defective proventriculus : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - defective proventriculus
Cytological map position - 58D1-2
Function - transcription factor
Symbol - dve
FlyBase ID: FBgn0020307
Genetic map position -
Classification - homeodomain protein
Cellular location - nuclear
|Recent literature||Yan, J., Anderson, C., Viets, K., Tran, S., Goldberg, G., Small, S. and Johnston, R. J., (2017). Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies. Development [Epub ahead of print]. PubMed ID: 28126841
How differential levels of gene expression are controlled in post-mitotic neurons is poorly understood. In the Drosophila retina, expression of the transcription factor Defective Proventriculus (Dve) at distinct cell-type-specific levels is required for terminal differentiation of color- and motion-detecting photoreceptors. This study found that the activities of two cis-regulatory enhancers are coordinated to drive dve expression in the fly eye. Three transcription factors act on these enhancers to determine cell-type-specificity. Negative autoregulation by Dve maintains expression from each enhancer at distinct homeostatic levels. One enhancer acts as an inducible backup ("dark" shadow enhancer) that is normally repressed but becomes active in the absence of the other enhancer. Thus, two enhancers integrate combinatorial transcription factor input, feedback, and redundancy to generate cell-type specific levels of dve expression and stable photoreceptor fate. This regulatory logic may represent a general paradigm for how precise levels of gene expression are established and maintained in post-mitotic neurons.
The gut epithelium of Drosophila is derived from the anterior and posterior primordia at both ends of the blastoderm embryo. These primordia are initially nonsegmental and fused into a single continuum. Secreted molecules, such as Decapentaplegic and Wingless, induce subsequent morphogenetic events that ultimately compartmentalize the primordia into morphologically distinct sectors. During this process, these signals also direct cells to distinct developmental paths thus establishing the functional organization of the midgut. The gene defective proventriculus (dve) is a target of Dpp and Wg during the establishment of two gut structures: the proventriculus (a foregut structure formed at the junction of the foregut and the midgut), and the central midgut (Nakagoshi, 1998). This overview will consider the roles of dve in the formation of these two structures.
The proventriculus is located at the caudal end of the esophagus and serves as a valve, regulating food passage into the midgut. It is composed of three tissue layers. The inner and outer layers consist of an ectodermal epithelial layer and the ensheathing visceral mesoderm, respectively. The third layer, intervening between the first two, is completely free of mesodermal tissues. This internal portion, called the cardiac valve (the proventriculus is also referred to as the cardia), is innervated by three axons from the proventricular ganglion, one of four major interconnected ganglia that together constitute the stomatogastric nervous system. The proventriculus develops at the junction of the foregut and the midgut. Initially, there is an outward buckling of the foregut tube, in a region that is free of visceral mesoderm, to form what is referred to as the keyhole structure. This area then undergoes further outward movement and will then fold back on itself and move inward to form the mature, multi-layered proventriculus (Pankratz, 1995).
The keyhole region expresses hh and wg: the activities of both these genes are essential for the subsequent posterior migration of the foregut, both toward and into the foregut, the anterior-most region of midgut (Pankratz, 1995). The anterior-most midgut, consisting of visceral mesoderm and endoderm (Pankratz, 1995), contributes to the outer layer of the proventriculus after stage 16 (Nakagoshi, 1998 and references). Thus the outer wall of the proventriculus is composed of inner endodermal and outer mesodermal tissues. The outer wall of the proventriculus, in particular the endodermal component, expresses dve (Nakagoshi, 1998).
The reduced body sizes of dve mutant larvae suggest that something in the way food is utilized is affected by the dve mutation. Colored yeast fed to heterozygous larvae stains red throughout the length of the gut, but the same colored yeast accumulates only in the proventriculus in dve mutant larvae. Consistent with this observation, the proventriculus is found to form aberrently in dve mutant larvae. In the wild type, cell movement leads to formation of the internal portion of the proventriculus during embryonic stages 16-17; cells of the foregut epithelium invaginate into the anterior-most midgut that normally expresses dve. In dve embryos, the cell migration is greatly delayed and the internalization is only temporary. As a result, dve larvae cannot form the three-layered structure of the proventriculus, the same failure that is observed in hh or wg mutant embryos. Since the dve-expressing anterior-most midgut constitutes the outer layer of the proventriculus, this dve phenotype suggests that dve activity is required for the functional development of outer layer cells and the consequent retention of the internal portion of the proventriculus. dve expression in the outer layer of the proventriculus is dependent on wingless expression in the keyhole structure. It is concluded that the Wg signal regulates dve expression during proventriculus development (Nakagoshi, 1998).
This discussion now turns from the role of dve in proventriculus development to dve's function in midgut development. The midgut consists of two germ layers: the visceral mesoderm and the endoderm. Cells of the portion of the middle midgut that are derived from the endoderm differentiate into four distinct cell types: copper, interstitial, large flat, and iron cells. These endodermal cell types are specified by Dpp and Wg, which are expressed in the adhering visceral mesoderm of the parasegments (PS) 7 and 8, respectively. Copper cells exhibit a unique morphology with banana shapes and exhibit UV light-induced fluorescence after copper feeding. These characteristics are specified by a homeotic gene, labial (lab), which is activated by the Dpp signal in the midgut. Two different thresholds of Wg define copper and large flat cells. However, it has been unclear how Lab confers the transcriptional regulation to specify copper cells. In the middle midgut, the dve gene is expressed in all precursors of the four distinct cell types; subsequent to this broad expression, dve is repressed only in copper cells. This repression is mediated by two Dpp target genes, lab and dve itself, and is also essential for the functional specification of copper cells. Thus, dve is involved in different developmental aspects of the midgut under the control of different extracellular signals (Nakagoshi, 1998).
