Table of contents

Wingless and segmentation

Specialized groups of cells known as organizers govern the establishment of cell type diversity across cellular fields. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for organizer function. Here cells differentiate into smooth cuticle or distinct denticle types. At parasegment boundaries, cells expressing Wingless confront cells co-expressing Engrailed and Hedgehog. While Wingless is essential for smooth cell fates, the signals that establish denticle diversity are unknown. wg mutants are shown to have residual mirror-symmetric patterns that are due to an Engrailed-dependent signal specifying anterior denticle fates. The Engrailed-dependent signal acts unidirectionally and Wg activity imposes this asymmetry. Reciprocally, the Engrailed/Hedgehog interface imposes asymmetry on Wg signaling. Thus, a bipartite organizer, with each signal acting essentially unidirectionally, specifies segmental pattern (Gritzan, 1999).

Reciprocal signaling between Wg- and En/Hh-expressing cells stabilizes one another’s expression, consolidating the parasegmental body plan. At this time, signaling is effective only locally, thereby restricting the expression of the other signal to a narrow strip of cells. This ensures that the bipartite organizer remains a line source rather than broadening during patterning. Ventrally, the organizer specifies half the fates as smooth cell types and compelling evidence demonstrates that Wg specifies these. This step occurs after Wg stabilizes En/Hh expression. In contrast, the identity of the signals responsible for specifying the diverse denticle cell types has been less clear. By bypassing the need for Wg input to En cells, row 2- to 4-type denticles are specified in the absence of Wg. This conclusion was confirmed by analyzing the control of Rhomboid expression in cells flanking the En domain, since Rhomboid is essential for the proper differentiation of row 1- 4 fates. Previous studies have indicated that denticle diversity arises early, around the time Wg stabilizes En/Hh expression. The data suggest that the stabilization of En/Hh expression establishes the conditions to generate denticle diversity, but that diversity is not specified until later as reflected in the induction of Serrate and Rhomboid (~7-8 hours AEL). Indeed, excess Hh delivered at late times can broaden Rho stripes and this still affects denticle diversity. Due to a lack of molecular markers, the exact posterior boundary of the En/Hh-dependent domain is unclear. However, since row 5 cell types are specified in embryos lacking all Hh/Smo signaling, En/Hh influence can only extend up to row 4. Consistent with this, Hh signaling sets the anterior Serrate expression boundary. Since Wg signaling sets Serrate’s posterior boundary, the Serrate domain defines a region of positional values within the segment where Hh and Wg cooperate in patterning. In fact, Serrate expression is perhaps a molecular marker for a default state, as its expression is almost global in cases where only row 5 cell types are specified, such as in wg;en doubly mutant embryos. Serrate would indeed be globally expressed in a wg;en;hh triple mutant (Gritzan, 1999).

The data suggest that both Wg- and the En/Hh-expressing cells establish a block so that each signal operates largely unidirectionally. Rho is repressed by Wg signaling and it is important to block activation of the Wg pathway from cells posterior to the En/Hh domain. This block seems to be Hh-dependent because Rho expression is greatly reduced in hh mutants but maintained in wg;smo double mutants. Also, Wg imparts asymmetry to signals from En cells. Without Wg function, Hh can signal more strongly to the anterior, as compared to wild type. Importantly, the cuticle pattern generated is also now symmetric relative to the En/Hh cells, strongly suggesting that a normally biased signal is now sent or received bidirectionally (Gritzan, 1999).

The signals expressed from an organizer are developmentally potent, because they confer pattern over a large cellular field. Thus, once the appropriate expression of these signals is established, an important facet to organizer function is the temporal and spatial restriction of signaling. In some cases, activation of a signaling pathway induces an inhibitor of that same pathway. For example, inhibition of signaling is crucial for proper fate specification by this parasegment organizer. In examining fate across the En domain, Rho-dependent activation of the Egfr pathway posterior to the En cells leads to the induction of the diffusible inhibitor, Argos. Argos attenuates Egfr activation in anterior En cells, allowing Wg signaling to win out, leading to proper fate specification of anterior En cells (Gritzan, 1999).

