Table of contents

Targets of DPP in dorsal closure

The pannier gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. Novel aspects of the regulation and function of pnr during embryogenesis are reported in this study. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but it has been found that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila (Heranz, 2001).

In early development, pnr is activated in response to dpp activity in a broad dorsal domain, which extends from parasegments 2/3 to the border between 13/14, although the borders are not strictly parasegmental. The control by dpp is consistent with the effect of brk mutations on early pnr expression. The original expression domain is substantially modified during embryogenesis. By germ band extension (stage 10) pnr activity is limited dorsally by the border between the epidermis and the amnioserosa, and laterally by the dorsal border of iro. It is not known which factor(s) is responsible for the loss of expression in the amnioserosa, although likely candidates are several genes specifically active in this region, such as Race, zen, hindsight or serpent. In addition, it is not known how the late expression is regulated at the lateral border. It is not achieved by iro, since the loss of the entire Iroquois complex does not affect pnr expression (Heranz, 2001).

Interestingly, whereas early pnr expression is under dpp control, the late expression is not. Late inactivation of the Dpp pathway, using a dominant negative form of thick veins, does not modify pnr expression. In addition, mutations at brk, which allow higher response levels to Dpp signaling fail to affect pnr expression in late development, although they affect early expression. This indicates that pnr expression is controlled independently in early and late development, and by different factors (Heranz, 2001).

Pnr has concrete functions connected with the specification of cardiac cells and embryonic dorsal closure. The involvement of pnr in dorsal closure is exerted through its activation of dpp in late embryogenesis, which is responsible for the formation of the Dpp stripe at the junction of the epidermis with the amnioserosa. Normal functioning of the Dpp pathway in this region is required for dorsal closure, suggesting that defects in dorsal closure observed in pnr mutant embryos is the result of the lack of the dorsal dpp stripe (Heranz, 2001).

There is evidence that this dpp expression requires function of the JNK kinase pathway, and it also requires pnr activity. The observation that in the absence of pnr activity the expression of puc, the end element of the JNK pathway is normal, indicates that in pnr mutants the JNK pathway is normally active. In turn, it shows that the activation of dpp in the dorsal stripe requires independent inputs from both the JNK pathway and pnr (Heranz, 2001).

One intriguing aspect of pnr function is that it is able to induce a developmental modification in all ectodermal structures along the DV body axis except in the amnioserosa, the most dorsal tissue. Even under conditions in which pnr is transcribed and translated in all the amnioserosa cells, it does not appear to elicit any developmental effect; none of the amnioserosa marker genes is affected by forcing pnr activity and the retraction of the germ band [a morphological indicator of the function of specific amnioserosa genes is also normal. Similarly, pnr is able to induce dpp activity all over the body except in the amnioserosa, where the presence of the Pnr protein appears to be inconsequential. This situation resembles the phenotypic suppression/posterior prevalence phenomenon discovered in the Hox genes specifying the AP body axis. It consists of a functional inactivation of a Hox protein by the presence of another normally expressed in a more posterior region of the body. It is conceivable that there might be a ‘dorsal prevalence’ in the DV axis, by which dorsal expressing genes are functionally dominant over the ventral expressing ones. It would be expected that genes specifying amnioserosa would be able to transform all structures since they would be ranking highest in the functional hierarchy (Heranz, 2001).

Targets of DPP in wing morphogenesis

The wing imaginal disc comprises the primordia of the adult wing and the dorsal thoracic body wall. During second larval instar, the wing disc is subdivided into distinct domains that correspond to the presumptive wing and body wall. Early activity of the signaling protein Wingless has been implicated in the specification of the wing primordium. Wingless mutants can produce animals in which the wing is replaced by a duplication of thoracic structures. Specification of wing fate has been visualized by expression of the POU-homeodomain protein Nubbin in the presumptive wing territory and by repression of the homeodomain protein Homothorax. Repression of the zinc-finger transcription factor Teashirt (Tsh) is the earliest event in wing specification. Repression of Tsh by the combined action of Wingless and Decapentaplegic is required for wing pouch formation and for subsequent repression of Hth. Thus, repression of Tsh defines the presumptive wing earlier in development than repression of Hth, which must therefore be considered a secondary event (Wu, 2002).

Wing patterning can be subdivided into at least three discrete stages. The earliest observable changes in gene expression patterns that indicate specification of the wing field are repression of Tsh and retraction of Vestigial expression. Wg signaling represses Tsh expression at this stage. Dpp contributes to repression of Tsh. At present, the time at which Dpp acts can only be addressed directly by comparing the effects of clones induced at different stages. Clones induced in late second or early third instar are ineffective, whereas clones induced in early second instar are able to repress Tsh. Interestingly, Wg and Dpp cooperate to repress Hth in the wing pouch, even though this occurs somewhat later that repression of Tsh. These observations support the view that Wg and Dpp act in conjunction to specify the wing field in a manner analogous to the way they cooperate in leg patterning (Wu, 2002).

Interestingly, Hth and Tsh can also repress the vestigial quadrant enhancer, which depends on Wg and Dpp signaling in phase 3. Homothorax, Tsh and Vestigial appear to form a loop of mutual repression at this stage, since Vestigial also represses expression of Hth. Together, these observations suggest that Wg and Dpp have a complex regulatory interaction with Hth. Their activities repress it in the pouch, perhaps through activation of Vestigial and Scalloped. At the same time, the outer rings of Wg expression are required for Hth expression in the wing hinge. It is suggested that regulation of Hth may be secondary to regulation of Tsh in specification of the wing field (Wu, 2002).

Decapentaplegic, through its receptors Thickveins and Punt target optimotor blind and spalt transcription in the wing imaginal disc. The range of DPP action is wide, affecting spalt and omb expression on both sides of the anterior-posterior compartment boundary. The finding of an extended range of action for DPP is unexpected, but still DPP diffusion away from its site of expression may be limited by its tendency to be sequestered by components of the extracellular matrix. spalt and omb respond differently to the DPP concentration gradient, with omb showing a wider range of response due to its greater sensitivity to low DPP concentrations (Nellen, 1996).

The broad domain of omb expression in the wing imaginal disc corresponds to a wedge of expression that extends anteriorly from midway between veins 1 and 2, across the compartment boundary, to midway between veins 4 and 5 in the posterior compartment. Reduced omb activity leads to deletion of a central domain of the wing (Lecuit, 1996).

An investigation has been carried out of how Drosophila imaginal disc cells establish and maintain their appendage-specific determined states. Ectopic expression of wingless (wg) induces leg disc cells to activate expression of the wing marker Vestigial (Vg) and to transdetermine to wing cells. Ectopic wg expression non-cell-autonomously induces Vg expression in leg discs; activated Armadillo, a cytosolic transducer of the Wg signal, cell-autonomously induces Vg expression in leg discs, indicating that this Vg expression is directly activated by Wg signaling. Ubiquitous expression of wg in leg discs can induce only dorsal leg disc cells to express Vg and transdetermine to wing. Dorsal leg disc cells normally express high levels of decapentaplegic (dpp) and its downstream target, optomotor-blind (omb). Ectopic omb expression is sufficient to dorsalize leg cells but is not sufficient to induce transdetermination to wing. Dorsalization of ventral leg disc cells, through targeted expression of either dpp or omb, is sufficient to allow wg to induce Vg expression and wing fate. Leg cells dorsalized by omb are competent to transdetermine to wing, as shown when wingless is expressed ubiquitously. Under these circumstances Vg is expressed in both dorsal and ventral regions. A non-autonomous effect of omb was observed on wg-induced Vg expression, suggesting that omb induces the expression of another signal that acts with wg to induce Vg expression (Maves, 1998).