The expression domains and regulation of labial and dve in the middle midgut were compared. It was found that there are two endodermal expression domains: one is located immediately adjacent to the visceral mesoderm, and the other in a more interior inner endoderm layer. Dpp has been shown to be sufficient to induce dve expression in the midgut without Wg. These results indicate that dve expression in the middle midgut does not depend on Wg but on Dpp. This is in contrast to dve expression during proventriculus development. lab is expressed under the control of Dpp as is dve, however, lab is regulated negatively by the Wg signal to generate a sharp posterior border. The expression of lab is observed in the endoderm just beneath the dpp-expressing visceral mesoderm of PS 7, but not in the inner endodermal cells. In contrast, dve is expressed more broadly throughout the inner endodermal layers, including presumptive interstitial cell precursors. Another difference in lab and dve expression is that dve expression is subsequently repressed in lab-expressing cells that become copper cells. The possibility that Lab might be involved in the repression of dve in copper cells was examined. In lab mutants, dve expression is not repressed in presumptive copper cells. This pattern of gene expression is similar to that of neighboring interstitial cells, which express dve continuously without lab expression in the wild type. Evidence is presented that it is unlikely that the lab mutation causes the transformation of copper cells into interstitial cells. Taken together, the repression of dve requires the activities of both Lab and Dve itself (Nakagoshi, 1998).
To determine whether the dve repression in copper cells is essential for the establishment of their correct identities, dve was overexpressed ubiquitously at stage 17. Strong heat shock-induced dve expression results in an abnormal morphology for copper cells. The typical banana shape is frequently lost, and the cells becoming circular, suggesting an abnormal cytoskeletal organization. To determine the effect of ectopic dve on the copper cell function, ubiquitous dve expression was induced using a milder heat shock. Under this condition, the copper cells appear to retain their normal morphology, however, the typical character of copper cells, UV light-induced orange fluorescence on copper feeding, is greatly reduced. This mild heat shock does not affect the posterior fluorescence attributable to iron cells, suggesting that the function of copper cells is specifically impaired by this treatment. Taken together with the results for dve mutants described above, both the loss of function and ectopic expression of the dve gene affect the morphology of copper cells, and ectopic dve expression impairs the function of copper cells without affecting their morphology. These results indicate that temporally restricted dve repression is essential for this functional specification, in addition to the dve gene, which is indispensable for copper cell development. This repression depends on Lab and Dve itself. Thus, the cross-regulation of the two Dpp target genes (dve and lab) specifies the functional identity of copper cells (Nakagoshi, 1998).
A pair of the Drosophila eye-antennal disc gives rise to four distinct organs (eyes, antennae, maxillary palps, and ocelli) and surrounding head cuticle. Developmental processes of this imaginal disc provide an excellent model system to study the mechanism of regional specification and subsequent organogenesis. The dorsal head capsule (vertex) of adult Drosophila is divided into three morphologically distinct subdomains: ocellar, frons, and orbital. The homeobox gene orthodenticle (otd) is required for head vertex development, and mutations that reduce or abolish otd expression in the vertex primordium lead to ocelliless flies. The homeodomain-containing transcriptional repressor Engrailed (En) is also involved in ocellar specification, and the En expression is completely lost in otd mutants. However, the molecular mechanism of ocellar specification remains elusive. This study provides evidence that the homeobox gene defective proventriculus (dve) is a downstream effector of Otd, and also that the repressor activity of Dve is required for en activation through a relief-of-repression mechanism. Furthermore, the Dve activity is involved in repression of the frons identity in an incoherent feedforward loop of Otd and Dve (Yorimitsu, 2011).
This study presents evidence that Dve is a new member involved in ocellar specification and acts as a downstream effector of Otd. The results also revealed a complicated pathway of transcriptional regulators, Otd-Dve-Ara-Ci-En, for ocellar specification (Yorimitsu, 2011).
Transcription networks contain a small set of recurring regulation patterns called network motifs. A feedforward loop (FFL) consists of three genes, two input transcription factors and a target gene, and their regulatory interactions generate eight possible structures of feedforward loop (FFL). When a target gene is suppressed by a repressor 1 (Rep1), relief of this repression by another repressor 2 (Rep2) can induce the target gene expression. When Rep2 also acts as an activator of the target gene, this relief of repression mechanism is classified as a coherent type-4 feedforward loop (c-FFL). During vertex development, Ara is involved in hh repression, and the Dve-mediated ara repression is crucial for hh expression and subsequent ocellar specification. However, the cascade of dve-ara-hh seems to be a relief of repression rather than a cFFL, because Dve is not a direct activator of the hh gene. Furthermore, dve RNAi phenotypes were rescued in the ara mutant background, suggesting that a linear relief of repression mechanism is crucial for hh maintenance (Yorimitsu, 2011).