In discs, signals emanate from compartment boundaries, which are inherited from the embryonic parasegment boundaries. For the compartment organizer, Hh locally induces a line source for a long-range morphogen, either Wg or Dpp, which each act symmetrically. Cells exposed to the same ligand concentration on opposite sides of the source, adopt the same positional value. However, the anterior compartment cell will select a different fate from the posterior compartment cell at the same positional value. This is because the posterior compartment expresses a unique transcription factor, En, and therefore is programmed intrinsically with a different response repertoire to the morphogen. In parasegments, 10 of 12 rows of cells are intrinsically equivalent anterior compartment cells, while the posterior En-expressing compartment only accounts for 2 cells. Thus, compartmental organization, with each compartment sporting unique transcription factors, can only make a small contribution toward distinguishing cell fate selection. Perhaps for this reason patterning cannot rely on induction of one longer-range morphogen. Instead, a bipartite organizer is used with each signal acting essentially unidirectionally. Equivalent cells to each side of the parasegment boundary develop differently because they are exposed to different signals (Gritzan, 1999).

The secreted proteins Wingless and Hedgehog are essential to the elaboration of the denticle pattern in the epidermis of Drosophila embryos. Signaling by Wingless and Hedgehog regulates the expression of veinlet (rhomboid) and Serrate, two genes expressed in prospective denticle belts. Thus, Serrate and veinlet (rhom) partake in the last layer of the segmentation cascade. Ultimately, Wingless, Hedgehog, Veinlet (an indirect activator of the Egfr) and Serrate (an activator of Notch) are expressed in non-overlapping narrow stripes. The interface between any two stripes allows a reliable prediction of individual denticle types and polarity, suggesting that contact-dependent signaling modulates individual cell fates. Attributes of a morphogen can be ascribed to Hedgehog in this system. However, no single morphogen organizes the whole denticle pattern (Alexandre, 1999).

Both Wingless and Hedgehog signaling pathways repress Serrate expression. Since both pathways are believed to activate transcription, it is imagined that they activate the expression of a repressor of Serrate. In addition, Serrate may also be negatively regulated by the transcriptional repressor Engrailed. In contrast to Serrate, veinlet is regulated both positively and negatively: it is repressed by Wingless and activated by Hedgehog. In addition to this vertical flow of information, regulatory interactions also exist between veinlet and Serrate. At the least, Serrate activates veinlet expression by way of the Notch pathway. This effect is purely non-cell autonomous. In contrast, Serrate appears to repress veinlet in a cell autonomous manner (indeed, in cells where it is expressed, Serrate represses the Notch pathway). However, it is also possible that whichever mechanism activates Serrate expression also represses veinlet expression. This would explain why the expression of Serrate and veinlet is always mutually exclusive (Alexandre, 1999).

The regulatory interactions summarized above are sufficient to explain the spatial pattern of both Serrate and veinlet expression. Non-autonomous repression of Serrate by Wingless and Hedgehog ensures that Serrate is expressed in stripes. The spread of Wingless toward the anterior defines the posterior edge of the domain of Serrate expression. Similarly, the anterior edge of the Serrate domain appears to be specified over three cell diameters by Hedgehog slightly further than expected since Hedgehog is thought to act only over 1-2 cells in Drosophila embryos. Expression of veinlet is activated by two different signals, Hedgehog at the anterior and Serrate at the posterior. Although Hedgehog signaling is symmetrical, it does not activate veinlet (rhom) expression anteriorly because there, Wingless represses veinlet expression. Likewise, Serrate activates veinlet expression but only on one side because of unilateral repression by Wingless (Alexandre, 1999).

These interactions display a clear temporal hierarchy. The secreted molecules Hedgehog and Wingless are expressed first and where they do not reach, Serrate expression is subsequently allowed. At stage 11, Hedgehog and Serrate activates veinlet expression in separate cells. Ultimately, this chain of interactions results in detailed patterns of gene expression (Alexandre, 1999).

