defective proventriculus

Transcriptional Regulation

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 wg^CX4 null embryos, dve expression is abolished in the stomodeum, whereas epidermal expression is normally detected. In hh^AC 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 ci^D 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 ci^D 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 hh^IJ35 and wg^IL114 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).

Targets of Activity

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 (Tkv^Q253D) 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 (dTCF^DN) 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).

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