can repress vnd in the intermediate and lateral columns but not in the medial columns. It is likely that some factors that are present in the intermediate and lateral columns but are absent
in the medial column help ind to repress vnd (Zhao, 2007).
In addition, this work demonstrates that
Egfr and vnd are sufficient to confer medial fate and that Egfr
and ind are sufficient to confer intermediate fate. Although loss-of-
function studies have shown that both Egfr and vnd are
necessary for NBs to acquire medial fate, it is not clear whether
Egfr functions solely through vnd. It has been shown that ectopic vnd expression results in partial transformation of lateral column into medial column. The current work shows that ectopic Egfr activity can induce the expression
of vnd and together Egfr and vnd fully transform the lateral
column into the medial column. Therefore, Egfr likely plays
additional roles in determining medial cell fate other than
maintaining vnd expression in the neuroectoderm. However, it
remains unclear whether Egfr contributes to the intermediate
column NB fate determination other than through its regulation
of ind and whether ind by itself is sufficient to confer intermediate
fate. Further studies are necessary to dissect the regulatory
mechanisms that control intermediate column NB fate specification.
In addition, while this work did not address the roles of
Dichaete and Sox-Neuro, it has been reported that ubiquitous
EGFR signaling activates Dichaete expression throughout the
neuroectoderm. Because Dichaete and SoxNeuro cooperates with vnd in the mediate column and ind in the intermediate column in NB fate specification, they are likely to act as co-factors with Vnd and Ind in embryos expressing Egfr over a prolonged period to specify NB fate in the lateral column (Zhao, 2007).
These experiments also underline the importance of temporal
regulation of gene expression during CNS patterning. This is
most notable with respect to the dynamic regulation of ind and
vnd expression by Egfr signaling. Previous work suggested that
the spatial dynamics of Egfr activity in the CNS account for the
transient nature of ind expression in the intermediate column.
Prior to NB formation Egfr activity is present in the intermediate
column and activates ind expression in this domain. Once NBs
begin to form Egfr activity disappears from the intermediate
column and ind expression is also lost from intermediate column
neuroectodermal cells. These data supported a simple
regulatory relationship in which the presence of Egfr activity is
necessary for ind expression in the intermediate column. However,
while Egfr is necessary to activate ind in the intermediate
column and sufficient to activate ind in the entire CNS, this study finds that ind expression turns over at its normal time even
in the presence of ubiquitous and prolonged Egfr activity in the
CNS. Thus, even though Egfr activity is necessary and
sufficient for the activation of ind, once activated ind expression
in the CNS appears to become independent of Egfr activity and other factors must regulate its temporally precise downregulation in the CNS (Zhao, 2007).
Similarly, vnd also exhibits differential sensitivity to Egfr
activity as a function of time. In contrast to ind, Egfr activity is
not necessary to activate vnd expression in the medial column,
however, Egfr activity is required later to maintain vnd expression
in this domain. Thus, vnd and ind exhibit opposite responses
to the Egfr signaling -- ind is activated but not maintained
by Egfr activity while vnd is maintained but not activated
by this pathway. It is interesting to note that vnd becomes
competent to respond to Egfr signaling about the time ind loses
its ability to respond to this signal. While the differential competency
of the vnd and ind promoters to Egfr signaling is essential
for proper DV patterning of the CNS, the molecular bases of
these differences remain unknown. Some of the specificity
likely resides within the promoters or regulatory regions of the
genes themselves. However, since both promoters are Egfr-responsive
albeit at different times additional levels of regulation
appear necessary to explain the complexity in regulation.
Alteration to higher order chromatin structure is known to play
a key role in controlling the competency of different promoters
to respond to specific signals and is a clear candidate to help mediate the differential responses of ind and vnd to Egfr-activity. However, how chromatin structure affects the ability of ind and/or vnd to respond to Egfr-activity
remains unexplored. Future work that addresses the influence
of modulation of chromatin structure on the ability of these and
other genes to respond differentially to the same inputs should
shed light on basic principles of gene regulation during development (Zhao, 2007).
Genetic studies indicate that the activities of Egfr and the
ac/sc genes converge to specify the fate of MP2 and possibly
other NBs. Additional work on genes that regulate NB fate
suggests that distinct convergent signals may play a general role
in NB specification. For example, the transcription factor Huckebein
is expressed in NB 4-2 and its associated proneural cluster and
helps promote the fate of some of the neurons that develop in the
4-2 lineage. However, in the absence of huckebein function, the 4-2 lineage retains many of its wild-type characteristics. Thus additional intrinsic
and extrinsic cues likely converge with huckebein to control the
fate of NB4-2 and enable it to elaborate its proper cell lineage.
Similar, albeit less detailed observations, have been made for
runt and msh. These genes are expressed in specific
NBs and the cell clusters from which they delaminate. Each gene
appears to regulate only a subset of the distinguishing characteristics
of the neuronal lineages that arise from their respective NBs
yet none of them appears deterministic for a specific NB fate.