Mapping the expression pattern of various genes onto the denticle pattern suggests simple correlations. These correlations have allowed the visualization of pattern where it was previously thought there was none, as in wingless mutants. It is now believed that wingless mutants make denticle type 3, 4 and 5 and not exclusively type 5 as has been suggested. The correlations provide a guide to understand various phenotypes such as those of patched mutants and wg-en-double mutants. In wg-en-double mutants, the correlation between gene expression and denticle type/polarity is particularly evident. Expression of veinlet is in circles surrounded by Serrate expression; this correlates with polarity reversal in the cuticle. Non-uniform gene expression shows that these embryos have more pattern than previously noted. For such embryos to be truly unpatterned, they would have to express Serrate uniformly as well as not express veinlet (rhom). This may occur in wg-en-hh- triple mutants since they may not contain any repressor of Serrate. It is presumed that the converse situation (Serrate 'off'and veinlet 'on' everywhere) would also lead to unpatterned embryos. This situation would prevail in wg-ptc-en-triple mutants. Although the correlations have good predictive value, they suffer from several limitations. (1) Denticle shape does not necessarily reflect an integer value. Indeed, unambiguous typing is not always possible and exact denticle shapes vary from segment to segment. (2) Causal relationships between the activation of a particular signaling pathway and a given denticle type still remain to be investigated. The various signaling pathways are predicted to control cytoskeletal behavior, which in turn affects denticle shape and cell polarity. Local polarity reversals indicate that individual cells are able to locate the source of a particular signal, suggesting that subcellular signaling complexes control the cytoskeleton directly. (3) The involvement of additional regulators cannot be excluded. In particular, it is possible that redundant regulators of the Notch and Egfr pathway contribute to the choice of denticle type. These could include Vein (another Egfr ligand), Delta (a Notch ligand) or possibly Fringe. vein is not required for embryogenesis suggesting that it does not play an important role if any. Possible contributions from Delta to denticle patterning are not readily assessed because of Delta’s earlier action in neurogenesis (Alexandre, 1999).

These results show that no single morphogen organizes the denticle pattern: patterning arises, at least initially, from the combined actions of Wingless and Hedgehog. Wingless is clearly not involved in the specification of denticle types (or diversity) across each belt since it does not act in this region of the epidermis. If it did, veinlet and Serrate would not be expressed because, as has been shown, they are both repressed by Wingless. Nevertheless, Wingless acts at a distance, over 3- to 5-cell diameters to set the boundaries of the Serrate expression domain and thus establishes conditions for subsequent juxtacrine signaling. Long-range Wingless action is also required for the asymmetric action of Serrate: Serrate does not activate veinlet (rhom) expression posteriorly because of the presence of Wingless there, 3- to 5-cell diameters from the site of wingless transcription. In this sense, Wingless modulates, at a distance, the outcome of local signaling. In neither of these activities is there evidence for concentration-dependent signaling. However, one cannot formally exclude the possibility that the specification of type 6 denticle requires low-level Wingless. Furthermore, the suggestion that Wingless is not a morphogen in the embryonic epidermis is at odds with studies of the first thoracic segment where various levels of Wingless signaling lead to the specification of distinct cuticular structures. Re-assessment of these phenotypes with early molecular markers might tell whether or not Wingless acts directly in a concentration-dependent manner in the embryonic epidermis (Alexandre, 1999).

The situation with Hedgehog is clearer since it has qualitatively distinct effects over a narrow strip of cells. It activates veinlet expression in adjoining posterior cells while its repressive effect on Serrate expression extends over three cell diameters. This suggests that, at high level, Hedgehog activates veinlet (near the source) while at both low and high levels it repress Serrate expression (further away from the source). In this sense, Hedgehog qualifies as a morphogen. Whether differential responses at different distances from the Hedgehog source reflect true concentration dependence remains to be assessed. It is noted that the repressive effect of Hedgehog on Serrate expression might take place early in development since, in wingless mutants, hedgehog expression decays around stage 10 and yet Serrate expression is still confined at the anterior. It is suggested that early Hedgehog has a repressive effect on Serrate expression that lasts at least until stage 11, when veinlet expression commences. It is therefore conceivable that the 3-cell-wide domain where Serrate is repressed at stage 11 originates by cell proliferation from a single row of cells that abut the Hedgehog source at early embryonic stages. According to this scenario, the effects of Hedgehog on Serrate and veinlet expression would both be occurring over one cell diameter. The apparent difference in range would reflect the difference in timing between these two effects and the intervening proliferation. This model is being tested by assessing the activity of a membrane-tethered form of Hedgehog (Alexandre, 1999).