The expression of vestigial during wing development is regulated through two enhancers: the second intron or boundary enhancer (vgBE), and the fourth intron quadrant enhancer (vgQE). These names reflect the patterns of expression directed by these regulatory regions: vgBE produces a thin stripe over the prospective wing margin, and vgQE produces a pattern in four quadrants that are complementary to the vgBE and which fill in the developing wing blade. Both, vgBE and vgQE, act as integrators of signaling systems that drive wing development and, in this manner, these regulatory regions determine the tempo and the mode of wing development (Klein, 1999 and references).

Notch signaling is required for the initial activity of the Quadrant Enhancer (vgQE) The activity of the vgQE can be detected first at the beginning of the third instar, several hours after the upregulation of the vgBE, when it closely outlines the realm of the growing wing. This enhancer is only expressed in the growing wing blade and thus provides a unique and most specific marker for wing blade tissue. A variety of experiments have shown that the vgQE receives a negative input from Notch signaling and a positive one from Dpp. The presence of an E(spl)-binding site in the sequence of the vgQE has led to the suggestion that this suppression by Notch is mediated by the E(spl) protein. However, no strong suppression of the activity of the vgQE is found if E(spl)-m8 is ectopically expressed, suggesting that the effect of Notch requires other mediators. Although the vgQE is suppressed in the domain of Notch activity, Notch signaling plays a non-autonomous role in its activation. For example, the vgQE is never active in Serrate (Ser) mutants in which wing development initiates normally but is aborted very early. Ectopic expression of Delta rescues the wing pouch and leads to the activation of the vgQE. Interestingly, this activity arises in regions devoid of Notch signaling. This result suggests that Notch signaling influences the activity of the vgQE in two ways: it represses the activity of the vgQE autonomously but it is also required for its activity in a non-autonomous way (Klein, 1999).

The interactions between Dpp and Wingless during wing development take place at the level of the vgQE and require the vg gene product. Ectopic expression of wg alone leads to ectopic expression of the vgQE in the posterior region of the domain of vgBE expression, over a region in which dpp is normally expressed. Expression of dpp under the same conditions leads to an enlargement of the wing blade along the AP axis, with the vgBE as a reference but without extending it into the notum. Coexpression of wg and dpp leads to a pronounced extension of the wing blade toward the notum. In some instances, the development of up to eight wing blades occurs, all with a common origin over a point ventral to the notum. All these wings express the vgQE and each of them has a defined margin. It is possible that these multiple wings are produced by the splitting of an initial primordium. In the wild type, the activity of the vgQE is initiated at the intersection of wg and dpp expression and radiates from this focus. The results presented here indicate that this overlap determines important parameters of the morphogenesis of the wing. For example, it is possible that the distance from this focus -- perhaps defined by the range of diffusion of the two molecules -- defines a threshold that contributes significantly to the determination of the size and shape of the wing blade. By tampering with these overlaps, one can alter the shape of the wing or, by generating a series of them, trigger the development of multiple wings from one primordium (Klein, 1999).

High levels of dpp expression, which are both necessary and sufficient for dorsal leg development, are required for wg-induced transdetermination. Thus, dpp and omb promote both dorsal leg cell fate as well as transdetermination-competent leg disc cells. In leg discs, antagonist interactions between Wg and Dpp normally prevent wg expression from overlapping with high levels of dpp expresssion and with omb expression. It is thought that the interaction between Wg and Dpp in transdetermination mimics the interaction between Wg and Dpp normally used to establish the wing disc primordium. It is suggested that Wg signaling directly activates the vg boundary enhancer during wing disc development, presumably in conjunction with Notch signaling through Suppressor of Hairless. Taken together, these results show that the Wg and Dpp signaling pathways cooperate to induce Vg expression and leg-to-wing transdetermination. A specific vg regulatory element, the vg boundary enhancer, is required for transdetermination. It is proposed that an interaction between Wg and Dpp signaling can explain why leg disc cells transdetermine to wing and that these results have implications for normal leg and wing development (Maves, 1998).

Overexpression of dpp causes an expansion of the wing along the AP axis and the width of the spalt domain is expanded relative to the DPP stripe. Cells with reduced Mothers against dpp (Mad) activity fail to express spalt, suggesting that cells must be able to transduce the dpp signal to express spalt (Lecuit, 1996).

It is suggested that the broader domain of omb expression relative to that of spalt could be generated by persistence of omb expression in the progeny of cells in which the gene was activated at an earlier time, when the cells were within the range of the DPP signal.

The reason for this belief comes from an experiment in which dpp was express ectopically. In this case spalt is expressed at a considerable distance away from the ectopic source of DPP while omb expression is more restricted to a region near the clone. These observations suggest that the effective range of DPP in activating omb is actually less than it is for spalt. Therefore, a domain of gene expression (and therefore of fate specification) may be defined by the history of a cell at least as much as by its direct responsiveness to secreted signals (Lecuit, 1996).

In wing development, decapentaplegic is expressed along the anteroposterior compartment boundary. Early wingless expression is involved in setting up the dorsoventral boundary. Interaction between dpp- and wg-expressing cells promotes appendage outgrowth. optomotor-blind expression is required for distal wing development and is controlled by both dpp and wg. Ectopic omb expression can lead to the growth of additional wings. Thus, omb is essential for wing development and is controlled by two signaling pathways (Grimm, 1996).

Different signals activate separate enhancers to control vestigial expression: initially, in the dorsal/ventral organizer through the Notch pathway, and subsequently, in the developing wing blade by Decapentaplegic, and by a signal from the dorsal/ventral organizer. Signal integration must be a general feature of genes like vestigial, that regulate growth or patterning along more than one axis (Kim, 1996).

Decapentaplegic, in addition to its role as a morphogen in structuring gene expression and positioning of veins in the larval wing disc, is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating DPP feedback loop. Ectopic expression of dpp activates rho and suppresses sog expression. It is thought that the dpp suppression of sog is indirect, acting through rhomboid. A network of gene interactions promote vein fates as EGF-R ligands Vein and Spitz are also involved in intervein and vein fates respectively. These data support the view that SOG is a dedicated DPP antagonist (Yu, 1997).

The family of TGF-beta signaling molecules play inductive roles in various developmental contexts. One member of this family, Drosophila Decapentaplegic (Dpp) serves as a morphogen that patterns both the embryo and adult. Daughters against dpp (Dad), whose transcription is induced by Dpp shares, weak homology with Drosophila Mad (Mothers against dpp), a protein required for transduction of Dpp signals. Dad is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule, and in fact ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad. In contrast to Mad or the activated Dpp receptor, whose overexpression hyperactivates the Dpp signaling pathway, overexpression of Dad blocks Dpp activity. Dpp target gene optomotor blind is absent in Dad-overexpressing cells. Expression of Dad together with either Mad or the activated receptor rescues phenotypic defects induced by either protein alone. Dad can also antagonize the activity of a vertebrate homolog of Dpp, bone morphogenetic protein, as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. It is concluded that the pattern-organizing mechanism governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Dad. This feedback loop appears to be conserved in vertebrate development (Tsuneizumi, 1997).

