Although dve is strongly expressed in the embryonic middle midgut, morphological defects are
not evident in this region in dve1 mutants; gut constriction normally occurs, and the arrangement of the stage-17 midgut appears to be normal. Cells that express dve at embryonic
stages develop into four types of larval midgut cells: copper, interstitial, large flat, and iron cells. The
expression of dve in these larval cells was monitored as the expression of lacZ that
is located in the dve1 enhancer-trap insertion (dve1-lacZ). beta-Galactosidase activity attributable to dve1-lacZ is observed in interstitial, large flat, and iron cells, but not in copper cells in heterozygous larvae. In dve mutant larvae there is ectopic dve1-lacZ expression in copper cells, in addition to a highly disorganized arrangement of these cells. These observations suggest that
dve activity is required to repress its own expression in copper cells. To determine whether dve could rescue this mutant phenotype of ectopic dve1-lacZ expression, dve was induced ubiquitously in the dve1 mutant background under the control of Gal4-UAS system. Weak dve induction in stage-17 embryos suppresses the ectopic dve1-lacZ
expression in larval copper cells, indicating that a mutant phenotype is caused specifically by
the lack of DVE gene product (Nakagoshi, 1998).
Little is known about the genes and mechanisms that pattern the proximodistal (PD) axis of the Drosophila wing. Vestigial (Vg) is instrumental in patterning this axis, but the genes that mediate its effects and the mechanisms that operate during PD patterning are not known. The gene defective proventriculus (dve) is required for a region of the PD axis encompassing the distal region of the proximal wing (PW) and a small part of the adjacent wing pouch. Loss-of-function of dve results in the deletion of this region and, consequently, shortening of the PD axis. dve expression is activated by Vg in a non-autonomous manner, and is repressed at the DV boundary through the combined activity of Nubbin and Wg. Besides its role in the establishment of the distal part of the PW, dve is also required for the formation of the wing veins 2 and 5, and the proliferation of wing pouch cells, especially in regions anterior to wing vein 3 and posterior to wing vein 4. The study of the regulation of dve expression provides information about the strategies employed to subdivide and pattern the PD axis, and reveals the importance of vg during this process (Kölzer, 2003).
Dve expression pattern was monitored during wing development by use of an anti-Dve antibody. The expression pattern of Dve was compared with that of Wg, which is expressed throughout wing development in a pattern that reveals the organization of the developing wing. Wg is initially expressed in a ventral domain during the second larval instar stage and defines the wing area or wing field. At this time, Dve is not expressed in the wing imaginal disc. At the beginning of the third larval instar stage, Wg expression resolves into a stripe along the future DV compartment boundary and a proximal ring-like domain. In the middle of third larval instar stage a second ring-like domain in the proximal region of the anlage is added. The two ring-like domains of Wg expression highlight the anlagen of the proximal and medial regions of the proximal wing, as deduced from X-Gal staining of adults carrying a wg-lacZ construct. Dve expression is initiated at the time when Wg resolves into a ring-like domain in the periphery and a domain along the DV boundary, and it becomes expressed in all cells inside the region framed by the ring-like domain of Wg. Dve continues to be expressed in a disc-like domain that fills the inside of the inner ring-like expression domain of Wg until the late third larval instar stage (Kölzer, 2003).
The anlage of the distal region of the PW, is located outside the wing pouch and inside the inner ring-like domain of Wg expression. Dve is expressed continuously in this region, and is present at the right place and time to control the development of this structure, which is absent in the mutants.
At the DV boundary, Dve is initially expressed, but it becomes downregulated soon after its initiation, with the exception of a short stretch at the anterior side. During the late third larval instar stage, it is also downregulated in the primordia of wing veins 3 and 4 (Kölzer, 2003).
The expression domain of dve has been mapped in relation to that of
other genes known to be involved in PD patterning of the wing, and in relation
to the ring-like domains of wg. The ring-like domains label the
region of the proximal and medial costa, as revealed by the X-Gal staining of
adult wings bearing a wg-lacZ insertion (Kölzer, 2003).
vestigial (vg) is required for all distal fates from the
medial costa distalwards. It is initially expressed in all pouch cells and its expression is controlled through the
vg-Quadrant enhancer (vg-QE). The expression domain of dve is larger earlier than that of the vg-QE. In addition, dve expression is initiated before the
vg- QE is activated, which indicates that dve expression is
initiated before the wing pouch forms (Kölzer, 2003).