To sum up, in the bald area of abdominal segments, one cell type forms in response to one signaling pathway while within denticle belts, a rich pattern of cell types arise from juxtacrine cell interactions initiated by the activation of distinct signaling pathways. Some of these pathways are controlled by the localized expression of segment polarity genes such as wingless and hedgehog, while others are regulated by downstream genes like veinlet and Serrate. Because wingless and hedgehog are expressed first, they are effectively at the top of the hierarchy and the knock-on effects of losing hedgehog or wingless function explain the 'organizer activity' of the parasegment boundary. Interestingly, the denticle Hedgehog originating from the parasegment boundaries of adjacent segments (and therefore, two parasegment boundaries) are needed to provide the signals that pattern a single denticle belt (Alexandre, 1999).

A test was performed to see whether naked cuticle nkd is regulated by Wg activity using gain- and loss-of-function experiments. In wg mutant embryos, nkd transcription initiates normally but is markedly reduced by stage 11. nkd transcript accumulates to higher levels and more broadly across the segment in nkd mutant embryos, presumably owing to the lack of negative feedback that Nkd protein normally provides to its own Wg-dependent expression. nkd expression is enhanced when Wg is ubiquitously expressed. Misexpression of either Wg or an activated form of the wg-signal transducer Armadillo (UAS-Arm S10) in wing, leg, haltere and antennal imaginal discs results in similar patterns of ectopic nkd transcription. ArmS10-induced nkd transcript obeys sharp boundaries consistent with a cell-autonomous induction of nkd by Wg (Zeng, 2000).

The abdomen of adult Drosophila consists of a chain of alternating anterior (A) and posterior (P) compartments which are themselves subdivided into stripes of different types of cuticle. Most of the cuticle is decorated with hairs and bristles that point posteriorly, indicating the planar polarity of the cells. This study has focused on a link between pattern and polarity. Previous studies have shown that the pattern of the A compartment depends on the local concentration (the scalar) of a Hedgehog morphogen produced by cells in the P compartment. Evidence is presented in this study that the P compartment is patterned by another morphogen, Wingless, which is induced by Hedgehog in A compartment cells and then spreads back into the P compartment. Both Hedgehog and Wingless appear to specify pattern by activating the optomotor blind gene, which encodes a transcription factor. A working model that planar polarity is determined by the cells reading the gradient in concentration (the vector) of a morphogen 'X' which is produced on receipt of Hedgehog, is re-examined. Evidence is presented that Hedgehog induces X production by driving optomotor blind expression. X has not yet been identified and data is presented that X is not likely to operate through the conventional Notch, Decapentaplegic, EGF or FGF transduction pathways, or to encode a Wnt. However, it is argued that Wingless may act to enhance the production or organize the distribution of X. A simple model that accommodates these results is that X forms a monotonic gradient extending from the back of the A compartment to the front of the P compartment in the next segment, a unit constituting a parasegment (Lawrence, 2002).

In clones mutant for arm or arrow, the expectation was that the Wg pathway in these two types of clones would be blocked. Two effects were noted. (1)The clones in the dorsal epidermis differentiated cuticle characteristic of the ventral epidermis: they made pleural hairs, and patches of sternite. Clones in all portions of the tergite, in both the A and P compartments, were transformed in this manner, indicating a general requirement for Wnt signaling to specify dorsal as opposed to ventral structures. Thus, in the wild type, all dorsal cells are probably exposed to at least low levels of Wg or some other Wnt protein. (2) Such clones affect polarity: in the tergites, the mutant clones were normal at the rear of the clone but reversed in the front, with reversal extending outside the clone. One explanation for these polarity changes could be that, in the tergites, Wg normally acts to enhance the production of X. Thus cells deficient in the Wnt pathway would produce less X than normal, giving a dip in the concentration landscape for X, causing reversed polarity at the front of the clone. In the eye, both arm- and arrow- clones cause equivalent polarity reversals and a similar resolution has been offered: it is suggested that Wg might regulate the production of a secondary polarizing factor also dubbed X (Lawrence, 2002).

