vein
Decapentaplegic has a prime function during endoderm induction in Drosophila. Dpp is secreted from the outer cell layer of the embryonic midgut (the visceral mesoderm), where Dpp's main source of expression in parasegment ps7 depends directly on the homeotic gene Ultrabithorax (Ubx). In the same cell layer, Dpp stimulates expression of another extracellular signal, Wingless (Wg), in a neighboring parasegment (ps8), which in turn feeds back to ps7 to stimulate Ubx expression. Thus, Dpp is part of a "parautocrine" feedback loop of Ubx (i.e., an autocrine feedback loop based partly on paracrine action) that sustains its own expression through Dpp and Wg. Dpp also spreads to the inner layer of the embryonic midgut, the endoderm, where it synergizes with Wg to induce expression of the homeotic gene labial (lab). To achieve this, Dpp locally elevates the endodermal expression levels of Drosophila D-Fos with which it cooperates to induce lab. Differentiation of various cell types in the larval gut depends on these inductive effects of Dpp and Wg. A cAMP response element (CRE) from the Ubx midgut enhancer has been shown to be necessary and to some extent sufficient to mediate the Dpp response in the embryonic midgut (Eresh, 1997). CREs are known to be signal-responsive elements, not only for cAMP signaling as described initially but also for other signals including ones acting through Ras. This prompted an investigation of whether any other signal may play a part in the Dpp response. This led to the discovery that the Drosophila epidermal growth factor receptor (Egfr) has a critical function during endoderm induction. Acting as a secondary signal with a permissive role in this same process is Vein, a neuregulin-like ligand that stimulates the epidermal growth factor receptor and Ras signaling. Dpp and Wg up-regulate vein expression in the midgut mesoderm in two regions overlapping the Dpp sources. This up-regulation depends on dpp and wg. Vein is thus a secondary signal of Dpp and Wg, and it stimulates homeotic gene expression in both cell layers of the midgut (Szuts, 1998).
EGFR expression is thought to be fairly ubiquitous in the embryo. However, vein transcripts are found in a highly restricted pattern, primarily in the embryonic mesoderm. In the midgut too, vein expression is spatially regulated, as follows: vein transcripts in the midgut are restricted to the visceral mesoderm. Initially, during stage 13, low levels of vein expression are seen at intervals throughout the midgut mesoderm. However, soon after the formation of the midgut epithelium, vein transcripts start to accumulate locally, and two main domains of prominent vein expression develop, one in the anterior and one in the middle midgut. Anteriorly, vein expression spans approximately ps2-ps4 and is strongest around the ps3/ps4 junction, that is, posterior to the gastric caeca. In the middle midgut, there is a fairly wide band of low vein expression spanning approximately ps6-ps10, with strongly up-regulated expression levels throughout ps7 (and trailing into anterior ps8). Posterior ps7 becomes the most prominent site of vein expression in the midgut. Finally, a narrow band with low levels of vein transcripts is seen at the posterior end of the midgut. The two main expression domains of vein overlap the two domains of Dpp expression in the visceral mesoderm (in ps3 and ps7), but each of them is considerably wider than the corresponding dpp domain. vein expression in the visceral mesoderm is severely diminished in dpps4 mutants. The prominent band of vein expression in ps7 is no longer seen, and expression in ps4 is reduced too. Instead, the strongest expression of vein in these mutants is seen at a novel location, at the ps5/ps6 junction around the incipient first midgut constriction (this ps5/ps6 expression is higher than in the wild type, and can be used to identify young dpp mutant embryos). It is concluded that dpp is required for the localized up-regulation of vein expression in the midgut (Szuts, 1998).
vein expression is also strongly diminished in wg mutants. vein expression can still be seen at moderate levels in the ps4 region, but vein expression is barely visible elsewhere in the midgut of these mutants. In particular, there are only traces of vein expression in the ps7/ps8 region, and expression at both midgut ends is almost undetectable. Clearly, wg plays an essential role as well in up-regulating vein expression. dpp and wg are sufficient to position the two domains of vein up-regulation. High mesodermal Wg causes very strong vein expression in ps2-ps7, significantly stronger than that caused in this region by mesodermal Dpp expression alone. This indicates that wg cooperates with dpp in positioning vein up-regulation. It is shown that neither Dpp for Egfr signaling is particularly effective in the absence of the other. Thus these two pathways are functionally interdependent and that they synergize with each other, revealing functional intertwining (Szuts, 1998).