Nub is involved in patterning the wing from the medial costa distalwards. The nub gene is expressed in a disc-like domain that is slightly larger than that of dve and that extends to the area between the two ring-like domains of wg expression. Examination of wing discs of early third instar larvae reveals that nub expression is initiated earlier than dve, and is always expressed in a larger domain than dve (Kölzer, 2003).
The boundary of the expression domain of rotund (rn)
falls between that of dve and nub. Its domain reaches the
proximal boundary of the inner ring-like domain of wg expression. By contrast, the expression domain of dve is larger than that of
the four-jointed (fj) gene, which is expressed in a similar
pattern to vg (Kölzer, 2003).
Defects in the morphology of mutant-wing discs can be observed by the late third larval instar stage. Because abnormal cell death is not observed in dve-mutant wings at earlier stages, the lack of the distal part of the PW in dve mutants could be caused by a failure in establishment of this region. However, overexpression of Dve
achieved through the Flp-out technique results in excessive proliferation of
cells in the region of the distal part of the PW. This suggests that Dve might
be required for the correct proliferation of the cells in this region. Hence,
the loss of the distal part of the PW in dve mutants could also be
explained by a failure in proliferation of the cells in the anlage of the
distal region of the PW (Kölzer, 2003).
Ectopic expression of Dve does not cause the more proximal
regions of the PW to become more distal, which indicates that other factors
are required in addition to Dve to establish the distal part of the PW. One of these factors is Nub, which is involved in the establishment of the medial as well as the distal area of the PW. However, neither ectopic
expression of Nub, nor a combination of Nub and Dve, consistently induces ectopic structures characteristic of the PW. Therefore, it is likely that a combination of Dve, Nub and other factors
is required for the establishment of the distal area of the PW and the
adjacent blade region (Kölzer, 2003).
Recent work has revealed that Nub seems to act in combination with Rn to
establish the medial part of the PW. Both factors cooperate to establish the
inner ring-like domain of wg expression. Thus, it appears that separate regions of the PW are
established independently through different combinations of transcription
factors (Kölzer, 2003).
dveP1738-mutant cell clones near the DV boundary of the wing have been shown to lead to the formation of ectopic bristles characteristic for the wing margin. Concomitant with these pattern disturbances, expression of wg was found in the mutant cell clones. Based on these observations, it has been proposed that Dve is required for the refinement of wg expression. However, in this study, no defects were found in the bristle pattern of flies, either homozygous for the same allele, or in other dve-mutant situations. Therefore, it is believed that the disturbances in the bristle pattern caused by the mutant clones are a result of the artificial apposition of Dve-expressing and non-expressing cells near the DV boundary, created by the induction of clones. These disturbances are thought not to reveal the biological function of Dve. In accordance with this conclusion is the observation that expression of Dve is suppressed along the DV boundary (Kölzer, 2003).
In addition to its function in pattern formation along the PD axis, this work shows that Dve is required for the proper proliferation of the wing
pouch cells. Interestingly, the requirement for Dve differs along the PD axis. In the area anterior to wing vein 3 or posterior to wing vein 4, dve-mutant cell clones contained only half as many cells as their
wild-type counterpart. Hence, the mutant cells trail their wild-type
counterpart by one cell cycle after 48 hours. In addition, in many cases
orphan wild-type clones without a mutant twin were found, which suggests that
the mutant cells had died. Cell death is a typical reaction for cells that are impaired in cell proliferation. Both observations indicate that dve-mutant cells have a slower proliferation rate than wild-type cells. It is likely it is the slower rate of proliferation that causes the size reduction observed in regions A1 and A3 of the dve-mutant wings. Proliferation of dve-mutant cells in the area A2 is also reduced, albeit to a lesser degree. The mutant clones contained 66% of the number of cells that their wild-type counterparts did. More importantly, no orphan
wild-type clones were observed: this indicates that the mutant cells do not undergo apoptosis in this region. Furthermore, the A2 area is of the same size in dve-mutant and wild-type wings. Hence, it appears that proliferation of dve-mutant cells is not as severely affected in A2 as it is in the other regions. This milder defect in proliferation of mutant cells in A2 seems to be compensated during later development. Altogether, these data suggest that Dve is required for the proliferation of all wing pouch cells, but the requirement for its activity varies along the AP axis (Kölzer, 2003).