Thus, it is proposed that Wg helps to produce X, but that Wg itself is not X. If Wg were X, both arm- and arrow- clones should not be able to transduce it, and hence, should have random polarity within the clone. Moreover, the effects on polarity should be cell autonomous. Yet, as has been seen, these clones behave as if they have caused an altered distribution of X, rather than any failure to transduce X. Similar arguments apply to sgg- clones. In this case, the Wg pathway should be constitutively activated in all cells within the clone, preventing them from detecting a gradient of Wg protein. However such clones are not randomly polarized, indicating that they can still respond to graded X activity (Lawrence, 2002).

It is useful to compare the roles of Omb and Wg on X production. Omb is apparently essential for X production: omb- clones at the back of A show reversed polarity that extends all the way to the posterior edge of the compartment. By contrast, in arm- and arrow- clones, reversal occurs only in the anterior portions of such clones. Thus, it is inferred that arm- and arrow- cells located at the back of A can produce some X, even though they cannot activate the canonical Wnt pathway. Thus, it could be that Hh drives X production mainly through Omb, but also adds to the level of X produced through the induction and action of Wg. The combination of both Omb and Wg activity might extend the reach of the X gradient to encompass the whole A compartment, and possibly also further forward into the neighboring P compartment (Lawrence, 2002).

None of the previous studies has helped gain an understanding of how the P compartment is patterned or how its cells are polarized. smo- clones have no phenotype in the P compartment, confirming that Hh has no function there. In the embryo and imaginal discs, Hh crossing over from the P compartment induces the expression of Wg and Dpp in line sources along the back of A. Both proteins then spread back into the P compartment where they act as gradient morphogens to control P growth and pattern. Wg and Dpp are also produced at the back of the A compartment in each abdominal segment (albeit in distinct dorsal and ventral domains). Hence, by analogy with the embryo and imaginal discs, these morphogens seem to be the most likely candidates to pattern the P compartment here as well. If so, it would be supposed that in the tergites, Hh induces Wg and this Wg moves posteriorly across the AP compartment boundary into the P compartment where it activates expression of omb, thus specifying the zone of hairy cuticle (p3) and distinguishing it from p2 cuticle, which is bald. This hypothesis was tested in the following experiments (Lawrence, 2002).

If Wg activates omb in anterior regions of the P compartment, blocking the Wnt pathway in cells in the P compartment should block expression of omb. Expression of omb was therefore monitored in arrow- clones. omb is sometimes, but not always, turned off autonomously in the clone. Conversely, ectopic activation of the Wnt pathway should transform bald cuticle (p2) at the back of P into hairy cuticle (p3) normally found at the front of P. Indeed, some clones lacking the sgg gene become hairy if situated in the bald areas of P, apparently causing a transformation from p2 to p3 cuticle. But, clones expressing either tethered Wg or activated Arm, which should behave similarly, have no clear effects. Even so the positive results with arrow and sgg give support to the hypothesis that Wg stratifies the P compartment by working through Omb (Lawrence, 2002).

Body structures of Drosophila develop through transient developmental units, termed parasegments, with boundaries lying between the adjacent expression domains of wingless and engrailed. Parasegments are transformed into the morphologically distinct segments that remain fixed. Segment borders are established adjacent and posterior to each engrailed domain. They are marked by single rows of stripe expressing cells that develop into epidermal muscle attachment sites. The positioning of these cells is achieved through repression of Hedgehog signal transduction by Wingless signaling at the parasegment boundary. The nuclear mediators of the two signaling pathways, Cubitus interruptus and Pangolin, function as activator and symmetry-breaking repressor of stripe expression, respectively (Piepenburg, 2000).

A cis-acting element of stripe has been identified that specifically directs gene expression in segment border cells during embryogenesis. This element was used to illuminate the molecular mechanism underlying segment border selection. The results show that Hedgehog (Hh) signaling can activate gene expression in two rows of cells, one on each side of the engrailed (en) expression domain. However, anterior Hh signaling causes the maintainance of wingless expression anterior to the PS boundary. Wg in turn antagonizes Hh-dependent gene expression and thereby prevents the formation of segment border cells anterior to the en domain. Hh and Wg activities relevant for the selection of segment border cells are mediated by functional binding sites of their nuclear mediators, Cubitus interruptus (Ci) and Pangolin (Pan), respectively within the sr cis-acing element. The data suggest that the segment border is established in response to the asymmetry of Wg signaling at the PS boundary (Piepenburg, 2000).

