wingless

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Wingless targets in the gut (part 1/2)

teashirt is necessary for proper formation of anterior and central midgut structures. ANTP activates tsh in anterior midgut mesoderm. In the central midgut mesoderm Ubx, abd-A, dpp, and wg are required for proper tsh expression. The control of tsh by Ubx and abd-A, and probably also by Antp, is mediated by secreted signaling molecules. By responding to signals as well as localized transcription regulators, the TSH transcription factor is produced in a spatial pattern distinct from any of the homeotic genes (Mathies, 1994).

Ultrabithorax and labial are a target of wingless signaling in the midgut. dishevelled, shaggy/zeste-white 3 and armadillo are required for transmission of the wingless signal in the Drosophila epidermis. These genes act in the same epistatic order in the embryonic midgut to transmit the wingless signal. In addition to mediating transcriptional stimulation of the homeotic genes Ultrabithorax and labial, they are also required for transcriptional repression of labial by high levels of wingless . Efficient labial expression thus only occurs within a window of intermediate wingless pathway activity. The shaggy/zeste-white 3 mutants reveal that wingless signaling can stimulate decapentaplegic transcription in the absence of Ultrabithorax, identifying decapentaplegic as a target gene of wingless. Since decapentaplegic itself is required for wingless expression in the midgut, this represents a positive feed-back loop between two cell groups signaling to each other to stimulate one another's signal production (Yu, 1996).

Drosophila wingless functions in the larval midgut, and acts at two different thresholds to pattern this tissue. Low wingless levels are required to promote the development of copper cells, highly differentiated cells of the larval midgut that are specified by the homeotic gene labial. In contrast, high wingless levels repress copper cell development and allow differentiation of an alternative cell type: large, flat cells. These two different developmental outcomes reflect labial expression, which is stimulated at low levels and repressed at high levels of wingless signaling. Thus, midgut cells respond differentially to distinct wingless thresholds in terms of both gene control and cellular differentiation (Hoppler, 1995).

In the embryonic midgut both Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways control the subcellular localization of Extradenticle protein. Exd protein is predominantly nuclear in endoderm cells close to the Dpp-and Wg-secreting cells of the visceral mesoderm, but is found in the cytoplasm in more distant endoderm cells. Both dpp and wg are required for the nuclear localization of Exd in the endoderm; ectopic expression of dpp and wg expands the domain of nuclear Exd (Mann, 1996). The requirement of homothorax for Exd's nuclear localization is apparent in many embryonic tissues, including the ectoderm, visceral mesoderm, and endoderm. This requirement is observed in cells where the signaling molecules Wg and Dpp contribute to Exd's nuclear translocation (e.g., the endoderm). It is suggested that Wg and Dpp both regulate expression of hth in the domains in which Exd nuclear function is required (Rieckhof, 1997).

Dpp has a prime function during endoderm induction in Drosophila. Dpp is secreted from the outer cell layer of the embryonic midgut (the visceral mesoderm), where Dpp's main source of expression in parasegment ps7 depends directly on the homeotic gene Ultrabithorax (Ubx). In the same cell layer, Dpp stimulates expression of another extracellular signal, Wingless (Wg), in a neighboring parasegment (ps8), which in turn feeds back to ps7 to stimulate Ubx expression. Thus, Dpp is part of a "parautocrine" feedback loop of Ubx (i.e., an autocrine feedback loop based partly on paracrine action) that sustains its own expression through Dpp and Wg. Dpp also spreads to the inner layer of the embryonic midgut, the endoderm, where it synergizes with Wg to induce expression of the homeotic gene labial (lab). To achieve this, Dpp locally elevates the endodermal expression levels of Drosophila D-Fos with which it cooperates to induce lab. Differentiation of various cell types in the larval gut depends on these inductive effects of Dpp and Wg. A cAMP response element (CRE) from the Ubx midgut enhancer has been shown to be necessary and to some extent sufficient to mediate the Dpp response in the embryonic midgut (Eresh, 1997).

CREs are known to be signal-responsive elements, not only for cAMP signaling as described initially but also for other signals including ones acting through Ras. This prompted an investigation of whether any other signal may play a part in the Dpp response. This led to the discovery that the Drosophila epidermal growth factor receptor (Egfr) has a critical function during endoderm induction. A secondary signal was discovered with a permissive role in this process, namely Vein, a neuregulin-like ligand that stimulates the epidermal growth factor receptor and Ras signaling. Dpp and Wg up-regulate vein expression in the midgut mesoderm in two regions overlapping the Dpp sources. This up-regulation depends on dpp and wg. Vein is thus a secondary signal of Dpp and Wg, and it stimulates homeotic gene expression in both cell layers of the midgut (Szuts, 1998).