The mutant analysis suggests strongly that Vein is the main, if not the only, ligand that stimulates Egfr in the embryonic midgut. This contrasts with other tissues, mainly of ectodermal origin, in which Spitz is the main Egfr ligand. Interestingly, Vein also has a major role during an inductive process between muscle and epidermis: Vein is secreted from muscle cells and triggers differentiation of the receiving epidermal cells into tendon cells. These functions of Vein during inductive processes between different cell layers suggest that the molecular properties of Vein are particularly suited to such processes that require the signal to cross basal membranes. Similarly, the extensive mesodermal expression of Vein may mean that this signal protein is particularly well-adapted to its production in this cell layer. Note that Vein is similar to mammalian neuregulins that appear to function in developmental contexts that involve communication between different cell layers (Szutz, 1998 and references). The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras, the secondary signal of Dpp. It is also shown that the Dpp response element in the labial enhancer comprises CREs and Mad binding sites. The results with the labial enhancer confirm the conclusions derived from the Ubx enhancer, namely that the response element to Dpp signaling is bipartite and contains Mad binding sites as well as CREs. The latter are critical in both cell layers for the signal response, whereas the former seem less criticial in the endoderm than in the visceral mesoderm. Perhaps this reflects the fact that lab is the ultimate target gene of the endoderm induction and that its enhancer clearly integrates a number of distinct positional inputs, some of which may be partially redundant (Szuts, 1998).
Why should there be this secondary signal whose role is entirely permissive, namely to assist the primary signal in implementing its tasks? Two kinds of answers are proposed. The first one is based on the observation that lack of Vein/Egfr signaling in the midgut appears to make cells sick and perhaps causes them to die. Therefore, Vein/Egfr signaling may serve as a "survival signal." Intriguingly, cell survival in embryos lacking vein or Egfr function appear to be affected preferentially near the two Dpp sources (where vein expression is up-regulated). Perhaps high levels of Dpp signaling can cause cell death; if so, vein signaling may be up-regulated to counteract a putative local deleterious effect of Dpp. A precedent for such a scenario may be found in the developing chick limb bud where the cell death-inducing properties of BMP (a TGF-beta-like signal) seem to be antagonized locally by a signal triggering the Ras pathway. However, although antagonistic effects between Egfr- and TGF-beta-type signaling have been observed, the evidence provided here suggests strongly that Vein/Egfr and Dpp both act positively in the embryonic midgut of Drosophila. Furthermore, they synergize with each other in the transcriptional stimulation of target genes. This observed synergy parallels cooperation between Ras and TGF-beta signaling during epithelial tumor progression. It is therefore thought unlikely that Vein functions in the midgut entirely as a survival signal near Dpp sources (Szuts, 1998).
The second kind of answer builds on the observations that indicate functional interdependence and synergy between the two signaling pathways in stimulating transcription of target genes. This could be beneficial for developmental systems in two ways: (1) if cells need to be costimulated by cooperating primary and secondary signals, this would serve to sharpen their signal response. This putative sharpening effect may be a contributory factor in sharp responses to signaling thresholds such as those observed in the Xenopus embryo.(2) The need for costimulation would safeguard against fortuitous and random stimulation of cells by any one signal, thus improving the reliability of their signal response. And although a requirement for the secondary signal is observed throughout the functional realm of the primary signal, it is envisaged that the role of the secondary signal is particularly critical in remote cells where the distribution of the primary signal becomes shallow, imprecise, and unreliable. Therefore, the secondary signal may provide primarily "remote stimulation." Whatever the case, it seems very likely that the use of a functionally coupled primary-secondary signal system results in a refinement and stabilization of positional information and in a degree of precision of this information that could not be conferred by one signal alone. Functional intertwining of a secondary and a primary signal may represent a mechanistic solution of how morphogens such as Dpp and activins work. Perhaps, signaling pathways do not function on their own in eliciting multiple different cellular responses, as envisaged by the purest version of the morphogen concept (Szuts, 1998).