Why do dve-mutant cells proliferate less? The observed cell death
of mutant cells in A1 and A3 gives a hint to the answer. Cell death is
probably not caused by a defect in the cell cycle machinery itself, since no
increased cell death was found in homozygous dve-mutant animals.
Furthermore, overexpression of Dve using the Flp-out technique does not lead
to an over-proliferation of pouch cells. Hence, it is probable that the mutant cells die as a result of being disadvantaged when in competition with normal cells for survival factors, as has been shown for cells heterozygous
for Minute mutations. In the case of the Minute mutations, the
survival factor is Dpp, which is also responsible for pattern formation along
the AP axis. The differential requirement of Dve along the AP axis
suggests that Dve might be required for the reception of Dpp in pouch cells.
However, one result argues against this possibility: Dve is required most in
cells that are far away from the source of Dpp (which is at the AP boundary).
However, these cells are not, or are only weakly, dependent on Dpp for their
survival. Hence, it is unlikely that dve-mutant cells cannot properly
receive Dpp (Kölzer, 2003).
dve expression is initiated shortly after the start
of wing development, during the early phase of the third larval instar stage.
It is expressed in a disc-like domain that fills the region inside the inner
ring-like domain of wg expression. Vg is required, and
is sufficient, for dve expression in the wing region. Importantly,
the data show that Vg activates the expression of dve
non-autonomously, which indicates that it must be mediated by a secreted
factor that is regulated by Vg. The expression of dve has been shown to be dependent on Dpp and Wg signals. Since vg is itself regulated by these signals, Vg may mediate the effect of these signals on the expression of dve (Kölzer, 2003).
Expression of dve at the DV boundary is
suppressed shortly after its initiation; Wg is required for this repression. In addition, Nub is another factor required for the repression of
dve expression. The data suggest that this suppression is important,
because forced expression of dve along the DV boundary
is deleterious for wing development. One gene affected by the forced
expression of dve is wg, which is required for the
development of the wing through maintenance of the expression of Vg in pouch
cells. Although the expression of other genes might be also affected, the loss of the expression of Wg is already sufficient to explain the loss of wing development upon forced expression of dve (Kölzer, 2003).
The wing imaginal disc is a single-cell layered epithelium and, thus, is a
two-dimensional structure. Therefore, establishment and patterning of the PD
axis must occur with the help of the existing AP and DV axes. The vg
gene is an important translator of the positional values of these axes in
corresponding PD values. vg is required for
the establishment of distal wing fates. This work gives insight into how Vg organizes the PD axis (Kölzer, 2003).
Vg is required for the establishment of
the medial part of the PW. During this process Vg induces the expression of
rn. Expression of rn is in turn required to set up the inner
ring-like expression domain of Wg, which subsequently organizes the formation
of the medial part of the PW. This work shows that Vg is further required for the establishment of the distal part of the PW. It shows that one crucial event during this process is the establishment of the expression of dve by Vg. Importantly, Vg induces
both parts of the PW in a non-autonomous manner. This indicates that Vg
controls the expression of a diffusible factor that induces the expression of
genes, such as dve and rn, in cells inside and outside of
its expression domain, in order to establish the corresponding regions of the
PW. Furthermore, the induction of expression of rn and
dve occurs independently of each other. The expression domain of
rn is larger than that of dve. Taking for granted that
expression of both genes is induced by the same diffusible factor, this
observation suggests that the factor might act in a concentration dependent manner. In this scenario the induction of rn expression would require less activity than the induction of dve. Expression of
nub has been shown to be lost in vg-mutant wing imaginal discs, suggesting that Vg is also required non-autonomously for
the activation of nub, in a yet larger domain than dve and
rn. However, these results are in conflict with earlier work that
reports that nub expression is not dependent on Vg function. Wg, but not Vg, has been shown to be able to induce ectopic
expression of nub in the notum of the wing imaginal disc.