Dpp, a TGFbeta, organizes pattern in the Drosophila wing by acting as a graded morphogen, activating different targets above distinct threshold concentrations. Like other TGFbetas, Dpp appears to induce transcription directly via activation of Mad. However, Dpp can also control gene expression indirectly by downregulating the expression of the brinker gene, which encodes a putative transcription factor that functions to repress Dpp targets. The medial-to-lateral Dpp gradient along the anterior-posterior axis is complemented by a lateral-to-medial gradient of Brinker, and the presence of these two opposing gradients may function to allow cells to detect small differences in Dpp concentration and respond by activating different target genes (Campbell, 1999).

Dpp controls patterning along the A-P axis in the wing of Drosophila by activating a number of downstream targets, including sal, omb, and vg. These targets are activated cell autonomously by Dpp signaling, and there is evidence, at least for vg, that Dpp induces gene transcription directly through activation of the SMAD Mad, which may act as a transcription factor. Expression of these three targets is also regulated negatively by brk: loss-of-function mutations in brk lead to ectopic and inappropriate levels of expression of sal, omb, and vg. Wing discs from the pupal lethal hypomorph brkXA are greatly enlarged along the A-P axis, phenocopying the ubiquitous expression of Dpp. These discs show expanded domains of sal, omb, and vg expression in the expanded wing pouch. Null mutants, including brkXA, are embryonic lethal, but mutant clones can result in outgrowths in adult wings when the clone is located in the anterior or posterior extremes of the wing. These outgrowths are comprised entirely of mutant tissue but are similar to outgrowths produced by misexpression of Dpp. Examination of such clones in wing discs reveals autonomous activation of sal, omb, and vg outside of their normal expression domains. Thus, Brk functions in the developing wing to repress the expression of Dpp targets such as sal, omb, and vg (Campbell, 1999).

Why is the indirect method involving Brk used to activate expression of Dpp targets? In other words, if Dpp can directly activate these genes via Mad, then why is this not sufficient? It is speculated that it is directly related to Dpp acting as a morphogen. Activation of sal, omb, and vg is not simply all or none, but each is induced above a distinct threshold concentration of Dpp, with sal requiring the highest level and vg the lowest. The gradient of Dpp will be transduced into a gradient of activated Mad, but it is possible that cells cannot perceive small differences in activated Mad reliably enough to faithfully define the expression domains of Dpp targets and that the introduction of the Brk intermediary provides the necessary information. This type of dual control of gene expression may turn out to be a common feature of many morphogen systems. The possibility is raised that other TGFßs may also use indirect mechanisms to control expression of target genes, possibly even Brk-related proteins, especially if they also induce multiple targets in a concentration-dependent manner. One relevant observation in this regard is that brk is also expressed in the early embryo where its expression also appears to be regulated by Dpp; null mutant embryos are partially dorsalized, suggesting it has a similar function here as in the wing. Unlike the wing, control of D-V patterning by TGFßs is probably a conserved feature of almost all animal embryos and strengthens the possibility that brk homologs will be typical regulators of TGFß target genes (Campbell, 1999).

decapentaplegic functions as a long-range morphogen in patterning of the embryo and the adult appendages. Dpp signals via the SMAD proteins Mad and Medea. In the absence of brinker (brk), Mad is not required for the activation of Dpp target genes that depend on low levels of Dpp. brk encodes a novel protein with features of a transcriptional repressor. brk itself is negatively regulated by Dpp. Dpp signaling might relieve brk's repression of low-level target genes either by transcriptional repression of brk or by antagonizing a repressor function of brk at the target gene promoters (Jazwinska, 1999a).

brk could be a transcription factor based both on its epistatic position in the pathway and on some features of the protein sequence. If brk specifically represses only the promoters of low- and intermediate-level target genes of Dpp, then loss of brk would lead to the activation of these genes at ectopic positions. At these positions, structures would form that correspond to low or intermediate levels of Dpp signaling, not because signaling has occurred, but instead because a specific subset of target genes had been activated in a signaling-independent way. If it is assumed that brk is a target gene-specific transcriptional regulator, then two models can be envisaged describing how Dpp regulates the target genes controlled by brk. In both models, the transcriptional control of brk by Dpp plays an important role. Dpp signaling is a potent repressor of brk transcription and seems to be required throughout wing development. As soon as Dpp signaling is abolished, strong brk expression can be seen at any position in the wing pouch. If brk is ectopically expressed in the center of the wing, then induction of omb and sal is suppressed even in regions of high Dpp signaling. All these observations suggest that Dpp signaling, at least in part, counteracts brk repression by reducing the amount of repressor. The promoter regions responsible for omb and low-level sal expression might even have only Brk-binding sites, so that their activation would be completely dependent on downregulation of brk expression. Alternatively, these promoters might integrate both the activation by SMAD proteins and repression by Brk (Jazwinska, 1999a).

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. The embryonic expression of dve depends on the Wg signal in the proventriculus and on the Dpp signal in the middle midgut. Whether or not dve expression in wing discs depends on these signals was examined. When the Wg signal is ectopically activated in a ring pattern around a wing pouch under the control of the 30A-Gal4 driver, dve is ectopically activated only in cells at the intersection of the ring with the A-P boundary, which normally expresses Dpp. In contrast, activation of the Dpp signal in the same ring pattern results in the ectopic expression of dve only at the D-V boundary, where Wg is normally expressed. Thus, the combined Wg and Dpp signals appear to induce dve expression in wing discs. To examine this possibility, flip-out recombination clones were generated that simultaneously express an activated form of the Dpp receptor (TkvQ253D) and that of a Wg signaling molecule (DeltaArm). Some of these clones induced ectopic Dve expression autonomously outside the compartment boundaries. These results strongly support the above notion that the combined activities of Wg and Dpp signals induce dve expression rather than other signals generated at compartment boundaries (Nakagoshi, 2002).

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 Drosophila, muscles attach to epidermal tendon cells are specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. The mechanisms underlying the specification of these domains of sr expression have been examined. The concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites (Ghazi, 2003).

wg controls sr expression by in segment border cells of the Drosophila embryo. Wg signaling restricts sr activation to a single row of cells. In the presumptive notum on the wing disc, hh expression is restricted to a very narrow region, which forms the posterior compartment. Its effects in the disc are mediated by dpp, which serves multiple functions. Dpp is required for induction of wg expression, as it positively regulates pnr, which in turn activates wg. However, once wg is induced, Dpp tightly restricts its domain. This antagonism is required for correct positioning of the DC bristles. This antagonism also defines domains of sr. It is unclear if dpp directly regulates sr, or its effect is by control of other genes. The similarity between sr phenotypes observed on expansion of wg expression, and in dpp mutants, is suggestive of its effects being mediated by wg only, but it is also possible that it influences sr expression directly (Ghazi, 2003).