Although there is a single EGF receptor in Drosophila, multiple ligands activate it. This
work explores the role of two ligands, Spitz and Vein, in the embryonic ventral ectoderm. Spitz is a
potent ligand, whereas Vein is an intrinsically weak activating ligand. Prior to gastrulation, vein is expressed in the
future neuroectoderm. This early phase of expression appears to cooperate with Spitz in the induction
of medial neuroblast cell fates. Following gastrulation at stage 9, vein expression in the
neuroectoderm becomes confined to three to four cell rows on either side of the midline. This pattern
gradually restricts, such that by stage 11 only one cell row on each side of the midline expresses vein. Secreted Spitz,
emanating from the midline, triggers expression of vein in the ventral-most rows of cells, by inducing
expression of the ETS domain transcription factor Pointed P1. In the absence of Vein, lateral cell fates
are not induced when Spitz levels are compromised. The positive feedback loop of Vein generates a
robust mechanism for patterning the ventral ectoderm (Golembo, 1999).
Expression of vein is observed in positions adjacent to the ventral midline, where activation of the
Egfr pathway by secreted Spitz emanating from the midline is maximal. This raised the possibility that
vein expression may be triggered by Spitz. In rhomboid mutant embryos in which active Spitz molecules are
not produced, no expression of vein is observed at stage 11, demonstrating that Rho/Spitz are indeed
necessary for vein expression in the ventral ectoderm. To test if Egfr activation is sufficient to induce vein expression in the embryo, the pathway was
ectopically activated at two different stages. Rho was ectopically expressed at stage 9 in the central
domain of the embryo by Kruppel-Gal4, and this gives rise to ectopic vein expression in the same region. At stage 11 secreted Spitz was expressed in the engrailed domains, resulting in the induction of
vein expression in the same pattern. These experiments demonstrate that high levels of Egfr
activation are sufficient for inducing vein expression (Golembo, 1999).
Induction of gene expression by Egfr in the ventral-most rows of cells occurs
for the argos, orthodenticle, and tartan genes. Induction is obtained through inactivation
of Yan, an ETS domain transcriptional repressor, and induction of Pointed P1, an ETS domain
transcriptional activator. To test if induction of vein by Egfr is also mediated by Pointed P1, vein expression was examined in pointed mutant embryos. Traces of expression are observed in the
midline in stage 10 embryos only, whereas no expression is displayed by the ventral-most and lateral
ectodermal cell rows at stage 11, thus showing that vein induction is defective in pointed mutants.
Elimination of Pointed activity can also be obtained in the following manner: an activated form of Yan,
in which the inactivating MAP kinase phosphorylation sites have been mutated, blocks the activity of Pointed by competing for the same DNA-binding sites. Indeed, when activated, Yan is ectopically expressed in the Kruppel domain the endogenous
expression of vein is abolished in that region. To examine if Pointed P1 is sufficient for
induction, vein expression was examined in embryos in which Pointed P1 was expressed in the Kruppel
domain. Indeed, expression of vein in the same domain is observed. These results
demonstrate that under conditions of ectopic expression, Pointed P1 is necessary and sufficient for
vein expression (Golembo, 1999).
Two different nested cell fates are induced by the Egfr pathway in the ventral ectoderm, depending
on the distance of the cells from the midline. The cell rows closest to the midline assume the
ventral-most fate, as reflected by the expression of target genes such as otd and argos. Intermediate
levels of Egfr activation induce lateral cell fates, reflected by the expression of FasIII, which is observed
in five rows of cells on each side of the midline. Both ventral-most and lateral markers are eliminated in
mutants for Egfr, as well as in mutants that abolish Spitz or its processing (spitz, rho, or Star).