Furthermore, expression of nub RNA was observed in vg-null
mutant wing imaginal discs. These data strongly suggest that Wg is required to
activate expression of nub. Hence, further work is necessary to
resolve the contradictions, and to determine whether Vg also plays a role
during activation of the expression of nub. Despite this uncertainty,
all of the mentioned genes are expressed in disc-like domains of different
sizes. Their expression leads to concentric areas with different combinations
of gene activities. It seems likely that a particular combination of these
genes establishes a specific part of the PW (Kölzer, 2003).
These data provide evidence that Vg controls the expression of fj,
within an expression domain that corresponds to the wing pouch. Fj is required
for the establishment of a proximal region of the wing pouch and also for
planar polarity of the wing. Furthermore, Vg regulates the expression of
Distal-less (Dll), which is required to pattern the wing
margin. Thus, Vg is involved in the patterning of the PD axis
inside as well as outside its expression domain (Kölzer, 2003).
It is widely accepted that pattern formation and cell proliferation are
closely connected during wing development. However, it has not been clear how
these processes are connected. The fact that expression of dve is
initiated by one of the central patterning factors, Vg, provides a possible
link (Kölzer, 2003).
The data presented in this study reveal how
patterning along the PD axis might occur with help of the two other existing
axes. Wing development starts at the cross-point of the
expression domains of Dpp and Wg in the ventral part of the wing disc. It appears that the combined activity of the two signals define the wing field. Although the activity of Wg is sufficient to establish the proximal-most pattern elements, the hinge and the proximal region, of the PW, the establishment of all distal regions requires the additional activity of vg. In the wing field, the Notch signalling pathway activates the expression of vg in cells at the future compartment boundary. In addition, Wg, perhaps in collaboration with Vg/Sd,
activates the expression of nub (Kölzer, 2003).
In the next step Vg induces the expression of wg in cells at the
DV boundary, in collaboration with the Notch pathway. In addition, Vg
activates an unknown diffusible factor that induces the expression of
dve and rn in disc-like domains of different sizes. All these domains are larger than that of Vg, and expression of the three genes is established
independently of one another. This fact suggests that the diffusible factor
might act in a concentration-dependent manner, as is typical for morphogens.
Dve and Rn act in collaboration with Nub to establish the medial and distal
parts of the PW (Kölzer, 2003).
When the expression of nub, rn and dve is initiated, Vg
is expressed in cells at the DV boundary. These cells will
later form the distal-most structure, the wing margin. The wing pouch is
formed by the progenies of cells at the DV boundary, and is therefore
intercalated between the margin and the anlagen of the PW. During its formation, the pouch will be further subdivided
through the combined activity of Vg and Wg. Both proteins generate gradients
that further subdivide the pouch along the DV axis (Kölzer, 2003).
In summary, the data suggest that pattern formation along the PD axis
occurs in several steps and uses a strategy similar to that observed during
leg development. It is initiated by the definition of the proximal (hinge
and the distal part of the PW) and the distal-most point (wing margin), with
help of the existing AP and DV axes. During development, the intermediate
pattern elements (first the anlagen of the medial and distal part of the PW,
then the wing blade) are intercalated stepwise with respect to these reference points (Kölzer, 2003).
Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg
Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. Evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints (Shirai, 2007).
To achieve specific developmental programs, antagonism between Notch and EGFR signaling has been widely observed. A graded activity of EGFR signaling from the distal tip of a leg disc is crucial for patterning the distal structure, and it should be converted into the segmental activation, which is critical for suppression of inappropriate joint formation. One possible explanation is that P-D patterning genes define the segment boundary, and thereby refine the Notch signaling pathway in the distal region of each segment, where EGFR signaling should be repressed. The expanded expression of argos-lacZ in Nts mutants strongly suggests that the Notch signaling pathway antagonizes EGFR signaling. Interestingly, a similar type of regulation has been reported for Caenorhabditis elegans vulval development. Thus, the antagonistic interaction between EGFR and Notch signaling establishes the complementary activation of these pathways in neighboring cells, and is crucial for both vulval cell fate determination and leg joint formation (Shirai, 2007).