EGFR expression is thought to be fairly ubiquitous in the embryo. However, vein transcripts are found in a highly restricted pattern, primarily in the embryonic mesoderm. In the midgut too, vein expression is spatially regulated, as follows: vein transcripts in the midgut are restricted to the visceral mesoderm. Initially, during stage 13, low levels of vein expression are seen at intervals throughout the midgut mesoderm. However, soon after the formation of the midgut epithelium, vein transcripts start to accumulate locally, and two main domains of prominent vein expression develop, one in the anterior and one in the middle midgut. Anteriorly, vein expression spans approximately ps2-ps4 and is strongest around the ps3/ps4 junction, that is, posterior to the gastric caeca. In the middle midgut, there is a fairly wide band of low vein expression spanning approximately ps6-ps10, with strongly up-regulated expression levels throughout ps7 (and trailing into anterior ps8). Posterior ps7 becomes the most prominent site of vein expression in the midgut. Finally, a narrow band with low levels of vein transcripts is seen at the posterior end of the midgut. The two main expression domains of vein overlap the two domains of Dpp expression in the visceral mesoderm (in ps3 and ps7), but each of them is considerably wider than the corresponding dpp domain. vein expression in the visceral mesoderm is severely diminished in dpp^s4 mutants. The prominent band of vein expression in ps7 is no longer seen, and expression in ps4 is reduced too. Instead, the strongest expression of vein in these mutants is seen at a novel location, at the ps5/ps6 junction around the incipient first midgut constriction (this ps5/ps6 expression is higher than in the wild type, and can be used to identify young dpp mutant embryos). It is concluded that dpp is required for the localized up-regulation of vein expression in the midgut (Szuts, 1998).

vein expression is also strongly diminished in wg mutants. vein expression can still be seen at moderate levels in the ps4 region, but vein expression is barely visible elsewhere in the midgut of these mutants. In particular, there are only traces of vein expression in the ps7/ps8 region, and expression at both midgut ends is almost undetectable. Clearly, wg plays an essential role as well in up-regulating vein expression. dpp and wg are sufficient to position the two domains of vein up-regulation. High mesodermal Wg causes very strong vein expression in ps2-ps7, significantly stronger than that caused in this region by mesodermal Dpp expression alone. This indicates that wg cooperates with dpp in positioning vein up-regulation. It is shown that neither Dpp for Egfr signaling is particularly effective in the absence of the other. Thus these two pathways are functionally interdependent and that they synergize with each other, revealing functional intertwining (Szuts, 1998).

The mutant analysis suggests strongly that Vein is the main, if not the only, ligand that stimulates Egfr in the embryonic midgut. This contrasts with other tissues, mainly of ectodermal origin, in which Spitz is the main Egfr ligand. Interestingly, Vein also has a major role during an inductive process between muscle and epidermis: Vein is secreted from muscle cells and triggers differentiation of the receiving epidermal cells into tendon cells. These functions of Vein during inductive processes between different cell layers suggest that the molecular properties of Vein are particularly suited to such processes that require the signal to cross basal membranes. Similarly, the extensive mesodermal expression of Vein may mean that this signal protein is particularly well-adapted to its production in this cell layer. Note that Vein is similar to mammalian neuregulins that appear to function in developmental contexts that involve communication between different cell layers (Szutz, 1998 and references).

The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras, the secondary signal of Dpp. It is also shown that the Dpp response element in the labial enhancer comprises CREs and Mad binding sites. The results with the labial enhancer confirm the conclusions derived from the Ubx enhancer, namely that the response element to Dpp signaling is bipartite and contains Mad binding sites as well as CREs. The latter are critical in both cell layers for the signal response, whereas the former seem less criticial in the endoderm than in the visceral mesoderm. Perhaps this reflects the fact that lab is the ultimate target gene of the endoderm induction and that its enhancer clearly integrates a number of distinct positional inputs, some of which may be partially redundant (Szuts, 1998).