Conversely, ectopic secreted Spitz or Rho is capable of expanding expression of both lateral and
ventral-most fates. To examine the role of Vein in the ventral ectoderm, vein null mutants were examined for the expression of marker genes. No defects in the expression of otd or FasIII were observed. These results indicate that at this level of resolution, the function of Vein is redundant. This is also consistent with normal levels of activated MAP kinase that are observed at stage 9 in vein mutant embryos. A role for Vein is revealed under conditions in which the level of Spitz is
compromised. Flies that are heterozygous for a null allele of Spitz are viable. Normal patterning of the
ventral ectoderm takes place in heterozygous spitz embryos, as monitored by the expression pattern of FasIII. Embryos were generated that are homozygous for the vein null allele and carry
only one functional copy of spitz. In these embryos, patterning of the ventral-most cells is normal, as
reflected by the expression of otd or FasIII. However, more lateral cells fail to
express FasIII. These results indicate that when Spitz levels are compromised in
heterozygous embryos, the cell row closest to the midline undergoes normal patterning. However, the
levels of Spitz may be too low to pattern the more lateral cells. Under these conditions, induction of
Vein appears to be critical to facilitate Egfr activation in these cells. Upon ectopic expression of Vein, the ventral-most markers are not induced at all or only intermittently,
thus reflecting the reduced inherent activity of Vein. To test directly the
biological activity of Vein in a controlled setting, it is necessary to eliminate endogenous signaling by
Spitz, as well as the presence of Argos. This can be achieved in embryos that are mutated for rhomboid, and
thus exhibit no expression of the ventral-most markers (such as argos) or lateral markers. Ectopic expression of Vein in rho mutant embryos is capable of restoring FasIII
expression, but did not induce otd expression. Thus, Vein is capable of
inducing the lateral cell fates in the absence of secreted Spitz (Golembo, 1999).
Two distinct mechanisms have been proposed for patterning by morphogens. In one case, a gradient of a single morphogen gives rise to distinct cell fates caused by the
induction of different levels of signaling in the same pathway. The complementary scenario involves a
relay mechanism: an initial induction by the primary morphogen induces the production of a relay factor
triggering another signaling pathway, to pattern the more distant cells. This work describes a
combination of the two models. Only the EGF receptor cascade patterns the ventral ectoderm.
However, the primary signal, Spitz, induces a relay mechanism by triggering expression of Vein,
another ligand of Egfr. Again, it is important to emphasize that although the restricted spatial
distribution of secreted Spitz is critical for correct patterning, Vein and Argos distribution may be more
uniform. Argos reduces the overall level of EGF receptor signaling, whereas Vein provides a lower
level of activation, capable of inducing only the lateral cell fates. vein expression is also induced by the EGF-receptor pathway in follicle cells within the dorsal-anterior corner of the egg chamber. It thus appears that Vein may provide a positive feedback loop in
several tissues that are patterned by EGF receptor activity (Golembo, 1999 and references).
Another facet of the activity of Vein that should be considered is its sensitivity to Argos inhibition. If
the lateral cells have not encountered sufficient levels of Spitz prior to the induction of Argos, their
capacity for activation in the presence of Argos is severely compromised, and relies on the continued
availability of secreted Spitz from the midline. The induction of Vein in the ventral-most cells, which
takes place in parallel to the induction of Argos, may help to overcome this problem. Vein is capable of
activating Egfr in the presence of Argos: in embryos heterozygous for spitz, the activity of Vein
induces the lateral fates. This takes place in a situation in which argos is expressed in the ventral-most cells. Vein itself is not capable of inducing expression of argos.
In conclusion, this work has revealed a powerful regulatory network, orchestrated by the sequential
utilization of Spitz and Vein, two ligands of the EGF receptor with different properties. Induction of
Vein takes place once the ventral-most cell fates already have been determined by high Spitz levels.
Vein expression prolongs the time window of activation of the Egfr pathway to ensure that the lateral
cell fates will be specified correctly. Vein is suited to this task, since it is a less potent ligand than Spitz,
capable of inducing only the lateral cell fates. Vein can activate Egfr even in the presence of Argos,
thus balancing the parallel negative-feedback loop of Argos (Golembo, 1999).