In photoreceptor R7, Dve acts as a key molecule in a cFFL. Dve (as a Rep1) represses rh3, and the transcription factor Spalt (Sal) (as a Rep2) represses dve and also activates rh3 in parallel to induce rh3 expression. Interestingly, Notch signaling is closely associated with the relief of Dve-mediated transcriptional repression in wing and leg disks. These regulatory networks may also be cFFLs in which Dve acts as a Rep1, although repressors involved in dve repression are not yet identified. In wing disks, expression of wg and ct are repressed by Dve, and Notch signaling represses dve to induce these genes at the dorso-ventral boundary. The Dve activity adjacent to the dorso-ventral boundary still represses wg to refine the source of morphogen. In leg disks, Dve represses expression of dAP-2, and Notch signaling represses dve to induce dAP-2 at the presumptive joint region. The Dve activity distal to the segment boundary still represses dAP-2 to prevent ectopic joint formation. Taken together, these results suggest that Dve plays a critical role as a Rep1 in cFFLs in different tissues. In the head vertex region, it is likely that the repressor activity of Dve is repressed in a cFFL to induce frons identity (Yorimitsu, 2011).
The homeodomain protein Otd is the most upstream transcription factor required for establishment of the head vertex. During second larval instar, Otd is ubiquitously expressed in the eye-antennal disk and it is gradually restricted in the vertex primordium until early third larval instar. Expression of an Otd-target gene, dve, is also detected in the same vertex region at early third larval instar. Otd is required for Dve expression, and the Otd-induced Dve is required for repression of frons identity through the Hh signaling pathway in the medial region. However, Otd is also required for the frons identity in both the medial and mediolateral regions (Yorimitsu, 2011).
This regulatory network is quite similar to the incoherent type-1 feedforward loop (iFFL) in photoreceptor R7. Otd-induced Dve is involved in rh3 repression, whereas Otd is also required for rh3 activation. iFFLs have been known to generate pulse-like dynamics and response acceleration if Rep1 does not completely represses its target gene expression. However, the repressor activity of Dve supersedes the Otd-dependent rh3 activation, resulting in complete rh3 repression in yR7. In pR7, Dve is repressed by Sal, resulting in rh3 expression through the Otd- and Sal-dependent rh3 activation. Thus, Dve serves as a common node that integrates the two loops, the Otd-Dve-Rh3 iFFL and the Sal-Dve-Rh3 cFFL (Yorimitsu, 2011).
In the head vertex region, Otd and Dve are expressed in a graded fashion along the mediolateral axis with highest concentration in the medial region. It is assumed that Otd determines the default state for frons development through restricting the source of morphogens Hh and Wg, and also that high level of Dve expression in the medial ocellar region represses the frons identity through an iFFL. It is likely that repression of dve by an unknown repressor X occurs in a cFFL and induces the frons identity in the mediolateral region (Yorimitsu, 2011).
Interlocked FFLs including Otd and Dve appear to be a common feature in the eye and the head vertex. However, other factors are not shared between two tissues. In R7, a default state is the Otd-dependent Rh3 activation, an acquired state is (1) Rh3 repression through the Otd-Dve iFFL and (2) Spineless-dependent Rh4 expression. In the vertex, a default state is Otd-dependent frons formation, an acquired state is (1) frons repression through the Otd-Dve iFFL and (2) Hh-dependent ocellar specification associated with En and Eya activation (Yorimitsu, 2011).
Both Otd and Dve are K50-type homeodomain transcription factors, and they bind to the rh3 promoter via canonical K50 binding sites (TAATCC). The Otd-Dve iFFL in the eye depends on direct binding activities to these K50 binding sites, but the iFFL in the vertex seems to be more complex. Although target genes for frons determination are not identified, the iFFL in the vertex includes some additional network motifs. For instance, in the downstream of Dve, Hh signaling is critically required for repression of the frons identity (Yorimitsu, 2011).
Since iFFLs also act as fold-change detection to normalize noise in inputs, interlocked FFLs of Dve-mediated transcriptional repression may contribute to robustness of gene expression by preventing aberrant activation. It is an intriguing possibility that, in wing and leg disks, Dve also serves as a common node that integrates the two loops as observed in the eye and the vertex. Further characterization of regulatory networks including Dve will clarify molecular mechanisms of cell specification (Yorimitsu, 2011).
defective proventriculus is composed of two homeodomains and is expressed in the Drosophila endoderm. dve expression can first be detected at the rostral tip of the anterior midgut primordium. This expression persists until late stages of embryogenesis and becomes confined to the outer endodermal wall of the developing proventriculus. In stage 12 embryos, dve is expressed in the migrating posterior midgut primordium and soon thereafter in an endodermal domain at the junction of the midgut and hindgut. In stage 13 embryos, when the anterior and the posterior midgut primordia have fused, dve expression is most prominent in the anterior, central and posterior portion of the midgut. Weak expression is detectable in the region from where the gastric caecae will bud out. In stage 14 embryos, the central expression domain broadens and finally covers the second and third midgut lobes in stage 16. Additional expression domains of dve include the tip cells of the Malpighian tubules, mesectodermal cells, nerve cells of the central and peripheral nervous system and a group of cells that lie below the pharynx (Fuß, 1998).