Why should there be this secondary signal whose role is entirely permissive, namely to assist the primary signal in implementing its tasks? Two kinds of answers are proposed. The first one is based on the observation that lack of Vein/Egfr signaling in the midgut appears to make cells sick and perhaps causes them to die. Therefore, Vein/Egfr signaling may serve as a "survival signal." Intriguingly, cell survival in embryos lacking vein or Egfr function appear to be affected preferentially near the two Dpp sources (where vein expression is up-regulated). Perhaps high levels of Dpp signaling can cause cell death; if so, vein signaling may be up-regulated to counteract a putative local deleterious effect of Dpp. A precedent for such a scenario may be found in the developing chick limb bud where the cell death-inducing properties of BMP (a TGF-beta-like signal) seem to be antagonized locally by a signal triggering the Ras pathway. However, although antagonistic effects between Egfr- and TGF-beta-type signaling have been observed, the evidence provided here suggests strongly that Vein/Egfr and Dpp both act positively in the embryonic midgut of Drosophila. Furthermore, they synergize with each other in the transcriptional stimulation of target genes. This observed synergy parallels cooperation between Ras and TGF-beta signaling during epithelial tumor progression. It is therefore thought unlikely that Vein functions in the midgut entirely as a survival signal near Dpp sources (Szuts, 1998).

The second kind of answer builds on the observations that indicate functional interdependence and synergy between the two signaling pathways in stimulating transcription of target genes. This could be beneficial for developmental systems in two ways: (1) if cells need to be costimulated by cooperating primary and secondary signals, this would serve to sharpen their signal response. This putative sharpening effect may be a contributory factor in sharp responses to signaling thresholds such as those observed in the Xenopus embryo.(2) The need for costimulation would safeguard against fortuitous and random stimulation of cells by any one signal, thus improving the reliability of their signal response. And although a requirement for the secondary signal is observed throughout the functional realm of the primary signal, it is envisaged that the role of the secondary signal is particularly critical in remote cells where the distribution of the primary signal becomes shallow, imprecise, and unreliable. Therefore, the secondary signal may provide primarily "remote stimulation." Whatever the case, it seems very likely that the use of a functionally coupled primary-secondary signal system results in a refinement and stabilization of positional information and in a degree of precision of this information that could not be conferred by one signal alone. Functional intertwining of a secondary and a primary signal may represent a mechanistic solution of how morphogens such as Dpp and activins work. Perhaps, signaling pathways do not function on their own in eliciting multiple different cellular responses, as envisaged by the purest version of the morphogen concept (Szuts, 1998).

Extracellular signals can act at different threshold levels to elicit distinct transcriptional and cellular responses. The transcriptional regulation of the Wingless target gene Ultrabithorax has been examined in the embryonic midgut of Drosophila. Ubx transcription is stimulated in this tissue by Dpp and by low levels of Wingless signaling. High levels of Wingless signaling can repress Ubx transcription. The response sequence within the Ubx midgut enhancer required for this repression coincides with a motif required for transcriptional stimulation by Dpp, namely a tandem array of binding sites for the Dpp-tranducing protein, Mad. Indeed, Wingless-mediated repression depends on low levels of Dpp, although apparently not on Mad itself. In contrast, high levels of Dpp signaling antagonize Wingless-mediated repression. This suggests that transcriptional activation of Ubx is subject to competition between Dpp-activated Mad and another Smad whose function as a transcriptional repressor depends on high Wg signaling. Wingless can repress its own expression via an autorepressive feedback loop that results in a change of the Wingless signaling profile during development (Yu, 1998).

Dpp and Wg signaling synergize in the visceral mesoderm to stimulate Ubx transcription, targeting distinct, albeit adjacent, response sequences in the Ubx midgut enhancer. Therefore, efficient stimulation of Ubx transcription by Wg depends on dpp. Wg-mediated repression also depends on dpp, but, remarkably in this case, the response sequence for Wg-mediated repression within the Ubx enhancer coincides with that for Dpp-mediated stimulation. Indeed, the WRS-R/DRS (Wingless response sequence mediating repression and Dpp response sequence) functions in two antipodal responses: it mediates efficient transcriptional stimulation when the signaling levels of Dpp are high and those of Wg are low, but it is also required for transcriptional repression when the Wg signaling levels are high and those of Dpp are low. This raises the possibility that the same factor may confer the two antipodal responses. However, this is unlikely to be the case since Mad itself, which binds to the DRS to mediate the positive response to Dpp, is apparently not required for the Wg-mediated repression (Yu, 1998).