The Hedgehog (Hh) and Epidermal growth factor receptor (Egfr) signaling pathways play critical roles in pattern formation and cell proliferation in invertebrates and vertebrates. In this study, a direct link between these two pathways is demonstrated in Drosophila. Hh and Egfr signaling are each required for the formation of a specific region of the head of the adult fruitfly. hh and vein (vn), which encodes a ligand of the Drosophila Egfr, are expressed in adjacent domains within the imaginal primordium of this region. Using loss- and gain-of-function approaches, it has been demonstrated that Hh activates vn expression. Hh activation of vn is mediated through the gene cubitus interruptus (ci) and this activation requires the C-terminal region of the Ci protein. wingless (wg) represses vn expression, thereby limiting the domain of Egfr signaling (Amin, 1999).
The dorsal head capsule, which lies between the compound
eyes, contains three morphologically distinct domains. The medial domain includes the ocelli and their
associated bristles, which lie on the triangular ocellar cuticle.
The mediolateral region contains the frons cuticle, which
consists of a series of closely spaced parallel ridges. The lateral
region is occupied by the orbital cuticle, which contains a
stereotypical pattern of bristles.
The head capsule forms primarily from the two eye-antennal
imaginal discs. Each half of the dorsal head derives from a
primordium in the disc immediately adjacent to the anlage of
the compound eye. During the
pupal stage, the two discs fuse at what will form the midline
of the dorsal head capsule (Amin, 1999).
hh is expressed within the medial domain of the dorsal head
capsule. Specifically, it is
expressed in the interocellar cuticle, which contains the small
interocellar bristles. Using a vn-lacZ strain, vn is found to be expressed in
the dorsal head capsule. vn expression lies primarily within
the mediolateral frons cuticle, near but not immediately
adjacent to the region of hh transcription.
The regions of hh and vn expression in the
dorsal head primordium of the eye-antennal disc are compared. Consistent
with its expression on the adult head capsule, hh is expressed
in the region of this primordium that lies between the precursor
cells of the ocelli. vn is expressed in the wing and haltere discs, but its expression in the eye-antennal disc has not been described.
Using both the vn-lacZ strain and in situ hybridization with
a vn probe, vn is also found to be expressed in the dorsal
head primordium. As on the adult head, vn
expression lies near that of hh. Double-labeling shows that
the domains of hh and vn expression are immediately adjacent
to each other. In situ hybridization reveals that vn
is also expressed at low levels in the morphogenetic furrow.
Eliminating Hh function during head development results in
the deletion of the entire medial domain, including the
interocellar cuticle and bristles, and the ocelli and their
associated bristles. This region is replaced by frons cuticle, which
is normally confined to the mediolateral region of the head
capsule. Ectopic hh expression generates ectopic medial
structures at more lateral positions. Hh is therefore necessary for the specification of the
medial domain and sufficient to direct more lateral regions of
the dorsal head towards a medial fate (Amin, 1999).
Particular combinations of
Egfr alleles cause a reduction in the size of the ocelli and the
loss of the two ocellar bristles, which flank the medial ocellus. Since vn is expressed within
the dorsal head primordium, the effects of
eliminating either vn expression or Egfr-mediated signaling
on head development were determined.
Examination of vn mutant clones shows that Vn is required for the development of
some, but not all, of the Hh-dependent medial head structures. The ocelli and ocellar bristles are deleted and the
postvertical bristles, which lie near the lateral ocelli, are also
lost. However, most of the interocellar cuticle is retained,
indicating that the vn dorsal head phenotype is less global than
that caused by loss of Hh function. Since vn encodes a ligand for Egfr, the
effects of eliminating Egfr-mediated signaling on head
development were examined. To do so, the GAL4/UAS system was used to express a dominant negative form of the Egfr (DN-DER) across the entire dorsal
head primordium. DN-DER expression eliminates
the same structures deleted in vn clones, suggesting that vn is
primarily responsible for activating Egfr signaling in this
region. As was the case for Vn, the interocellar cuticle is
retained in the absence of Egfr signaling (Amin, 1999).
Since the hh mutant phenotype is more extensive than either
the vn or Egfr phenotypes, a test was made to determine whether Hh acts
upstream of the Egfr pathway. Using a temperature-sensitive
hh allele (hhts2), Hh function was eliminated
during the third instar larval stage. Loss of Hh eliminates or
strongly reduces vn expression in both the dorsal head
primordium and the morphogenetic furrow.