Mutant analysis reveals that dve activity is required at the foregut/midgut boundary for the development of the proventriculus. This organ regulates food passage from the foregut into the midgut and forms by the infolding of ectoderm and endoderm-derived tissues. The endodermal outer wall structure of the proventriculus is collapsed in the mutants leading to a failure of the ectodermal part to invaginate and build a functional multilayered organ. Lack-of-function and gain of-function experiments show that the expression of this homeobox gene in the proventriculus endoderm is induced in response to Wingless activity emanating from the ectoderm/endoderm boundary, whereas its expression in the central midgut is controlled by Dpp and Wingless signaling emanating from the overlying visceral mesoderm (Fuß, 1998).
hedgehog, wingless and decapentaplegic define through their restricted expression a signaling center at the boundary of the forgut ectoderm and the midgut endoderm where proventriculus morphogenesis occurs. This boundary become established at the posterior margin of the keyhole structure, formed from cells that migrate out of a mesoderm free zone of the foregut epithelium. The keyhole tissue folds back on itself to generate a structure called the cardiac valve, which is subsequently pushed as an extension of the eosophagus into an endodermal sac-like midgut chamber which forms the outer wall of the proventriculus. wg is initially transcribed in an expression domain that includes the ectodermal region from which the keyhole will form and extends slightly beyond it into the proventriculus. The striped dve expression domain extends from the ectoderm/endoderm boundary toward the posterior and overlaps at its anterior margin with the wg expression domain. With the onset of keyhole formation, the wg expression domain becomes split into two domains: one lies at the anterior border of the keyhole in the ectodermal forgut cells and the other in endodermal cells posterior to the keyhole. Both expression domains of wg persist in the developing proventriculus until very late stages of embryogenesis. dve expression continues to overlap the domain of wg until the end of embryogenesis. dve is required for the maintenance of the posterior wg domain. wingless is required for dve activation in both the anterior and posterior dve expression domains (Fuß, 1998).
These results are consistent with the argument that dve is a newly identified target of the Wg signal that mediates the coordination of epithelial morphogenesis upon signal reception in the proventriculus. In wg mutants, dve expression in the endodermal part of the proventriculus is absent and upon ectopic expression of wg, the dve expression domain becomes ectopically activated, both in the ectoderm and the endoderm. The interaction of wg and dve fit well with the expression pattern of the genes during gut development. wg is initially expressed in a domain that overlaps the ectoderm/endoderm boundary and dve becomes induced, overlapping wg in neighboring endodermal midgut cells. Upon keyhole formation, when the wg domain splits, dve overlaps with the posterior wg domain until late stages. This overlap seems to be important, since in dve mutants, posterior wg is not maintained, pointing toward the possibility of an autoregulatory feedback loop between wg and dve. If wg of dve are not expressed in this region, the proventriculus endoderm collapses and the invagination of the ectodermal into the endodermal tissue is defective (Fuß, 1998).
When the anterior and posterior midgut primordia fuse, dve is expressed in the central part of the endoderm region that underlies parasegments 7 and 8 of the visceral mesoderm in which dpp and wg are expressed. Once the second midgut constriction starts to build at the border of parasegments 7 and 8, dve expression expands towards both sides into the endoderm region underlying the parasegments 6-9. After the formation of the second constriction, dve expression persists in the second and third midgut lobes. In wg mutants, dve is still expressed in the central region of the embryo but the expression only weakly expands towards the posterior, as compared to wild type embryos. In dpp mutants there is a lack of dve expression in the anterior region of the dve domain parasegment 7 of the visceral mesoderm where dpp is expressed in wild type embryos. At later stages, dve expression is found only in the third midgut lobe instead of the second and third lobes, both of which display dve expression in wild type embryos. No expression of dve is found in schnurri mutants, which encode a transcription factor mediating the Dpp signal. Dpp can activate dve in the complete absence of wg expression and it is not the combination of both which is critical for dve expression. Ubiquitous expression of Wg in the visceral mesoderm leads to a repression of dve in the central region of the embryos. These results provide evidence that the Dpp signaling pathway eminating from the visceral mesoderm plays a pivital roe in activating central dve expression. The mutant analysis suggests that Wg is required to maintain dve in the posterior part of the central dve domain (Fuß, 1998).
Because the dve-expressing domain in the presumptive proventriculus initially overlaps with the wingless- and hedgehog-expressing domains, the possibility was examined that Wg and/or Hh regulate the expression of the dve gene. In wgCX4 null embryos, dve expression is abolished in the stomodeum, whereas epidermal expression is normally detected. In hhAC mutant embryos, dve is normally expressed. Thus, Wg rather than Hh is required to activate the dve gene in the presumptive proventriculus. The keyhole structure also expresses cubitus interruptus (ci). In ciD mutants, the expression of wg and hh is not maintained, leading to the absence of a keyhole structure. The maintenance of wg expression also requires hh activity. In ciD mutants, dve expression is never detected in the presumptive proventriculus until after stage 13; it begins to be detectable at stage 14, despite the absence of a keyhole structure. dve expression expands ectopically into the presumptive first gut lobe after stage 15. Similar ectopic expression of dve is observed in hhIJ35 and wgIL114 mutant embryos. The ectopic dve expression in these mutants suggests that the Wg signal is also required to define the posterior border of late dve expression in the presumptive proventriculus, in addition to the initial activation of the dve gene (Nakagoshi, 1998).