Secreted signaling molecules such as Wingless (Wg) and Decapentaplegic (Dpp) organize positional information along the proximodistal (PD) axis of the Drosophila wing imaginal disc. Responding cells activate different downstream targets depending on the combination and level of these signals and other factors present at the time of signal transduction. Two such factors, teashirt (tsh) and homothorax (hth), are initially co-expressed throughout the entire wing disc, but are later repressed in distal cells, permitting the subsequent elaboration of distal fates. Control of tsh and hth repression is, therefore, crucial for wing development, and plays a role in shaping and sizing the adult appendage. Although both Wg and Dpp participate in this control, their specific contributions remain unclear. In this report, tsh and hth regulation were analyzed in the wing disc; Wg and Dpp act independently as the primary signals for the repression of tsh and hth, respectively. In cells that receive low levels of Dpp, hth repression also requires Vestigial (Vg). Furthermore, although Dpp is required continuously for hth repression throughout development, Wg is only required for the initiation of tsh repression. Instead, the maintenance of tsh repression requires Polycomb group (PcG) mediated gene silencing, which is dispensable for hth repression. Thus, despite their overall similar expression patterns, tsh and hth repression in the wing disc is controlled by two very different mechanisms (Zirin, 2004).

In the course of a screen for mutations affecting the PD axis of the wing, an allele of the Drosophila Smad4 homolog Med was isolated. Like other Dpp pathway mutations, Medadro clones located in the wing pouch cell autonomously de-repress hth. This is evident even in late-induced clones, demonstrating the continuous role of Dpp signaling in shaping the wing blade/hinge subdivision during larval development. By contrast, no de-repression of tsh was detected resulting from any manipulations of the Dpp pathway (Zirin, 2004).

The ability of ectopic Dpp activity to repress tsh in early-induced proximal clones was interpreted to suggest that Wg and Dpp cooperate to repress tsh in the early pouch. However, because Dpp is dispensable for tsh repression, this model must be an over-simplification. It is concluded that Wg, not Dpp, must be considered the primary repressor of tsh in the wing. The lack of synergy between the two pathways is reminiscent of the regulation of Dll, which is activated in the leg by the combined activities of Wg and Dpp, but requires only Wg for its expression in the wing pouch (Zirin, 2004).

In the absence of Dpp signaling, wing pouch cells co-express hth, nub and Dll, but not tsh. This combination of factors is normally found only in the distal hinge (DH), suggesting a transformation from pouch to DH when the Dpp pathway is compromised. The expression of the Iro-C genes, normally restricted to the notum, extends to the distal limit of the tsh domain in dpp mutant discs, leading to the hypothesis that Dpp signaling is essential for the separation of wing and body wall. However, because loss of Dpp signaling transforms wing pouch to DH, an alternative view is proposed in which Dpp further divides an already extant appendage/trunk subdivision by repression of hth in the pouch and Iro-C in the proximal hinge (PH). According to this proposal, the distal limit of tsh expression, initiated by early Wg expression and maintained by PcG silencing, denotes the boundary between the appendage and the body (Zirin, 2004).

The results suggest that repression of hth in the wing disc occurs only in cells with a history of vg expression and continuous Dpp input. Consistent with this, ectopic vg expression in the medial DH and loss of brk in the lateral DH both result in hth repression. The requirement for vg can be separated into two distinct stages. The first stage occurs in the second instar, when vg expressed at the DV compartment boundary determines which cells are competent to repress hth in response to Dpp signaling. Thus, both vg or Dpp-pathway mutant clones induced at this early stage fail to repress hth (Zirin, 2004).

In the third instar, vg expression is required for hth repression only at the lateral edges of the wing pouch, whereas Dpp signaling is required at all positions along the AP axis. Accordingly, the boundary between the lateral hinge and pouch is dictated by the threshold of Dpp activity that permits the Vg-dependent repression of hth. Wg signal transduction is also required to repress hth in pouch cells far from the AP boundary. However, the requirement for Wg signaling in this part of the wing pouch could be due to its role in vg activation. Alternatively, it is possible that Wg and Vg are independently required to repress hth in these cells (Zirin, 2004).

A model that encompasses these observations is that Vg and Dpp activate another factor that directly represses hth. This factor would be activated in Vg-positive cells by Dpp signaling beginning in the late second instar. By the third instar, high levels of Dpp signaling would be sufficient to maintain its activation, with additional input by Vg and Wg required only at the lateral regions of the pouch. Even further from the source of Dpp, in the lateral hinge, high levels of Brk would prevent expresssion of this factor, thus allowing hth expression despite the presence of Vg. This model is consistent with the idea that Brk is a transcriptional repressor and Vg is a transcriptional activator. There is also precedent for the idea that early vg expression predisposes cells to a particular Dpp response, which was also proposed for the activation of the vgQE (Zirin, 2004).

The above model does not apply to PH cells, which have a distinct response to Dpp signaling. For example, Medadro clones located near the AP boundary of the PH ectopically expressed vg. tsh is an attractive candidate for mediating this switch in response to Dpp signaling, since it is expressed in the PH but not the DH, and is reported to bind Brk in vitro. However, the absence of reagents to readily examine tsh loss-of-function clones prevents this idea from being tested (Zirin, 2004).

If tsh repression marks a fundamental subdivision along the PD axis, then the maintenance of tsh repression is crucial for the maintenance of this subdivision. Although Wg signaling is clearly required for the initiation of tsh repression, it is dispensable by the time the DV margin is established. The elbow-no ocelli (el-noc) gene complex has been identified as a target of both Dpp and Wg that is necessary for tsh repression in the wing. However, tsh de-repression is observed in el-noc loss-of-function clones induced only in first or early second instar larvae. tsh repression must therefore be maintained by a wg- and el-noc-independent mechanism. The possibility of redundant Wg- and Dpp-mediated tsh repression was ruled out by making clones doubly mutant for both signaling pathways. Such clones upregulate hth, and lose Dll expression, but show no ectopic tsh expression. Thus, neither of the two major long-range signaling systems of the wing pouch is involved in the maintenance of tsh repression (Zirin, 2004).

Instead, analysis of Su(z)12daed and Pc mutant clones indicates that the maintenance of tsh repression is mediated by a heritable silencing mechanism. By inducing Pc mutant clones in third instar discs, it was demonstrated that this ectopic tsh expression represents a failure to maintain rather than a failure to establish repression. The weak hth levels observed in some PcG mutant clones may be due to the ability of tsh to upregulate hth. This interpretation is supported by the fact that hth expression is seen only in large Pc mutant clones, and only in cells expressing the highest levels of tsh. The general absence of hth expression in PcG mutant clones, together with the ectopic hth expression resulting from late Dpp pathway disruption, points to the need for continuous signaling input to maintain hth repression. By contrast, tsh requires PcG gene activity, but not continuous Wg or Dpp input, to maintain its repression during the third instar (Zirin, 2004).

At this stage the possibility that the affects of PcG mutant clones on tsh repression described here are indirectly due to the de-repression of another factor cannot be ruled out. It is suggested that this is unlikely, however, in part because the spatial distribution of tsh de-repression in PcG mutant clones differs significantly from reports of Hox gene de-repression. Additionally, the ectopic tsh expression in Pc mutant clones is repressible by Nrt-Wg, indicating that tsh is still subject to regulation by Wg signaling (Zirin, 2004).