To determine whether Hh can induce vn expression outside
the dorsal head primordium, ectopic hh expression was induced
using the Flp recombinase technique. Hh is found to be capable of activating vn in other regions
of the eye disc. A disc-specific enhancer
from the dpp gene was used to induce ectopic hh expression using the
GAL4/UAS system. This
enhancer drives reporter gene expression at the posterior and
lateral margins of the third instar eye disc as well as in a portion
of the antennal anlage. Ectopic hh expression induced
by this enhancer severely disrupts eye-antennal disc
morphology. It also induces a band of ectopic vn
expression anterior to the region of hh transcription. Combined
with the previous results, these experiments demonstrate that
Hh is necessary for vn expression in the dorsal head
primordium, and is sufficient to induce ectopic vn expression in
other regions of the disc (Amin, 1999).
To test whether Hh is also required to activate Egfr-mediated
signaling, a monoclonal antibody that
specifically recognizes the active, dually phosphorylated form
of mitogen-activated protein kinase (dp-ERK) was used. dp-ERK is expressed
at high levels in the morphogenetic furrow, and at lower levels
in ommatidia posterior to the furrow. When the anti-dp-ERK signal is allowed to
develop for longer periods, weaker expression appears in cells
within the dorsal head primordium. Eliminating Hh
function reduces or eliminates dp-ERK expression both in
these cells and in the furrow. Hh mediated induction of the Egfr pathway has been shown to be medated by Cubitus interruptus. Expression of an N-terminal fragment of Ci with repressor
activity reduces or eliminates vn
expression in the dorsal head analage. On the contrary, expression of the Ci155 activator
increases the intensity and extent of vn expression
and causes the ocelli to increase in size and fuse (Amin, 1999).
wingless is broadly expressed throughout the early eye-antennal disc,
where it confers a default state of head cuticle. Later, wg expression becomes restricted to the
primordia of the orbital cuticle and ptilinium, and to a portion
of the antennal anlage. Just as hh expression is medially adjacent to that of vn
on the adult head capsule, wg expression abuts vn in the frons
both laterally and anteriorly. Loss
of Wg signaling causes the deletion of both the frons and
orbital cuticles. To determine whether Wg participates in vn regulation, a temperature-sensitive allele was used to eliminate Wg function
during second instar development. In contrast to Hh, Wg negatively regulates vn. Loss of Wg activity
during this time window expands the domain of vn expression
in the dorsal head primordium and induces ectopic vn
expression in other regions of the eye-antennal disc (Amin, 1999).
Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely
unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings
are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation,
the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).
Col regulates vn transcription in the AP organizer. This expression of vn is required for formation of L4 vein. The loss of this vein in col1 mutants could not, however, be rescued by expressing high level of Vn in the AP organizer, suggesting that a second signal dependent upon Col is also required. The specific loss of L4 vein is observed in conditions of reduced levels of Dpp signaling caused by ubiquitous expression of either Tkv or TkvDN. Together with this, the col1 wing phenotype and Col requirement for downregulation of Dpp signaling in the AP organizer suggests a role for Dpp signaling in formation of L4 vein. Indeed, expressing a dominant-negative form of Tkv (TkvDN) in the AP organizer results in L4 loss. At first sight, it may appear contradictory that either upregulation (in col mutants) or downregulation (by expressing TkvDN) of Dpp signaling in the AP organizer leads to the preferential loss of posterior L4 vein. In both cases, however, there is increased sequestering of Dpp in the AP organizer, which limits its range of diffusion and signaling in posterior cells. Therefore attenuation of Dpp signaling in the AP organizer and increased signaling in posterior flanking cells appears to be required in addition to Vn activity for formation of L4 vein. By modulating Dpp signaling and vn transcription in cells receiving high doses of Hh, Col thus links Hh short-range activity to both positioning of the anterior L3 vein and formation of the posterior L4 vein (Crozatier, 2002).