abdominal-A (abd-A) mutants were used to examine the dependence of dve expression on Wg in the midgut. In abd-A mutants, the expression of Ultrabithorax (Ubx) and dpp in the visceral mesoderm expands throughout the posterior midgut and endogenous wg is not activated. In abd-A mutants, dve expression also expands posteriorly. These three observations are consistent with the notion that Dpp is sufficient to induce dve expression in the midgut without Wg. To confirm this, an examination was performed of the effect of ubiquitous wg expression on dve expression in the visceral mesoderm. The ubiquitous activation of the Wg signal using shaggy null mutations induces the expression of Ubx and dpp only anteriorly, but not posteriorly, in the midgut. The ubiquitous wg expression in the whole visceral mesoderm induces ectopic dve expression only anteriorly, indicating that this induction appears to be indirect and mediated by ectopic dpp activation. It is noteworthy that the ectopic dve induction is especially strong in the anterior-most region that is normally responsive to Wg. Taken together, these results indicate that dve expression in the middle midgut does not depend on Wg but on Dpp, which is in contrast to dve expression during proventriculus development (Nakagoshi, 1998).
In the middle midgut, Dpp and Wg signals from the visceral mesoderm control gene expression on both the anterior and posterior sides of the middle constriction. Hence an examination was made to see if dve expression depends on Dpp or Wg signals in this region. In thick veins (tkv) mutants, which lack the functional type I receptor for Dpp, dve expression is completely absent in the middle midgut, whereas the expression in other parts of the midgut is not affected. This indicates that dve expression in the middle midgut requires the Dpp signal. In contrast, the ubiquitous expression of dpp throughout the visceral mesoderm, using the combination of UAS-dpp and 24B-Gal4 driver, induces dve expression throughout the underlying endoderm. This dependence of dve expression on Dpp is quite similar to the dependence of lab expression on Dpp (Nakagoshi, 1998).
Two distinct transcription factors, Schnurri (Shn) and Mad, have been reported to mediate the Dpp signal. The expression of Defective proventriculus protein in the middle midgut is absent in shn mutants. In addition, the ubiquitous mesodermal expression of dpp in the shn mutant background fails to induce endodermal dve expression, indicating that dve induction requires shn activity in the midgut endoderm. In contrast, dve expression is not affected in Mad mutants, whereas lab expression is completely absent in the same mutant background (Nakagoshi, 1998).
Pattern formation during animal development is often induced by extracellular signaling molecules, known as morphogens, which are secreted from localized sources. During wing development in Drosophila, Wingless (Wg) is activated by Notch signaling along the dorsal-ventral boundary of the wing imaginal disc and acts as a morphogen to organize gene expression and cell growth. Expression of wg is restricted to a narrow stripe by Wg itself, repressing its own expression in adjacent cells. This refinement of wg expression is essential for specification of the wing margin. A homeodomain protein, Defective proventriculus (Dve), mediates the refinement of wg expression in both the wing disc and embryonic proventriculus, where dve expression requires Wg signaling. These results provide evidence for a feedback mechanism that establishes the wg-expressing domain through the action of a Wg-induced gene product (Nakagoshi, 2002).
The dve gene is essential for proventriculus development and is initially triggered by the Wg signal in its primordium. The expression of wg and dve initially overlaps, but then segregates into adjacent but exclusive domains. The function of dve in regulating wg expression was examined. In the absence of dve, wg expression expands posteriorly to the region that normally expresses dve. This indicates that dve defines the posterior border of wg expression by repressing its expression. Thus, Wg refines its expression domain via dve gene activation in the embryonic proventriculus (Nakagoshi, 2002).
Wg refines its own expression along the D-V boundary in wing imaginal discs. The above result of the dve function in the embryonic proventriculus led to an examination of the possibility that dve acts in the refinement of wg expression at the D-V boundary. The expression of dve in wing discs was examined. At early to mid-third larval instar (48-24 h before pupariation, BP), dve expression begins throughout the prospective wing pouch, which overlaps partly with wg expression. The coexpression of dve and wg is obvious until mid-third instar, and subsequently, dve is excluded from the D-V boundary at mid- to late third instar (24-12 h BP). At this stage, dve is expressed complementary to wg. The activation of wg and repression of dve are both mediated by Notch (N) signaling at the D-V boundary. At late third instar (12-0 h BP), dve expression is reduced in the distal region, whereas it remains strong at the proximal region of the wing pouch. The significance of this repression is unclear (Nakagoshi, 2002).
The embryonic expression of dve depends on the Wg signal in the proventriculus and on the Dpp signal in the middle midgut. Whether or not dve expression in wing discs depends on these signals was examined. When the Wg signal is ectopically activated in a ring pattern around a wing pouch under the control of the 30A-Gal4 driver, dve is ectopically activated only in cells at the intersection of the ring with the A-P boundary, which normally expresses Dpp. In contrast, activation of the Dpp signal in the same ring pattern results in the ectopic expression of dve only at the D-V boundary, where Wg is normally expressed. Thus, the combined Wg and Dpp signals appear to induce dve expression in wing discs. To examine this possibility, flip-out recombination clones were generated that simultaneously express an activated form of the Dpp receptor (TkvQ253D) and that of a Wg signaling molecule (DeltaArm). Some of these clones induced ectopic Dve expression autonomously outside the compartment boundaries. These results strongly support the above notion that the combined activities of Wg and Dpp signals induce dve expression rather than other signals generated at compartment boundaries (Nakagoshi, 2002).