In the embryo, Hox genes are repressed in some segments by the transient presence of the gap genes. This initial repression is then maintained by the PcG proteins through a heritable silencing mechanism. A model of tsh repression follows this general outline, whereby Wg signaling is required transiently to establish the limits of the tsh expression domain. PcG proteins subsequently maintain the tsh silenced state, while the appendage is further subdivided along the PD axis. Similar mechanisms may be important for tsh regulation in other tissues, as is suggested by tsh de-repression in PcG mutant clones in the eye disc (Zirin, 2004).

tsh and hth repression are distinct events during the development of the wing imaginal disc. The requirement for PcG activity in tsh, but not hth, repression points to the primacy of tsh repression in determining appendage versus trunk fate. PcG regulation ensures a strict and inflexible pattern of gene expression, ideal for defining the fundamental divisions of the disc. Within the specified appendage domain, Wg and Dpp signaling can then modify the shape and size of the hinge and wing blade through continuous input into transcription factors that control patterning and growth. In the absence of Tsh, Hth is an essential mediator of this process, since it promotes hinge development at the expense of wing pouch growth. The complexity of hth relative to tsh regulation may, therefore, reflect the greater need for plasticity in the response of hth to the Wg and Dpp morphogen gradients (Zirin, 2004).

The co-regulator dNAB interacts with Brinker to eliminate cells with reduced Dpp signaling

The proper development of tissues requires morphogen activity that dictates the appropriate growth and differentiation of each cell according to its position within a developing field. Elimination of underperforming cells that are less efficient in receiving/transducing the morphogenetic signal is thought to provide a general fail-safe mechanism to avoid developmental misspecification. In the developing Drosophila wing, the morphogen Dpp provides cells with growth and survival cues. Much of the regulation of transcriptional output by Dpp is mediated through repression of the transcriptional repressor Brinker (Brk), and thus through the activation of target genes. Mutant cells impaired for Dpp reception or transduction are lost from the wing epithelium. At the molecular level, reduced Dpp signaling results in Brk upregulation that triggers apoptosis through activation of the JNK pathway. This study shows that the transcriptional co-regulator dNAB is a Dpp target in the developing wing that interacts with Brk to eliminate cells with reduced Dpp signaling through the JNK pathway. Both dNAB and Brk are required for cell elimination induced by differential dMyc expression, a process that depends on reduced Dpp transduction in outcompeted cells. A novel mechanism is proposed whereby the morphogen Dpp regulates the responsiveness to its own survival signal by inversely controlling the expression of a repressor, Brk, and its co-repressor, dNAB (Ziv, 2009).

NAB proteins comprise a family of transcriptional co-regulators implicated in various developmental processes in different organisms. Drosophila NAB was found to be required for determining specific neuronal fates in the embryonic CNS and for wing hinge patterning. This study shows that dNAB induces cell elimination through induction of the JNK pathway, which in turn triggers Caspase-3-mediated apoptosis. dNAB acts as a co-repressor that physically interacts with Brk to induce apoptotic cell elimination. This conclusion is based on several lines of evidence. First, dNAB-induced apoptosis is completely nullified by removal of Brk. Second, epistatic analysis placed dNAB in the Dpp signaling pathway downstream of the receptor complex and of brk transcriptional repression and upstream of Brk. Third, dNAB physically associates with Brk through its NCD2 domain in vitro. Fourth, dNAB enhances the killing activity of Brk in the presumptive wing blade region and is required for elimination of Dad-overexpressing cells, a process that is completely dependent upon Brk function. Finally, ectopic expression of dNAB represses the expression of Dpp/Brk target genes (Ziv, 2009).

Competitive interactions occur between cells differing in their levels of dMyc, such that cells expressing more dMyc both outgrow neighboring cells and induce their death. This competitive behavior correlates with, and can be modulated by, the activation of the Dpp survival signaling pathway, showing that dMyc-induced cell competition relies on Dpp signaling. The fact that dNAB, similar to Brk, is crucial for dMyc-induced cell competition strongly supports a role for dNAB as an effector of cell elimination of underperforming cells with reduced Dpp signaling (Ziv, 2009).

Elimination of underperforming cells takes place only during early larval stages. Clones generated later, during the third instar larval stage, persist to adulthood. Consistently, using double staining of wing discs with antibodies directed against Brk and dNAB, it was found that the two do not overlap in the second instar larval stage [60 hours after egg laying (AEL)] and only slightly overlap during the third instar (80 hours AEL). These findings suggest that the Brk-dNAB complex is active in cell elimination only during early development. This might indicate that either another factor required for complex activity is present only during early development, or that a factor is present during later stages that inhibits the complex. Alternatively, intensive growth/proliferation might be required for the execution of the killing activity of the complex (Ziv, 2009).

The morphogen Dpp acts through a well-characterized transduction pathway to simultaneously regulate growth, survival and patterning. To a large extent, Dpp signaling acts through negative regulation of brk expression. This implies that a complete answer to how the Dpp signal directs different cellular and developmental processes requires an understanding of how Brk executes its transcriptional repression functions. The finding that dNAB is a Brk co-repressor is in accordance with recent results showing that overexpression of Brk forms that cannot bind either Gro or CtBP results in repression of sal, omb and vg, and that Brk contains additional co-repressor-binding domains. On contrast to Gro, a known co-repressor of Brk, the function of dNAB is not required for Dpp-dependent patterning. However, Gro does not play a similar role to that of dNAB in promoting JNK-mediated cell killing. These findings imply that the choice of Brk co-repressor determines the specificity of target gene repression, thereby modulating different Dpp outputs. Mechanistically, this could be achieved in a number of ways: for example, dNAB or Gro association might alter the DNA-binding specificity of Brk, or the promoters of Brk target genes might be differentially responsive to dNAB and Gro. In addition, the fact that Gro is ubiquitously expressed throughout the developing wing, and that Dpp induces dNAB expression in the center of the wing disc while restricting Brk expression to lateral regions, provide another means for differentially modulating Dpp outputs (Ziv, 2009).

Based on these findings, a molecular model is proposed to explain how the morphogen Dpp regulates the cellular response to its own survival signal in the developing wing by inversely controlling the expression of two key factors, Brk and dNAB. In the center of the wing disc, Dpp represses brk and induces dnab expression, so that in situations in which Dpp signaling activity is abnormally reduced, the resulting local increase in the levels of Brk, which complexes with dNAB, activates the apoptotic pathway. Thus, the Dpp signal sensitizes cells in the center of the wing disc to the apoptotic effect associated with reduced Dpp signaling by maintaining dNAB expression. In lateral regions of the wing disc, where Brk expression is normally higher, apoptotic cell elimination is attenuated, at least in part owing to a lack of dNAB. Thus, by invoking dNAB as a Dpp effector molecule that sensitizes cells to the levels of Brk, it can be at least in part explained why cells in the center of the wing disc, near the Dpp source, are more susceptible to cell elimination induced by reduced Dpp signaling, and why high levels of Brk in the periphery do not necessarily bring about apoptosis (Ziv, 2009).

Given that dNAB appears to play no role in Dpp-mediated patterning, it is proposed that dNAB functions in the wing to prevent developmental errors and discontinuities along the Dpp signaling gradient. This mechanism might be a general feature of morphogen gradients that functions to avoid the accumulation of detrimental developmental mistakes that would otherwise lead to embryonic malformation, and is potentially important in cancer, where tumor cells overexpressing oncogenes such as Myc may act as super-competitors. Thus, the molecular principles underlying such developmental fail-safe mechanisms are clearly of biomedical interest (Ziv, 2009).