In vertebrates, the early process of body segmentation, i.e., somitogenesis, takes place sequentially from head to tail. Somites are generated from the presomitic mesoderm (PSM), the unsegmented paraxial mesoderm at the tail end of the embryo. A 'clock and wavefront' model has been proposed to explain the mechanism of sequential somite formation. Oscillated gene expression, i.e., the clock, driven by Wnt and Notch signaling in the posterior PSM is translated into the segmental units in the wavefront, which is generated in the anterior PSM in response to the decreased activities of graded Wnt and FGF signaling from the tail end. As an embryo grows caudally, the wavefront moves backwards at a constant rate. Thus, the segment boundary is set at the interface between the Notch-activated and -repressed domains in the anterior PSM (Shirai, 2007).
During vertebrate somitogenesis, it has been shown that the interface between the Notch-activated and Notch-repressed domains is generated on suppression of Notch activity through induction of the lunatic-fringe (Lfng) gene in the segment boundary. This refinement is under the control of the basic helix-loop-helix type transcription factor Mesp2, which is expressed in the rostral half of the anterior PSM, indicating that rostral-caudal polarity within a somite is important for restricted Notch activation. The results indicate that the restricted Notch activation during Drosophila leg segmentation also occurs at the segment boundary rather than the center of each segment, suggesting that a conserved mechanism in both Drosophila legs and vertebrate somites underlies the activation of Notch signaling adjacent to the segment boundary (Shirai, 2007).
A Dve-expressing region straddles the fold of the segment boundary, and the following observations indicate that Dve has joint-suppressive activity: (1) dve mutant legs resulted in extra joint formation and (2) forced expression of dve in the presumptive joint region suppressed joint formation. Thus, the mechanism of joint development can be explained as follows; Notch-mediated Dve repression on the proximal side to the intersegmental fold relieves the above joint-suppressive activity, leading to normal leg joint formation. This is reminiscent of the abdomen-suppressive activity of Hunchback, which is relieved by Nanos to induce the abdominal structure. In contrast, Dve expression on the distal side to the fold should be maintained to suppress inappropriate joint formation, because dve mutation leads to extra joint formation with reverse polarity. It appears that Dve activity is only induced to suppress joint formation and that temporally regulated Dve repression is crucial for normal leg joint formation, because dve mutations did not affect normal joint formation (Shirai, 2007).
Extra joints with reverse polarity (reverse joints) are derived from mutants deficient in the PCP or EGFR signaling pathway. Previous reports have suggested a model in which the Notch signal activation proximal to the Notch ligand-expressing domains is blocked by these signals, only allowing the Notch signal activation in a distally adjacent region, i.e., the distal region of each segment. Based on the expression pattern of the Notch ligand Ser, it is assumed that the center of a segment is highly potent for receiving Notch signaling. This idea can explain the reverse polarity of extra joints, because Ser activates the Notch signaling pathway in two different directions: from proximal to distal for normal joints, and distal to proximal for extra joints. However, it seems unlikely that ectopic activation of Notch signaling is restricted at the center of a segment. A Notch-target gene, dAP-2, is autonomously activated in response to ectopic Notch signaling, and, in pk mutants, ectopic dAP-2 expression has expanded on the distal side to the intersegmental fold, the most proximal but not the central region in a segment. Furthermore, the joint-suppressive activity of Dve is also required to repress dAP-2 expression on the distal side to the intersegmental fold. These results suggest that reverse joints are derived from the distally adjacent region to the intersegmental fold (Shirai, 2007).
Based on the results, a model is proposed in which joint-forming activity is generated from the intersegmental fold in a bidirectional manner, and that an inappropriate signal having reverse polarity is blocked by Dve activity, and the PCP and EGFR signaling pathways. In this model, Dve activity is required to suppress Notch target genes, such as dAP-2, involved in joint formation. This is very similar to the situation observed in wing discs, where the Notch target gene wg is repressed by Dve in regions adjacent to the Notch-activated D-V boundary. It is an intriguing possibility that the vertebrate somite boundary generates similar bidirectional signals, and that the inhibition of either one is closely linked to the rostral-caudal polarity within a somite. Further characterization of Drosophila leg segmentation is needed to determine whether this model is applicable to vertebrate somitogenesis or other segmentation processes (Shirai, 2007).