The expression pattern of dve raises the possibility that repression of dve along the D-V boundary is mediated by N signaling, which plays a pivotal role in the activation of margin-patterning genes, such as wg and cut. To examine this possibility, the N signal was ectopically activated along the A-P boundary by expressing N ligands Delta (Dl) and Serrate (Ser) under the control of patched (ptc)-Gal4. Dl and Ser trigger N signaling in the dorsal half and the ventral half of wing discs, respectively. In ptc-Gal4/UAS-Ser discs, N signaling is ectopically activated along the ventral A-P boundary and results in the ventral expression of the margin-patterning genes wg and cut. In these discs, dve expression was found to be repressed along the ventral A-P boundary. In ptc-Gal4/UAS-Dl discs, N signaling is ectopically activated along the dorsal A-P boundary, leading to dorsal dve suppression. Thus, the ectopic N signal can repress dve expression. In order to determine whether or not N indeed suppresses Dve expression along the D-V boundary of wing discs, mosaic clones were generated lacking N activity. The loss of N activity results in the ectopic activation of Dve in the region where Dve expression is normally suppressed along the D-V boundary. These results indicate that N signaling represses dve along the D-V boundary of wing discs to create a domain in which Dve is absent and wg is activated (Nakagoshi, 2002).
Wg signaling appears to be necessary for dve repression along the D-V boundary. The action of Wg, which is up-regulated by N at the D-V boundary, might explain N-mediated repression of dve. To examine the cell autonomy for N-mediated repression of dve, dve expression was examined in N mutant clones at later stages. N mutant clones crossing the D-V boundary cause the derepression of Dve, with varying levels of dve expression within clones. This suggests that there is some nonautonomous effect on dve repression. Mutant mosaic clones were generated for zeste-white 3 (zw3), in which Wg signaling is constitutively active. Partial repression of Dve was observed in zw3 mutant clones at early to mid-third instar. At later stages, when N signaling is strongly activated along the D-V boundary, ectopic Dve repression in zw3 mutant clones is more evident outside the D-V boundary. N-mediated dve repression thus depends largely on Wg signaling that is activated by N. At the late third instar, expansion of Dve repression at the distal region also depends on Wg and Dpp signaling. Inhibition of these signals by expressing a dominant-negative form of Wg signaling molecule (dTCFDN) or a negative regulator for Dpp signaling (dad) along the A-P boundary results in elevated expression of Dve. These results support the notion that N-mediated repression of Dve has a nonautonomous effect, although Wg signaling alone is insufficient for complete repression at early stage. It is inferred that N-mediated events along the D-V boundary modulate the Wg and Dpp signaling, or another secreted signaling molecule, such as Spitz, might be involved in dve repression because the Spitz ligand is up-regulated along the D-V boundary. Indeed, inhibition of EGF signaling by expressing a dominant-negative form of Drosophila EGF receptor along the A-P boundary results in derepression of Dve (Nakagoshi, 2002).
This study suggests that the combined activities of Wg and Dpp induce the initial dve gene expression in wing discs. However, the continuous input of these signals might be unnecessary for its maintenance, the possibility that the perdurance of signaling molecules in tkv, arm, or dsh mutant clones was enough to maintain Dve expression cannot be excluded. Interestingly, these clones exhibit a rather high level of dve expression when they are made at the D-V boundary. Expression of a dominant-negative form of dTCF along the D-V boundary also elevates dve expression. The adults of such animals had notched wings resembling those caused by ectopic dve expression along the D-V boundary. Similar wing phenotypes have been observed by inhibiting the Dpp signal along the D-V boundary. These results suggest that Wg and Dpp signals cause repression of dve at the D-V boundary after the initial induction (Nakagoshi, 2002).
In both wing discs and the proventriculus, the initially overlapped expression of wg and dve becomes segregated into complementary patterns via the ability of Dve to suppress wg gene expression, which leads to refinement of the border of the wg-expressing domain. In wing discs, several different mechanisms limit wg expression to the D-V boundary: (1) restriction of N activation to the D-V boundary, which requires the Fringe function, and a positive feedback loop between Ser and Dl expression; (2) the inhibition of N signaling in Ser- and Dl-expressing cells in a dominant-negative manner; (3) the local suppression by the Wg signal itself near the D-V boundary. This study has revealed a feedback mechanism by which a Wg-induced gene product refines the source of wg expression to shape a morphogen gradient. Refinement of wg expression is important to specify the structure of the wing margin. In dsh mutant clones that abut the D-V boundary, wg expression expands. The dve mutant clone encompassing the D-V boundary also allows the expansion of wg expression, as observed in dsh mutant clones. The action of Dve in the refinement of wg expression appears to attenuate N-mediated gene expression. How is the Dve function related to the Wg signaling cascade in this process? When dsh mutant clones are created so as to abut the D-V boundary, dve expression is still observed in such clones. These observations for dsh clones suggest that Dve activity in the absence of the Wg signal input is insufficient to refine wg expression. However, when dve mutant clones are created adjacent to the D-V boundary, wg expression is expanded within the clones, but Wg-dependent accumulation of Ser seems to be normal. Thus, the Dsh-mediated Wg signal also appears to be insufficient to refine wg expression in the absence of Dve. Taken together, both Wg signaling and Dve appear to be necessary for the refinement of wg. There might be some interaction between Dve and Wg signaling downstream of N (Nakagoshi, 2002).