Thus, it is speculated that convergent regulation of NB fate by
multiple intrinsic and extrinsic factors is a general theme in CNS development and that classical double and triple mutant analyses will be essential to reveal convergent pathways involved in NB as well as neuronal specification (Zhao, 2007).
The epidermal growth factor receptor (EGFR) and Hippo signaling pathways control cell proliferation and apoptosis to promote tissue growth during development. Misregulation of these pathways is implicated in cancer. Understanding of the mechanisms that integrate the activity of these pathways remains fragmentary. This study identifies bantam microRNA as a common target of these pathways and suggests a mechanistic link between them. The EGFR pathway acts through bantam to control tissue growth. bantam expression is regulated by the EGFR pathway, acting via repression of the transcriptional repressor Capicua. Thus EGFR signaling induces bantam expression by alleviating the effects of a repressor. bantam in turn acts in a negative feedback loop to limit Capicua expression.
bantam appears to be a transcriptional target of both the EGFR and Hippo growth control pathways. Feedback regulation by bantam on Capicua provides a means to link signal propagation by the EGFR pathway to activity of the Hippo pathway and may play an important role in integration of these two pathways in growth control (Herranz, 2012).
The ability of the EGFR pathway to drive tissue growth resides in its ability to coordinately stimulate cell proliferation and suppress apoptosis. Understanding how coordinated control is achieved depends on identification of the effector mechanisms that mediate these outputs along with the connections to other growth regulatory pathways. The results show that the bantam miRNA is a critical target of the EGFR pathway. Further, a mechanism is outlined by which bantam serves as a link between the EGFR and Hippo pathways (Herranz, 2012).
In Drosophila, EGFR pathway effectors include the transcription factors Pointed, Tramtrack, and Yan, and the HMG-box repressor Capicua. Capicua has an important role in early embryonic patterning and as a negative growth regulator. Although several Capicua targets involved in embryonic patterning have been identified, how Capicua regulates tissue growth was unknown. These results identify bantam as an important target of Capicua required to mediate EGFR-dependent tissue growth (Herranz, 2012).
A key finding of this study is the regulatory feedback relationship between bantam and Capicua. Each represses the activity of the other. Viewed from the perspective of the EGFR/MAPK pathway alone, the outcome of this relationship would be signal amplification, with downregulation of Capicua levels by bantam reinforcing direct MAPK-induced turnover of Capicua protein. This adds a new mechanism to the repertoire of positive and negative feedback loops affecting EGFR pathway activity. These feedback mechanisms are thought to be important in disease, and their regulation is complex. Relatively little is known about Capicua in cancer, although one recent study reports mutants in the human Cic protein in oligodendroglioma (Bettegowda, 2012; Herranz, 2012 and references therein).
An alternative logic for the relationship between bantam and Capicua may be seen in the fact that it links the output of the EGFR pathway to the output of the Hippo pathway, mediated through transcriptional regulation of bantam by Yorkie. EGFR signaling via MAPK and bantam cooperate to downregulate Capicua protein levels. Thus the transcriptional output of the Hippo pathway via Yorkie can be seen as potentiating EGFR signaling by 'lowering' the effective threshold of MAPK activity needed to reduce Capicua to a given level. Alternatively, the lack of sufficient Yorkie activity would lower bantam activity and thereby raise the threshold of EGFR activity required to reach an effective level of Capicua downregulation. This provides a mechanism to ensure coordination of the growth regulatory pathways. Signaling via the Hippo pathway has also been shown to induce the EGFR ligand amphiregulin to promote tissue growth in a nonautonomous manner. Thus, there appear to be multiple levels of crosstalk between these pathways (Herranz, 2012).
Considerable evidence is emerging linking miRNAs to robustness of regulatory feedback networks. It is intriguing that miRNAs are now implicated in regulation of all three of the known transcriptional effectors of EGFR signaling. miR-7 acts in two feed-forward loops downstream of EGFR to control photoreceptor specification and differentiation in the Drosophila eye. EGFR acts via the transcription factors Yan and Pointed. Yan is a direct target of miR-7. Yan also represses miR-7 transcription directly as well as indirectly. In the same cells, the ETS-1 factor Pointed-P1 activates miR-7 to repress Yan as well as acting directly to repress Yan. Use of interlinked motifs is thought to provide stability to the cell differentiation program controlled by EGFR. The current findings link bantam to regulation of a third EGFR transcriptional effector, Capicua, in addition to its regulation by the Hippo pathway. Coordination of diverse growth control inputs by miRNAs might contribute to robustness (Herranz, 2012).
back to Targets of Activity part 1/2
EGF receptor
:
Biological Overview
| Evolutionary Homologs
| Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
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