Thus it is proposed that the two antipodal responses are conferred by two distinct factors: by Mad and by a hypothetical protein WR. It is further proposed that WR is a Mad-related protein, i.e. a Smad, since WR acts through Mad-binding sites and since its function as a repressor depends on dpp. It is envisaged that WR, like Mad itself and other Smads, is activated by Dpp signaling through phosphorylation by ligand-bound membrane receptors, an event that promotes their subsequent translocation to the nucleus. In this scenario, Dpp enables WR (which also needs to be activated by high Wg signaling) to occupy the Mad-binding sites within the Ubx enhancer. Once bound to this enhancer, WR dominantly represses Ubx transcription, overriding the activating function of Arm-Pangolin and other transcriptional activators bound to the same enhancer (Yu, 1998).

How is WR's repressor function activated by high Wg levels? It is presumed that high Wg signaling regulates, directly or indirectly, the availability of WR as an enhancer-binding protein: either high Wg signaling controls a post-transcriptional event (e.g. it may promote WR's association with Armadillo, or WR's translocation into the nucleus), or it simply activates transcription of the WR gene. The latter possibility of indirect regulation, which involves transcriptional coupling, is favored because it accomodates readily the dependence of Wg-mediated repression on arm and Pangolin. Whatever the case, it is emphasized that high Wg signaling controls the activity of the protein WR (possibly a Smad), which also requires Dpp signaling. Thus, WR is a common target for two signaling pathways and represents a point of convergence between them (Yu, 1998).

This model readily explains how high Dpp levels antagonize WR, namely by promoting maximal levels of nuclear Mad which now competes with WR for binding to the Ubx enhancer. The outcome of this competition is the transcriptional activation or repression of target genes, depending on the prevalence of Mad or WR. This may illustrate a general principle, namely that the response sequence for the positive effect of one signal is also the response sequence for the negative effect of an antagonistic signal. Such a layout provides a sharp flipping of the response from positive to negative in an area where cells are experiencing increasingly more of one signal and increasingly less of the antagonizing one (Yu, 1998).

Medea is the Smad4 homolog that is known to be the common oligomerization partner for pathway-specific Smads. Furthermore, Medea binds to the same DNA sequences as Mad. This raises the possibility that Medea is an oligomerization partner of WR: while Medea, together with Mad, is expected to activate transcription, together with WR it may repress transcription. A precedent for this scenario is the Myc/Mad/Max system, in which Mad (a bHLH protein that happens to have the same name as the Dpp transducer Mad) is a common dimerization partner for either Myc, a transcriptional activator, or Max, a transcriptional repressor. In addition to antagonism, there is also synergy between Wg and Dpp in the embryonic midgut. This synergy apparently results from cooperation between the nuclear target factors activated by the two signals, i.e. between Arm-Pangolin and Mad/CRE-binding proteins. Other examples of apparent synergy between Wg and Dpp are the leg and wing imaginal discs, where these signals act together in central disc regions to stimulate expression of homeobox genes. But the two signals also antagonize each other in leg discs, as well as in eye discs. Although it is conceivable that the developmental context determines the synergy or antagonism between Dpp and Wg, the situation in the midgut suggests that the decisive factor in each case may be the levels of signaling (Yu, 1998 and references).

It is interesting that Wg signaling can repress its own expression when signaling levels reach a critically high level. This indicates a negative feedback loop, which could account for two observations: (1) Wg signaling shifts its own expression towards the anterior over time. It is not known at present whether this shift has any biological significance. (2) Wg has the potential for switching itself off over time. This is actually observed, since Wg expression becomes undetectable by the end of embryogenesis. Clearly, Wg's negative feedback loop is capable of changing the Wg signaling profile as development procedes. There are negative feedback loops for other signaling pathways in Drosophila. For example, the epidermal growth factor (EGF) receptor inhibits itself eventually, after signaling has reached a critical level, by switching on expression of an inhibitory ligand, Argos. In the ovary, this negative feedback loop causes splitting of a single signaling peak into twin peaks. Furthermore, Hedgehog signaling in the eye imaginal disc is repressive at high Hedgehog levels, but stimulatory in cells, further away from the signaling source, which experience lower Hedgehog levels. Perhaps such 'hard-wired' negative feedback loops in signaling pathways are fairly universal, and serve to stop these pathways from escalating out of control. If so, this would be akin to feedback inhibition of metabolic pathways, which provides homeostatic control (Yu, 1998 and references).

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