In Drosophila, wings and halteres are the dorsal appendages of the second and third thoracic segments, respectively. In the third thoracic segment, homeotic selector gene Ultrabithorax (Ubx) suppresses wing development to mediate haltere development. Halteres lack stout sensory bristles of the wing margin and veins that reticulate the wing blade. Furthermore, wing and haltere epithelia differ in the size, shape, spacing and number of cuticular hairs. The differential development of wing and haltere, thus, constitutes a good genetic system to study cell fate determination. Down-regulation of Egfr/Ras pathway is critical for haltere fate specification: over-expression of positive components of this pathway causes significant haltere-to-wing transformations. RNA in situ, immunohistochemistry, and epistasis genetic experiments suggest that Ubx negatively regulates the expression of the ligand vein as well as the receptor Egf-r to down-regulate the signaling pathway. Electromobility shift assays further suggest that Egf-r is a potential direct target of Ubx. These results and other recent findings suggest that homeotic genes may regulate cell fate determination by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways (Pallavi, 2006).
To identify potential targets of Ubx and thereby mechanism of its function, a gain-of-function genetics strategy was employed. Ubx-GAL4 driver, is expressed in the entire anterior compartment of the haltere imaginal disc. Ubx-GAL4 is also a null allele of Ubx and exhibits characteristic dominant phenotype; the presence of wing-type sensory bristles in the capitellum of the haltere. This GAL4 driver provides a fortuitous sensitive background to carry out large-scale screens for identifying the suppressors and enhancers of Ubx function, which otherwise may be less efficient in a wild type background. Indeed, over-expression of Vestigial (Vg), a pro-wing gene and a target of Ubx function, in the developing haltere results in very high degree of haltere-to-wing homeotic transformations, A candidate gene screen was employed to identify downstream targets of Ubx, in which various genes known to be involved in wing development were ectopically expressed in the developing haltere using the Ubx-GAL4 driver. Criterion for defining haltere-to-wing transformation in this study was the presence of wing-type sensory bristles, although increase in haltere size and enhanced pigmentation was frequently observed. For comparison between different genotypes, the degree of transformation was estimated by counting the number of sensory bristles on the haltere capitellum. UAS stocks for a large number of such wing patterning genes were crossed to Ubx-GAL4 driver and were scored for enhancement and suppression of the dominant phenotype of heterozygous Ubx. Progeny for many of the crosses resulted in early embryonic or early larval lethality, reflecting the fact that the GAL4 driver expresses at early stages during embryonic development. Nevertheless, strong haltere-to-wing transformations were observed upon over-expression/mis-expression of most of the positive components of the Egfr/Ras pathway. For example, the bristle number in the capitellum of the Ubx-GAL4 haltere increased when positive components such as Vn or Egf-r were over-expressed and over-expression of negative components such as Aos completely suppressed the heterozygous Ubx phenotype (Pallavi, 2006).
A significant finding of this study is the down-regulation of Egfr/Ras pathway in haltere discs by Ubx. Earlier reports suggest that a short-range signal originating from the D/V boundary activates Egfr/Ras pathway in a zone of cells on the edges of the D/V boundary and that this activation is essential for vg transcription (Nagaraj, 1999). Egfr/Ras pathway has also been implicated in the developmental events along the A/P axis: in wing vein specification. Consistent with the down-regulation of both A/P and D/V signaling events in haltere discs, expression of most of the Egfr/Ras pathway components is repressed in the entire haltere pouch. Observations on the strengths of haltere-to-wing transformations (at the margin bristle level) in different genetic backgrounds establish the specificity of genetic interactions between Egfr/Ras pathway and Ubx during haltere development (Pallavi, 2006).
The abovementioned results on the down-regulation of Egfr/Ras pathway in haltere discs by Ubx are consistent with the previously reported genetic screen for the modifiers of homeotic genes, which indicates that Ras1 activity modulates functions of the homeotic loci Sex combs reduced (Scr) and Ubx. For example, haploinsufficient (haltere-to-wing) phenotype of Ubx109/+ is significantly enhanced in Gap1−Ubx109/Gap1− adults (Gap1 is a negative regulator of the Egfr/Ras pathway). This effect of Gap1 is reversed in Gap1−Ubx109/Gap1−Rase1b individuals due to the antagonistic roles of Gap1 and Ras1 in the Egfr/Ras pathway (Pallavi, 2006).