N-mediated activation of wg together with Vg function is important for disc growth. In addition, repression of dve at the D-V boundary largely depends on N-mediated Wg signaling and is also crucial for disc outgrowth and patterning of wing discs. Complementary pattern of dve and wg expression at mid- to late third instar appears to be important for wing patterning. How do these events organize growth and patterning? Studies involving flip-out Nact clones might provide an insight in this issue. These experiments suggested that the two types of Nact clones arise from a difference in the level of N signaling within clones: lower N signaling-clones express dve but not wg and cut, and higher N signaling-clones express wg and cut but not dve. The second type of clone appears to mimic the situation at the D-V boundary. It is remarkable that the on and off states of dve expression within the clones are tightly correlated with the induction of N-target gene expression. Considering the ability of Dve to repress wg, this observation makes it possible to hypothesize a threshold of N-mediated signaling that defines both wg activation and subsequent dve repression; N-mediated signaling over this threshold can repress dve and results in the sustained expression of wg. Thus, it establishes a complementary pattern of dve and wg expression at the D-V boundary of wing discs at mid- to late third instar. This threshold also appears to define nonautonomous induction of cell growth. By utilizing the cold-sensitivity of Gal4 to drive gene expression, different levels of N signaling were induced. These experiments also suggest the notion that the level of N signaling that represses dve is important for disc outgrowth. Thus, the model assuming a threshold of the N-mediated signal repressing dve might provide a clue for understanding the coordination between cell growth and patterning through shaping of the Wg stripe (Nakagoshi, 2002).
The segmentation of the proximal-distal axis of the Drosophila leg depends on the localized activation of the Notch receptor. The expression of the Notch ligand genes Serrate and Delta in concentric, segmental rings results in the localized activation of Notch, which induces joint formation and is required for the growth of leg segments. This study reports that the expression of Serrate and Delta in the leg is regulated by the transcription factor genes dAP-2 and defective proventriculus. Previous studies have shown that Notch activation induces dAP-2 in cells distal and adjacent to the Serrate/Delta domain of expression. Serrate and Delta are ectopically expressed in dAP-2 mutant legs, and Serrate and Delta are repressed by ectopic expression of dAP-2. Furthermore, Serrate is induced cell-autonomously in dAP-2 mutant clones in many regions of the leg. It was also found that the expression of a defective proventriculus reporter overlaps with dAP-2 expression and is complementary to Serrate expression in the tarsal segments. Ectopic expression of defective proventriculus is sufficient to block joint formation and Serrate and Delta expression. Loss of defective proventriculus results in localized, ectopic Serrate expression and the formation of ectopic joints with reversed polarity. Thus, in tarsal segments, dAP-2 and defective proventriculus are necessary for the correct proximal and distal boundaries of Serrate expression and repression of Serrate by defective proventriculus contributes to tarsal segment asymmetry. The repression of the Notch ligand genes Serrate and Delta by the Notch target gene dAP-2 may be a pattern-refining mechanism similar to those acting in embryonic segmentation and compartment boundary formation (Ciechanska, 2007).
The expression of the Dve protein exhibits a pleiotropic pattern including three separate midgut domains: anterior-most, middle, and posterior-most regions. Because defects in a lethal dve allele are evidently observed in the gut, dve expression was examined during gut development. At stage 10, dve expression is first observed in the invaginated stomodeum, where hh and wg are expressed under the control of the winged-helix transcription factor Forkhead. Anti-Dve staining was compared with wg gene expression, which was monitored with wg-lacZ. The expression of dve and wg overlaps initially in the stomodeum, and is then segregated into nonoverlapping but adjacent regions; the mesoderm-free keyhole structure expresses wg; dve is solely expressed in the anterior-most region of the midgut, which becomes the outer layer of the proventriculus later, at stage 16 (Nakagoshi, 1998).
From stage 13 onward, dve is also expressed in two other parts of the endodermal midgut; one is the middle region where the anterior and posterior midgut primordia are fused, and the other is the posterior-most region. The middle region expressing dve corresponds to both the anterior and posterior sides of the middle constriction. At stage 16, this region becomes the second and third gut lobes. Dve-expressing cells in this region include copper cell precursors that coexpress labial (Nakagoshi, 1998).
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).
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).
The Drosophila male accessory gland has functions similar to those of the mammalian prostate gland and the seminal vesicle, and secretes accessory gland proteins into the seminal fluid. Each of the two lobes of the accessory gland is composed of two types of binucleate cell: about 1,000 main cells and 40 secondary cells. A well-known accessory gland protein, sex peptide, is secreted from the main cells and induces female postmating response to increase progeny production, whereas little is known about physiological significance of the secondary cells. The homeodomain transcriptional repressor Defective proventriculus (Dve) is strongly expressed in adult secondary cells, and its mutation resulted in loss of secondary cells, mononucleation of main cells, and reduced size of the accessory gland. dve mutant males had low fecundity despite the presence of sex peptide, and failed to induce the female postmating responses of increased egg laying and reduced sexual receptivity. RNAi-mediated dve knockdown males also had low fecundity with normally binucleate main cells. This study provides evidence that secondary cells are crucial for male fecundity, and also that Dve activity is required for survival of the secondary cells. These findings provide new insights into a mechanism of fertility/fecundity (Minami, 2012).