Identification of genes affecting wing patterning through a loss-of-function mutagenesis screen and characterization of med15 function during wing development

The development of the Drosophila wing depends on the correct regulation of cell survival, growth, proliferation, differentiation, and pattern formation. These processes, and the genes controlling then, are common to the development of epithelia in many different organisms. To identify additional genes contributing to wing development a genetic screen was performed in mosaic wings carrying clones of homozygous mutant cells. Twelve complementation groups were obtained corresponding to genes with a proven role in wing formation such as smoothened, thick veins, mothers against dpp, expanded, and fat and 71 new complementation groups were obtained affecting the pattern of veins and the size of wing. One of these groups mapped to the mediator15 gene (med15), a component of the Mediator complex. Med15 and other members of the Mediator complex were shown to be required, among other processes, for the transcription of decapentaplegic target genes (Terriente-Félix, 2010).

The complementation group formed by the 77A and 133A1 mutants was analyzed in some detail. These mutations are alleles of med15, a gene encoding one component of the Mediator complex. Thus, they fail to complement other med15 alleles, and med15133A1 is associated with a stop codon that could truncate the protein in the N-terminal region after the KIX domain. The Mediator multiprotein complex promotes the transcription of inducible genes, acting as a link between the RNApolII holoenzyme and several sequence-specific transcription factors. The human homolog of Med15, MED105, is included in all Mediator complexes identified so far and forms part of a module named the tail that is the main target for the transcriptional activators. Thus, Med15 homologs can bid to different transcription factors such as Gcn4 and Gal4 in Saccharomyces cerevisiae, and, more interesting from the perspective of these data, to Smad2/3 and Smad4 in Xenopus. Other members of the Mediator complex that were previously analyzed are kohtalo and skuld (Med12 and Med13, respectively), which form part of the conserved Cdk8 module. Interestingly, mouse Cdk8 and Cdk9 phosphorylate Smad proteins, regulating their transcriptional activity and turnover (Alarcón). However, kohtalo and skuld are required for sensory organ development, for some aspects of Notch and Hedgehog signaling, and for the transcription of Wingless downstream genes (Terriente-Félix, 2010).

Med15 mutations result in smaller than normal wings and loss of mainly the L2 vein. They also affect the fusion between the left and the right hemithorax and leg morphogenesis. The reduction in the level of expression of other components of the Mediator complex, most notably med20, med27, and med30, also results in smaller than normal wings and failures in vein differentiation, in addition to causing some levels of cell death. Although these phenotypes were similar, they are not identical, which might indicate specific requirements of these subunits or, alternatively, a different degree in the effectiveness of each interference RNA used. Mutant med15 cells display specific defects in gene expression, suggesting a requirement limited to particular enhancer-promotor interactions. In particular, the expression of spalt, a direct target of Dpp signaling, is compromised in med15 mutant cells. There are no known transcriptional targets of TGFβ signaling in the wing, and consequently it could not be determined directly whether the activity of this pathway is diminished in med15 mutants. A direct requirement of Med15 for the transcription of TGFβ target genes is nonetheless suggested by the similar phenotypes of wing size reduction observed in med15 mutants and in baboon mutations (Terriente-Félix, 2010).

Dpp-induced Egfr signaling triggers postembryonic wing development in Drosophila

The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings (Paul, 2012).

Deciphering gene regulatory networks (GRNs) is critical for understanding the causation of development, and a large network that explains endoderm development in the sea urchin has been elaborated . The Drosophila early wing disc provides a tractable system for GRN analysis because it is a relatively small field of cells and many genes involved in the process are known. This study has developed knowledge of the GRN involving Egfr signaling activated by its ligand Vn. It is a key circuit because Vn/Egfr signaling induces the ap and iro-C genes, which are required for development of the major territories of wing and body wall beginning in the first instar. The spatial and temporal control of vn expression involves two major inputs: initiation by paracrine Dpp signaling and maintenance by a positive Vn/Egfr autocrine feedback loop. These are direct, positive, early acting inputs into the vn promoter. The Dpp signal emanates from the peripodial epithelium and is unidirectional, activating vn only in the disc proper. This directionality is important because ectopic activation of Egfr in the peripodial epithelium antagonizes its development and transforms the cells into columnar cells characteristic of the disc proper, which develop into body wall structures. It is not known why Dpp signaling is apparently only active in cells across the lumen from the expression domain in the first instar. It is possible some factor required for Dpp signaling is differentially expressed or Dpp signaling is mediated by cellular processes as has been proposed for the early eye disc. There are also negative inputs that operate slightly later in development and function to limit the spatial extent of vn expression. Productive Vn/Egfr signaling is confined to the future body wall because pntP2 expression focuses proximally as development proceeds. There is also an early acting negative input from Wg signaling that has been defined genetically. wg expression begins in distal cells in the first instar wing disc, which is consistent with a role in repressing vn expression in cells that will become the future wing (Paul, 2012).

The direct feedback loop involving vn expression is an example of a regulatory circuit governing a 'community effect,' a term coined by Gurdon to describe the change in transcription that occurs when cells are isolated from their neighbors. This phenomenon suggested that proximity to other cells serves as a mechanism to sustain expression of particular genes important for a collective cell fate. The term has been used to describe a developmental event that is the result of a ligand inducing its own expression in a neighboring cell. In this way, the signal is propagated through a field of cells, which, as a 'community,' then express a similar repertoire of target genes. Examples of this type of subcircuit operating in pattern formation have been described that involve TGF-β, FGF, and Wnt ligands. This study reports a case in which an EGF ligand is implicated. Adding an additional ligand class to the list supports the idea that the strategy is widespread in development. In a normal developmental context, a secreted EGF ligand, such as Vn, can be produced as part of a positive feedback loop provided there are tight controls that limit the operation of the loop both spatially and temporally. Clearly, if there were no limits, a runaway situation would result and too many cells would become part of the 'community,' or the state of activity would be perpetuated beyond a certain developmental window. Indeed this seems to be the case in some disease contexts involving EGF ligands, including neuregulin (the vertebrate equivalent to Vn), where autocrine loops sustain the continued growth of cancer cells (Paul, 2012).

It is widely thought that flight contributed significantly to the success of insects and consequently the evolution of the wing is of great interest. There are two major theories; the first holds that wings derived from a proximal branch of the leg and the second that wings derived from a paranotal lobe extending from the dorsal body wall. In a combination of these ideas, it has been proposed that the wing may have a leg origin but that the ability to form a flat wing-like structure depends upon proximity to the dorsal-lateral boundary in the side body (the paranotal lobe in wingless forms). Dorsal appendages such as the tracheal gill or stylus are like the Drosophila wing in that they express wg and vg and may represent evolutionary precursors to the wing. Unlike the wing, however, they do not form close to a region where ap is also expressed, which may be essential for an outgrowth to form a flat structure like the wing. Vn/Egfr signaling is upstream of ap and therefore a prerequisite for wing formation. This study trace the circuit regulating ap expression back earlier to the induction of vn in a transient stripe triggered by Dpp signaling. This initiates broad Vn/Egfr/PntP2 signaling that extends distally and ap is induced throughout the dorsal compartment, where, together with wg and vg, it acts to produce the wing. Continued Egfr activation, however, blocks wing development, and hence the domain of active signaling is shortly thereafter restricted to proximal cells by the absence of PntP2 in distal cells. In proximal cells where PntP2 persists, a feedback loop is established and body wall development is promoted. The changing spatial domain of vn (from a stripe to a proximal wedge) establishes a prepattern that is permissive for both wing and body wall development. It will be interesting to determine if Egfr signaling underlies body wall formation in other species and if a similar transient spatial extension of this activity correlates with winged morphs (Paul, 2012).