All the components of the Egfr/Ras pathway tested so far are differentially expressed between wing and haltere discs. This suggests the utility of developing wings and halteres as assay systems to identify novel components of Egfr/Ras pathway. Indeed, enhancer-trap screens and microarray analyses to identify genes that are differentially expressed between wing and haltere discs have resulted in the identification of CG32062 (Drosophila homologue of human ataxin-2 binding protein) and Mapmodulin (Drosophila homologue of human Inhibitor-1 of protein phosphatase-2A) as potential modulators of Egfr/Ras pathway (Pallavi, 2006).
The activation of dpERK1/ERK2 and aos in the haltere pouch by the ectopic expression of vn or Egf-r suggests that Ubx regulates Egfr/Ras pathway at ligand as well as receptor levels. Clonal analysis of Ubx function (loss of Ubx in haltere discs and gain in wing discs) demonstrates that Ubx controls vn expression in a cell-autonomous manner. Inability of ectopic Hh to activate vn expression in haltere discs suggests that Ubx functions downstream of Hh to repress vn expression. Putative Ubx-binding sites are present in the cis-regulatory regions of both vn and Egf-r, further suggesting their direct regulation by Ubx. Indeed, electromobility shift experiments suggest that, at least, Egf-r is probably a direct target of Ubx function. Thus, it is likely that Ubx independently down-regulates both vn and Egf-r. Ubx-mediated down-regulation of vn and Egf-r appears to be critical; over-expression of normal or the activated form of Egf-r induced stronger phenotypes when expressed in aos heterozygous background than in Ubx heterozygous background. However, Ubx may exert some influence on the pathway downstream of the receptor, since the strength of the phenotypes induced by the over-expression of Vn or Egf-r was stronger in Ubx heterozygous background. At dpERK1/ERK2 level too, the activation was stronger when Egf-r was over-expressed in Ubx heterozygous genetic background than in the wild type background (Pallavi, 2006).
It is interesting to note that Ubx regulates Egfr/Ras pathway at the level of the receptor itself. Although Egfr/Ras pathway is auto-regulated at several levels including the transcription of Egf-r itself, external factors regulating Egfr/Ras pathway during various developmental events mostly act at the level of the ligand/s or downstream effectors such as MAPK or transcription factors Yan, Pointed, etc. This study has found that the receptor itself is a direct target of Ubx indicating a novel mode of regulation of this pathway (Pallavi, 2006).
Specification of the larval oenocyte has been shown to be dependent on the regulation of just one principal target Rho by the homeotic gene abdominal A. Similarly, Hox proteins AbdA and AbdB specify the lineage of the embryonic NB6-4 neuroblast in abdominal segments by down-regulating CycE. Differential expression of CycE is both required and sufficient to generate segmental differences in NB6-4 lineage. This study reports that down-regulation of Vn and Egf-r is critical for Ubx-mediated suppression of wing margin bristles in the haltere. These results suggest that one common mechanism by which homeotic genes may regulate cell fate determination is by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways. In contrast, Wingless and Decapentaplegic signaling pathways, which regulate more complex traits such as wing growth and shape, are regulated by Ubx at multiple levels in the hierarchy of those pathways (Pallavi, 2006 and references therein).
Although absence of veins in the haltere could be attributed to down-regulation of Egfr/Ras pathway, activation of sensory bristle development in Ubx+/Ubx+ halteres over-expressing positive components of Egfr/Ras pathway suggests a role for this pathway in cell fate specification in the wing margin. So far, no direct role for Egfr/Ras pathway has been assigned in the specification of sensory bristles of the wing margin, although it is known to specify macrochaete of the notum. Indeed, preliminary investigations suggest that Wg pathway induces EGFR/Ras pathway expression in cells immediately adjacent to the D/V boundary, and the latter pathway is required and sufficient to specify sensory organs of the wing margin (Pallavi, 2006).
The bristle development in the transformed halteres appears to be organized in two parallel rows when various components of Egfr/Ras pathway are over-expressed in Ubx heterozygous background, while the bristles are positioned in a disorganized way when phenotypes are induced in wild type background. This could be due to partial de-repression of D/V signaling in Ubx heterozygous background, which may allow appropriate positioning of the zone of margin bristle development (Pallavi, 2006).
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