How repeating striped patterns arise across cellular fields is unclear. To address this the repeating pattern of Stripe (Sr) expression across the parasegment (PS) was examined in Drosophila. This pattern is generated in two steps. Initially, the ligands Hedgehog (Hh) and Wingless (Wg) subdivide the PS into smaller territories. Next, the ligands Hh, Spitz (Spi), and Wg each emanate from a specific territory and induce Sr expression in an adjacent territory. The width of Sr expression is determined by signaling strength. Finally, an enhancer trap in the sr gene detects the response to Spi and Wg, but not to Hh, implying the existence of separable control elements in the sr gene. Thus, a distinct inductive event is used to initiate each element of the repeating striped pattern (Hatini, 2001).

The repeating pattern of Stripe (Sr) expression across the parasegment (PS) is generated by inductive inputs from three spatially localized ligand sources. The ligands, Hh, Spi, and Wg, emitted by En, Ve, and Wg territories, respectively, control Sr expression in cells adjacent to each ligand source. There are three notable features to this regulation: (1) each ligand-producing territory induces Sr expression in the adjacent territory; (2) the induction is asymmetric, either anterior or posterior to each source; (3) the induction is initiated at the high level of signaling achieved near the source, limiting expression of Sr to a narrow row of cells. Because these same ligands act more broadly in cuticle cell fate specification, these results also suggest that the ligands and signaling territories operate in a fundamentally distinct way in order to construct a repeating striped pattern. These observations reveal a strategy used to generate a repeating striped pattern across a cellular field that may be used generally (Hatini, 2001).

Each Sr row is initiated adjacent to a different ligand source. The induction of Sr was limited to a narrow row of cells at each position. Manipulating either the ligand level, or the sensitivity of cells to a specific signaling pathway, leads to a broadened territory of Sr induction. Thus, local activity gradients of Hh, Spi, and Wg are each generated, and a threshold for activation of Sr is only surpassed in cells adjacent to each source. The gradient landscape of Spi and Wg is sculpted using the inducible antagonists Argos and Naked, respectively. Although how the activity landscape for Hh is sculpted was not specifically addressed, the Hh pathway also makes use of an inducible antagonist. It is likely that Hh spread is limited by binding to the Hh receptor Ptc, which is upregulated by Hh input (Hatini, 2001).

To generate the repeating striped tendon pattern, the Sr gene must be able to respond to each of three different ligands. To account for this, it is expected that the Sr promoter is modular, and each Sr row is induced via a separable, cis-acting response element. An enhancer trap P-insertion in the sr gene (sr03999) provides evidence for this since it detects the response to Spi and Wg, but not to Hh, implying that the P-insertion separates response elements in the sr gene. A separable Sr promoter element controlling Hh-dependent expression has been identified. Although this element operates only in dorsal and lateral epidermis, and not ventrally where Sr is expressed in repeating striped pattern, this observation strongly suggests that the control elements will be modular. Furthermore, in this dorsal/lateral element, the presence of functional, consensus Cubitus interruptus (Ci) DNA binding sites suggests direct regulation of Sr by the Hh signaling pathway. The obvious analogy is to the modularity of regulatory regions of certain pair-rule genes, which are able to integrate non-periodic information in order to generate periodicity. Note that the induction of the sr gene is limited to cells bordering each ligand source, even though each of the signals can act across several cell diameters. It is predicted that a given Sr response-element is configured to sense and respond only to a particularly high threshold level of each ligand (Hatini, 2001).