Bridging Decapentaplegic and Wingless signaling in Drosophila wings through repression of naked cuticle by Brinker: Dpp negatively regulates the Wg target gene Distal-less (Dll)

Wnts and bone morphogenetic proteins (BMPs) are signaling elements that are crucial for a variety of events in animal development. In Drosophila, Wingless (Wg, a Wnt ligand) and Decapentaplegic (Dpp, a BMP homolog) are thought to function through distinct signal transduction pathways and independently direct the patterning of the wing. However, recent studies suggest that Mothers against Dpp (Mad), the key transducer of Dpp signaling, might serve as a node for the crosstalk between these two pathways, and both positive and negative roles of Mad in Wg signaling have been suggested. This study describes a novel molecular mechanism by which Dpp signaling suppresses Wg outputs. Brinker (Brk), a transcriptional repressor that is downregulated by Dpp, directly represses naked cuticle (nkd), which encodes a feedback inhibitor of Wg signaling, in vitro and in vivo. Through genetic studies, this study demonstrates that Brk is required for Wg target gene expression in fly wing imaginal discs and that loss or gain of brk during wing development mimics loss or gain of Wg signaling, respectively. Finally, it was shown that Dpp positively regulates the expression of nkd and negatively regulates the Wg target gene Distal-less (Dll). These data support a model in which different signaling pathways interact via a negative-feedback mechanism. Such a mechanism might explain how organs coordinate inputs from multiple signaling cues (Yang, 2013).

This study has shown that Brk directly represses nkd expression. The direct repression of nkd by Brk is underscored by three observations. First, a Brk site was identified in the intronic region of nkd, which Brk physically occupies in vitro. Second, ChIP analysis shows that Brk binds a DNA region near this Brk site in embryos in a manner inversely related to Wg activity. Third, reporter analysis in Kc cells indicates that Brk represses Arm-dependent activation of an intronic WRE containing this Brk site, but only when the Brk site is intact. In addition, genetic analyses has shown that the repression of nkd by Brk is functionally significant. In the developing wing, it was found that the loss of brk de-represses nkd and downregulates Wg target proteins, such as Dll and Sens. Conversely, ectopic brk inhibits nkd expression and markedly enhances Dll expression. Furthermore, removal of nkd prevents the loss of Dll in brk clones whereas co-expression of nkd abolishes the expanded Dll caused by ectopic brk. In adult wing, the loss and gain of brk phenotypically resembles the loss and gain of Wg signaling, respectively. Consistent with a repressive role of Dpp cascade on brk, it was found that ectopic Dpp signaling enhances nkd and inhibits Wg signaling). These results support a model in which Dpp signaling increases the expression of Nkd, a Wg inhibitor, by the downregulation of Brk, and thereby inhibits the Wg outputs. In another words, nkd might fall into a class of Dpp targets, which are de-repressed upon the activation of Dpp signaling. This study has thus uncovered a previously unsuspected molecular mechanism underlying the interaction between Wg and Dpp signaling pathways in Drosophila wing development (Yang, 2013).

Until recently, little has been known about the cross-interaction between Wg and Dpp signaling in Drosophila wings, in spite of the fact that the fly wing has served as an excellent model system for the dissection of the molecular basis of these signaling transduction pathways. This is in contrast to Drosophila leg imaginal discs, in which mutual repression between Wg and Dpp signaling has long been suspected. However, several studies have indicated that manipulation of Dpp signaling levels in the wing sometimes leads to phenotypes resembling those caused by loss or gain of Wg activity. Notably, ectopic Dpp signaling increases notches in the wing, which is characteristic of reduced Wg signaling. However, the underlying mechanism for this effect of Dpp is not clear. Recently, independent research groups have suggested that Mad, the key effector of Dpp signaling, might play a role in the regulation of Wg target gene expression in fly wings. The molecular basis for their findings has mainly been the physical interaction between Mad and TCF, similar to the findings in mammals, in which several Smad proteins interact with members of the lymphoid enhancer binding factor 1/TCF family of DNA-binding HMG box transcription factor. It remains to be determined whether the role of Mad is direct or indirect because the reporter assays in these studies were performed with TOPFlash or similar constructs in mammalian cell culture, which might not always accurately represent the complicated situation of the in vivo regulation of Wg target genes. Furthermore, manipulation of Mad expression in wing discs influences Dll expression in different directions. Although these intriguing discrepancies can be explained by the physical interaction between Mad and TCF, the current model offers an alternative interpretation based on the negative regulation of nkd by Brk, which might suggest an indirect role of Mad in Wg signaling. For example, the current model could provide an explanation for the previous finding that ectopic Dpp signaling, caused by Mad, Medea, TkvQD, etc., results in notched wings (Yang, 2013).

The role of Brk in Wg signaling has been previously documented in Drosophila. It has been suggested that brk is able to antagonize Wg signaling based on the activity of a midgut-specific Ubx reporter gene in which physical interactions among Brk, Teashirt and CtBP have been described. In leg discs, Wg signaling may directly repress Dpp morphogen expression via an Arm-TCF-Brk complex, offering a direct model for the cross-talk between Wg and Dpp. However, the current studies have indicated a positive role for Brk in Wg signaling through an indirect action. In addition to the repression of Dpp targets, the roles of Brk in Wg signaling described in these different models exemplify the pleiotropic actions of brk throughout development and might provide the molecular basis for tissue-specific consequences of developmental signaling pathways (Yang, 2013).

nkd was first identified as a Drosophila segment-polarity gene, mutation of which gives rise to major deficits in fly embryonic development. Its expression appears to be universally induced by Wg in fly embryos and larval imaginal discs. It is interesting that although the loss of nkd in embryos has an effect similar to gain of wg, decreased nkd function in fly wings shows little impact. However, none of the nkd alleles used in these studies has been well characterized at the molecular level. Given the complexity of nkd transcriptional regulation, it could be that these mutant forms of nkd still possess residual function in the wing. Alternately, overexpression of nkd blocks ectopic Wg signaling in the eyes and generates PCP phenotypes in the wing through a direct interaction with Dsh. Consistent with these observations, this study found that loss of brk can cause a dramatic increase of nkd expression in certain areas of the wing imaginal disc, leading to wing notches and PCP defects. The current findings suggest that nkd may indeed play roles, at a certain level, in both canonical and noncanonical Wg signaling in fly wings. However, a closer examination of nkd function in fly wings is needed (Yang, 2013).

In conclusion, this study found that Brk influences Wg signaling by directly repressing nkd expression and could serve as a node for cross-talk between the Wg and Dpp signaling pathways. Wnt-BMP cross-interactions have been implicated in many developmental and disease processes). For example, a Wnt-BMP feedback circuit mechanism is important for inter-tissue signaling dynamics in tooth organogenesis in mouse. The findings may therefore add new insights into cell differentiation and human cancer (Yang, 2013).

