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Wingless targets in the gut (part 2/2)

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 defective proventriculus 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. 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 (Nakagoshi, 1998).

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. Because the dve-expressing domain in the presumptive proventriculus initially overlaps with the wg- and hh-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. 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).

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).

Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signaling from a specialized subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to secrete the EGF ligand, Spitz. The response to EGF signaling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signaling by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of pointedP2 is induced by Wingless signaling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. Both signaling and responsive cells are set aside in each tubule primordium from a proneural gene-expressing cluster of cells, in a two-step process: (1) a proneural cluster develops within the domain of Wingless-activated, pointedP2-expressing cells to initiate the co-expression of seven up; (2) lateral inhibition, mediated by the neurogenic genes, acts within this cluster of cells to segregate the tip cell precursor, in which proneural gene expression strengthens to initiate rhomboid expression. As a consequence, when the precursor cell divides, both daughters secrete Spitz and become signaling cells. Establishing domains of cells competent to transduce the EGF signal and divide ensures a rapid and reliable response to mitogenic signaling in the tubules and also imposes a limit on the extent of cell division, thus preventing tubule hyperplasia (Sudarsan, 2002).

It was therefore asked whether early expression of pnt as well as svp is required to prime the mitogenic response in tubule cells. pntP2 is initiated in the posterior side of each tubule during stage 10. This domain is characterized by high levels of wg expression, which are required for the normal development of AS-C expression in the PNC, during the time it develops within this domain. The domain of wg and pntP2 expression is slightly wider than the PNC and pntP2 expression is initiated well before Egfr activity is required for tubule cell divisions. The expression of pntP2 persists in this posterior domain when the tip mother cell is specified. In wgCX4 mutant embryos, tubule expression of pntP2 is completely abolished, showing that Wg signaling is required to initiate its expression. Conversely, the overexpression of wg, using a hs-wg construct, results in expansion of pntP2 expression to the anterior side of the tubule primordium and elevation of expression to high levels. Thus, Wg is necessary and sufficient to activate the expression of pntP2 in the tubules (Sudarsan, 2002).

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).

Endoreduplication cycles that lead to an increase of DNA ploidy and cell size occur in distinct spatial and temporal patterns during Drosophila development. Only little is known about the regulation of these modified cell cycles. Fore- and hind-gut development have been investigated and evidence is presented that the knirps and knirps-related genes are key components to spatially restrict endoreduplication domains. Lack and gain-of-function experiments show that knirps and knirps-related, which both encode nuclear orphan receptors, transcriptionally repress S-phase genes of the cell cycle required for DNA replication and that this down-regulation is crucial for gut morphogenesis. Furthermore, both genes are activated in overlapping expression domains in the fore- and hind-gut in response to Wingless and Hedgehog activities emanating from epithelial signaling centers that control the regionalization of the gut tube. These results provide a novel link between morphogen-dependent positional information and the spatio-temporal regulation of cell cycle activity in the gut Fuß, 2001).

The kni and knrl expression domains in the developing foregut and hindgut partially overlap with the expression domains of wingless and hedgehog, which define signaling centers that control morphogenetic movements during the regionalization of the gut. To investigate whether kni/knrl expression and consequently also the restriction of the endoreduplication pattern in the gut is coordinated the Wg and Hh signaling cascades, expression studies in various lack and gain-of-function situations were performed. In hh mutants, kni expression is only mildly reduced in the developing fore- and hind-gut expression domains. In early wg mutants, kni fails to be expressed in the esophagus primordium and is strongly reduced in the developing small intestine and rectum. wg mutant embryos lack a foregut at later stages and have a strongly reduced hindgut. Ectopic expression of hh in all the hindgut cells using the UAS-Hh effector and the 14-3fkh driver line does not alter the kni or knrl expression domains in the hindgut, even when the Hh dose is increased by using effector lines with multiple UAS-Hh transgene insertions. However, if the same experiment is carried out in engrailed mutants, kni/knrl can be induced ectopically in all the hindgut cells. In wild-type embryos, engrailed is expressed in the dorsal part of the large intestine and exerts a repressing function on kni/knrl expression that apparently cannot be overcome by ectopic Hh activity. However, ectopic wg expression in all the hindgut cells using the UAS-Wg effector and the 14-3fkh driver line does result in ubiquitous induction of kni and knrl expression. engrailed expression in the hindgut of these embryos is repressed under these conditions. To investigate whether ectopic Wg expression in the hindgut interferes with DNA replication activity required for endoreduplication, BrdU incorporation was examined. BrdU incorporation is absent in the hindgut of such embryos. Consistent with this result, S-phase genes such as RNR2 are transcriptionally repressed upon ectopic Wg expression in all the hindgut cells using the 14-3fkh-Gal4 driver and UAS-Wg. As has been observed for ectopic kni/knrl expression in the hindgut, the size of the hindgut cells are reduced in these embryos Fuß, 2001).

Table of contents

wingless continued: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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