The ligands controlling Sr expression emanate from each of three territories across the PS. These territories are established by the primary organizing signals, Wg and Hh. In the earliest step, cross regulation between Wg and En/Hh-expressing cells stabilizes each ligand's expression and consolidates these two territories. In addition, through negative regulation, both Wg and Hh limit the expression of Ser to a central territory within the PS. Finally, signals from the En/Hh territory induce Ve expression in two cell rows just posterior to the En/Hh territory. The exact width of the Ve-expressing territory is adjusted as local input from the Ser territory induces a third Ve-expressing cell row. Thus, Hh and Wg act indirectly by defining and limiting each other's expression territory, as well as that of downstream ligands. All of these ligands then organize the repeating pattern. Three ligands induce Sr expression at specific positions across the PS, while the role of Ser reveals a particularly interesting spatial cue. Although the second row of Sr is induced in the anterior-most row of Ser-expressing cells, Ser expression is not necessary for this. Rather, Ser dictates the spacing between Sr row 1 and 2, because it defines the breadth of the Ve territory and thereby the position of the first non-Ve cell that can induce Sr in response to Spi-Egfr signaling (Hatini, 2001).

Sr expression is induced asymmetrically relative to each ligand source. For instance, Hh induces Sr posterior to the En territory, but not in the En territory or anterior to it. Wg imparts asymmetry to Hh/En signaling, and thereby prevents Ve expression anterior to the En/Hh territory. In exactly the same way, via antagonism of Hh signaling, Wg appears to block Sr expression anterior to the En/Hh territory, because the removal of Wg function allows Sr expression anterior to the En/Hh territory. The Wg signaling pathway imposes asymmetry to the dorsal/lateral Sr regulatory element via consensus Pangolin DNA binding sites. This principle is likely to extend to the ventral control of Sr for the generation of one element of the repeating striped pattern (Hatini, 2001).

One reason why Hh signaling cannot induce Sr expression in the En cells is that En represses expression of the Hh signal transducer, Ci. Nevertheless, it is still necessary to explain why signals from both the Ve and Wg territories cannot induce Sr in the En cells, even though each signal definitely acts on these cells to specify cuticle fate. A clue comes from the observation that when the En territory is not maintained Sr is induced symmetrically relative to the Wg or to the Ve sources. Thus, it is proposed that the En protein prevents Sr induction by Wg or Egfr inputs by repressing Sr expression. This is supported by the observation that activating Wg signaling at high levels in En cells still does not lead to Sr expression. To explain why Sr is not induced by Spi in the Ve territory, nor by Wg in the Wg territory, it is inferred that there is a specific block to autocrine signaling in each territory. Interestingly, this block is specific to Sr induction, and not to other outcomes of signaling, such as cuticle fate specification. It suggests a lack of an activator essential to induce Sr expression or expression of a repressor that blocks such a response in the Ve and Wg territories (Hatini, 2001).

The same ligands establish strikingly distinct patterns across the same cellular field. While the cuticle pattern comprises a diversity of cell types, the Sr expression pattern reflects the near-periodic specification of the same cell type. These distinct outcomes arise because the same ligands act in a fundamentally different manner in these two processes. As an example, Wg specifies smooth cuticle in a broad region anterior to the Wg territory, in the Wg territory, and posterior to the Wg territory (in anterior En/Hh cells). However, as is shown in this study, Wg induces Sr only anterior to the Wg territory in a narrow region, and not in the Wg territory or posterior to it. Also, Spi, through Egfr function, induces denticles over a broad region, both in the Ve territory and anterior to it in a subset of En/Hh cells. However, Egfr function induces Sr only in a narrow region posterior to the Ve territory. Thus, despite the broad effects of Wg and Egfr on cuticle pattern, the effect of Wg and Egfr in building the repeating striped pattern is constrained to a narrow region of cells. As a final example of the distinction between control of denticle pattern and control of repeating striped pattern, in the same row of cells, cuticle fate is specified by Spi while tendon fate is specified by Hh input. The unique effects of these ligands on Sr expression are crucial for the establishment of the repeating striped pattern. Thus, the information encoded in the signaling territories is decoded in different ways to achieve both repeating pattern and cell-type diversity across the same field (Hatini, 2001).