Mutant males for the longer transcript, dve-A, showed low fecundity together with loss of secondary cells and reduced size of accessory glands. It has been reported that greatly reduced size of accessory glands results in sterility, and also suggested that there is a minimum size to maintain fertility. If a male is selected for larger size of accessory glands with 16 generations, the selected males have good fecundity. However, they have only about 1.4-fold larger size compared to the control males, suggesting that there is also a maximal size of accessory gland not to waste energy. Thus, the size of accessory glands should be controlled in an appropriate range and binucleation seems to be the best strategy to provide highly plastic change of the siz. Although reduced size of dve-A mutant accessory glands may have some effects on fecundity, dve KD males had similar size of accessory glands to the dve-A heterozygous controls and dve KD males showed low fecundity with loss of secondary cells. Thus, it is most likely that the low fecundity in dve-A mutant males is due to the absence of mature secondary cells. This is consistent with independent findings that Abd-B is required for maturation of secondary cells and for maintaining female postmating response (personal communication to Minami from M. F. Wolfner and F. Karch). Although it cannot be excluded that some defects in dve mutant main cells affect the fecundity, SP was normally expressed and transferred into the female reproductive tract. Thus, the following mechanisms should be considered for SP activation to induce long-term postmating response; (1) secondary cell products cooperate in parallel with SP-mediated signaling; (2) secondary cell products enhance SP binding to its receptor; (3) secondary cell products stabilize SP binding to sperm; and (4) secondary cell products are involved in modification and/or stabilization of SP secreted from main cells. Interestingly, egg laying of females mated with dve mutant or dve KD males was gradually reduced over time, suggesting that the last two interpretations, stabilization of SP by secondary cell products, are more plausible. Identification of unknown factors secreted from secondary cells will provide new insights into a mechanism that is crucial for activation of seminal fluid functions (Minami, 2012).
It seems likely that Dve functions are crucial to inhibit cell death of secondary cells, and an intriguing possibility is that inactivation of the Dve activity is closely linked to the regulated cell death to adjust the number of secondary cells. Dve and the special AT-rich sequence binding proteins (SATBs) belong to the cut superclass of homeobox genes and have an evolutionarily conserved compass domain. It is reported that SATB1 is cleaved and inactivated by Caspase 6 in response to the apoptotic signaling pathway. Expression of the BCL2 gene, which is a key regulator to inhibit apoptosis, is finely tuned by a variety of stimuli and activated through SATB1-mediated chromatin looping. Thus, SATB1 is required for cell survival through inhibition of programmed cell death. The functional similarity between Dve and SATB1 for inhibition of cell death raises a possibility that an evolutionarily conserved CMP plays important roles to inhibit cell death. The CMP of SATB1 is characterized as a PDZ-like domain (amino acids 90 to 204) involved in protein-protein interaction, and the Caspase 6-dependent cleavage of SATB1 at amino acid position 254 disrupts the dimerization of SATB1. Further characterization of CMP-interacting proteins will clarify the underlying mechanism and provide new insights into a regulatory mechanism of fecundity/fertility (Minami, 2012).
Search PubMed for articles about Drosophila defective proventriculus
Ciechanska, E., Dansereau, D. A., Svendsen, P. C., Heslip, T. R. and Brook, W. J. (2007). dAP-2 and defective proventriculus regulate Serrate and Delta expression in the tarsus of Drosophila melanogaster. Genome 50(8): 693-705. PubMed Citation: 17893729
Fuß, B. and Hoch, M. (1998). Drosophila endoderm development requires a novel homeobox gene which is a target of Wingless and Dpp signalling. Mech. Dev. 79(1-2): 83-97. PubMed Citation: 10349623
Kölzer, S., Fuss, B., Hoch, M. and Klein, T. (2003). defective proventriculus is required for pattern formation along the proximodistal axis, cell proliferation and formation of veins in the Drosophila wing. Development 130: 4135-4147. 12874133
Minami, R., et al. (2012). The homeodomain protein defective proventriculus is essential for male accessory gland development to enhance fecundity in Drosophila. PLoS One 7(3): e32302. PubMed Citation: 22427829
Pankratz, M. J. and Hoch, M. (1995). Control of epithelial morphogenesis by cell signaling and integrin molecules in the Drosophila foregut. Development 121(6): 1885-1898. PubMed Citation: 7601002
Nakagoshi, H., et al. (1998). A novel homeobox gene mediates the Dpp signal to establish functional specificity within target cells. Genes Dev. 12: 2724-2734. PubMed Citation: 9732270
Nakagoshi, H., et al. (2002). Refinement of wingless expression by a Wingless-and Notch-responsive homeodomain protein, Defective proventriculus. Dev. Bio. 249: 44-56. 12217317
Shirai, T., et al. (2007). Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg. Dev. Biol. 312(1): 147-56. PubMed Citation: 17950268
Yorimitsu, T., Kiritooshi, N. and Nakagoshi, H. (2011). Defective proventriculus specifies the ocellar region in the Drosophila head. Dev. Biol. 356(2): 598-607. PubMed Citation: 21722630
date revised: 30 November 2012
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