The generation of near-periodic Sr pattern across the PS is conceptually similar to the two-step process that is used in establishing the periodic body plan through pair-rule gene expression in syncitial embryos. Initially, primary pattern organizing centers are established at the boundaries of a field of naive nuclei or cells. In the first step, these centers establish patterned expression of secondary organizing genes across the field, subdividing the field into distinct gene expression territories. In the second step, the information encoded in these territories is used to initiate a repeating striped gene expression pattern. In the embryo, Bicoid together with Hunchback and Nanos organize expression of the gap genes. Territories of gap gene expression are then used to establish the periodic pattern of primary pair-rule gene expression. In the PS, Wg and Hh organize overall parasegmental pattern by first defining each other's territory and then the territories of secondary regulatory genes, Ser and Ve. The signaling territories are then used to establish near-periodic expression of Sr. Note that although the conceptual similarity is striking, the mechanisms generating these two repeating patterns are distinct. The pair-rule gene expression pattern is established in a unique, syncitial system by diffusion of transcriptional regulators in a common cytoplasm, whereas Sr expression is established across an epithelial monolayer by communication between cells via inter-cellular signaling systems. In addition, while a balance between diffusible activators and repressors determines pair-rule gene expression at any point along the syncitial embryo, the unique properties of the signaling territories across the PS determine Sr expression. In particular, the juxtaposition of pairs of territories, one that sends a signal with one that can initiate Sr expression in response to the signal, is utilized to initiate Sr expression adjacent to the boundaries between these territories (Hatini, 2001).

A near-periodic striped pattern of veins is produced in the developing wing disc. Emerging evidence suggests that the two-step strategy may also apply to this system, and that unique properties of putative signaling territories are used to initiate wing veins adjacent to at least two territories across the wing blade. In the first step, combined action of Hh and Dpp establishes different territories of downstream regulatory genes across the A/P axis of the future wing blade. While Hh establishes a territory of Ptc expression adjacent to the compartment boundary, Dpp acts more broadly across the wing and establishes two nested territories of Spalt and Optomotor-blind expression. In the second step, veins are induced adjacent to at least two territories, suggesting that an unknown ligand emanates from one territory and induces the vein in the adjacent territory. The possibility that the two-step process described here for the PS is used across the wing blade suggests that this may be a general strategy for creating repeating striped patterns across other cellular fields (Hatini, 2001).

During embryonic epidermal morphogenesis, Wg activity results in cells adopting a smooth apical surface, suggesting a direct effect of the Wg pathway on cytoskeletal remodelling. Consistent with this interpretation, dAPC-1 and dAPC-2, two paralogous proteins that display overlapping roles in the down regulation of Wg signalling through shuttling between nucleus and cytoplasm, associate with the cytoskeleton in different cell contexts. In the epidermis, dAPC-2 (E-APC) localizes at the basis of actin bundles that support cell extensions. Since dAPC2 mutant embryos lack ventral denticles, dAPC2 has been suggested to be a direct effector of Wg on cytoskeleton dynamics. However, shavenbaby (svb) is shown to be the determinant of cytoskeletal reorganization that leads to F-actin bundling during epidermal morphogenesis. Furthermore, Svb expression is necessary and sufficient (regardless to Wg activity) to localize dAPC-2 protein at the base of the apical actin-rich extensions. This shows that dAPC-2 is not primarily directed to actin bundles through Wg signalling. It also strongly suggests that the loss of denticles in dAPC2 embryos results from overactivity of the Wg pathway, which represses svb transcription. This interpretation is further supported by the fact that, as in case of ectopic activation of Wg signalling, dAPC2 mutations do not prevent formation of dorsal trichomes, which is dependent upon svb activity. Thus, dAPC2 and svb display two distinct functional interactions during denticle morphogenesis: (1) cytoplasmic dAPC2 acts to inhibit the signalling activity of ß-catenin that represses svb transcription; (2) when svb is expressed in epidermal cells, svb activity triggers F-actin bundling and redirects a pool of dAPC2 protein to the base of microfilament bundles. These data demonstrate that, rather than acting directly on F-actin dynamics, the Wg pathway acts through a ß-catenin/TCF-dependent signalling pathway culminating, in the nucleus, in the regulation of svb transcription during epidermal morphogenesis. A growing accumulation of evidence also supports the theory that APC proteins have Wnt independent roles in cytoskeletal regulation during the Drosophila development, such as spindle anchoring in syncytial embryos, cell adhesion, and larval brain development. Further elucidation of a putative Wg-independent function of dAPC2 in F-actin dynamics during epidermal differentiation now awaits uncoupling of its signalling activity from its association with the cytoskeleton (Delon, 2003).

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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