Targets of DPP in the notum

The morphogen gradient of Wingless provides positional information to cells in Drosophila imaginal discs. Elucidating the mechanism that precisely restricts the expression domain of wingless is important in understanding the two-dimensional patterning by secreted proteins in imaginal discs. In the pouch region of the wing disc, wingless is induced at the dorsal/ventral compartment boundary by Notch signaling in a compartment-dependent manner. In the notum region of the wing disc, wingless is also expressed across the dorsal/ventral axis, however, regulation of notal wingless expression is not fully understood. Notal wingless expression is established through the function of Pannier, U-shaped and Wingless signaling itself. Initial wingless induction is regulated by two transcription factors, Pannier and U-shaped. At a later stage, wingless expression expands ventrally from the pannier expression domain by a Wingless signaling-dependent mechanism. Interestingly, expression of pannier and u-shaped is regulated by Decapentaplegic signaling that provides the positional information along the anterior/posterior axis, in a concentration-dependent manner. This suggests that the Decapentaplegic morphogen gradient is utilized not only for anterior/posterior patterning but also contributes to dorsal/ventral patterning through the induction of pannier, u-shaped and wingless during Drosophila notum development (Tomoyasu, 2000).

A hierarchy of the activity of these genes during notum development is presented. dpp is initially induced at the dorsal region of the A/P compartment boundary by Hh signaling. Dpp signaling induces two target genes, pnr and ush. Analyses of pnr expression in put-ts and tkva12 cells suggest that different thresholds are set for the induction of these genes: low levels for pnr and high levels for ush. wg is induced by Pnr where ush is not expressed. Simultaneously, the Pnr-Ush complex represses wg expression at the dorsal-most region of the presumptive notum. In the later stage, the wg expression domain expands ventrally from the pnr expressing region and wg starts to be expressed in the non-pnr-expressing cells. During this process, Wg signaling plays a crucial role and this separation does not occur in the Wg signaling mutants. The Pnr-Ush complex acts as a repressor for the induction of wg and of DC enhancer-lacZ expression (DC enhancer is an enhancer of the achaete-scute proneural gene complex that activates gene expression in the dorsocentral area). It is interesting that Ush does not simply inhibit Pnr function but switches the activator function of Pnr to a repressor function. Based on the result that the extra doses of Pnr cannot revert the repressor activity of Pnr-Ush, it has been proposed that the activator function of Pnr and the repressor function of the Pnr-Ush complex do not simply compete with each other on the notal wg enhancer element. However, it also seems to be possible that Pnr and the Pnr-Ush complex compete for the binding site at the notal wg enhancer, but the ability of Pnr-Ush complex to bind this site may be greater than that of Pnr. It is also worth noting that FOG-1, a mammalian homolog of Ush, represses the transactivation of alpha-globin and EKLF promoter by GATA-1, but enhances the transactivation of NF-E2 p45 promoter by GATA-1 in a culture cell system. Dorsocentral (DC) bristles are ectopically formed but postvertical bristles on the head are missing in a loss-of-function allelic combination for ush or in pnrD1 heterozygous flies. These observations suggest that the Pnr-Ush complex acts as a repressor for the DC enhancer, but acts as an activator for the enhancer of postvertical bristles. Only a cis-regulatory element of the DC enhancer has been analyzed at the nucleotide level. Additional studies of the molecular analyses of the cis-regulatory elements of both wg and DC or other enhancers of the achaete-scute complex seem to be necessary in order to reveal the functions of Pnr and Ush (Tomoyasu, 2000).

Generally, at least two different coordinate axes are necessary for positional specification in a two-dimensional field. Morphogen gradients of Dpp and Wg provide this axial information during Drosophila imaginal disc development. In both wing and leg discs, Dpp is induced at the A/P compartment boundary by Hh signaling. In the leg disc, wg is also induced by Hh signaling. Mutual repression between Dpp and Wg signaling separates each expression territory, localizing dpp in the dorsal and wg in the ventral regions abutting the A/P border (a compartment-independent manner). In contrast, wg is induced by Notch signaling only at the D/V compartment boundary in the wing pouch (a compartment-dependent manner). Then, secreted Dpp and Wg proteins provide positional information along the A/P and D/V axes, respectively, to establish Cartesian-like coordinates in the pouch field. Relative positions of dpp and wg expression domains in the notum are more similar to those in the wing pouch (in both cases, the expression domains are orthogonal). However, a D/V compartment boundary does not exist in the notum. The results described here reveal that another compartment-independent mechanism acts to pattern the presumptive notum. Namely, the D/V axis, provided by Pnr, Ush, and Wg, is initially established by the Dpp gradient, which mainly contributes the positional information along the A/P axis. One of the key issues of this patterning model is that Dpp signaling seems to act preferentially along the A/P axis of the notum. This is because two target genes, pnr and ush, are induced farther from the Dpp source along the A/P axis than along the D/V axis. One possible explanation for this phenomenon is that the diffusion of Dpp protein may be positively regulated along the A/P axis. However, such asymmetric induction is not observed on the dad induction; dad is one of the Dpp signaling targets in the wing disc. This suggests that diffusion of Dpp protein is not directionally regulated in the notum region. An alternative explanation would be that an effective range of Dpp morphogen gradient is established in a relatively short range. Cells that respond to Dpp would proliferate or migrate preferentially along the A/P axis. pnr mRNA is detected mainly in the posterior-dorsal region of the presumptive notum. GFP expression of UAS-gfp pnrmd237 is seen along the entire dorsal side of the presumptive notum. This difference between the staining pattern of pnr mRNA and GFP expression of UAS-gfp pnrmd237 in the late third larval stage seems to be caused by a long half-life of gal4 and/or gfp products, suggesting that cells that once have expressed pnr mRNA proliferate preferentially along the A/P axis. However, it seems to be difficult to explain the determination of pnr and ush expression domains only by the Dpp morphogen gradient. The existence of Tkv*-insensitive cells for inducing pnr and ush indicates that some regional subdivision may occur independently of Dpp signaling. Discontinuous expression of dpp in the A/P border of the notum also suggests the existence of a Dpp-independent subdivision. D/V subdivision of the presumptive notum seems to be achieved by several parallel mechanisms, including Dpp signaling (Tomoyasu, 2000).

Because Drosophila is a holometabolous insect, it should destroy larval tissues and replace them with a different population of cells to form the adult structure during the pupal stage. Thus, formation of epidermal structure should occur reiteratively during embryogenesis and metamorphosis. Patterning of larval epidermal structure takes place during embryogenesis; however, patterning of adult structure is mainly performed in larval stage imaginal discs. The Dpp morphogen gradient has been shown here to regulate pnr and ush expression to pattern the presumptive notum, which forms the dorsal structure of the adult, in the wing imaginal disc. pnr and ush are necessary for the formation of amnioserosa, the dorsal structure of the embryo, and both pnr and ush expressions are also positively regulated by Dpp in a concentration-dependent manner during embryogenesis. Furthermore, dorsal closure during embryogenesis and thorax closure in metamorphosis is also analogous, because both processes are regulated by the same signaling cascade, JNK signaling. These similarities between embryogenesis and metamorphosis presumably reflect the evolutionary history of the development in holometabolous insects (Tomoyasu, 2000).

During development, the imaginal wing disc of Drosophila is subdivided along the proximal-distal axis into different territories that will give rise to body wall (notum and mesothoracic pleura) and appendage (wing hinge and wing blade). Expression of the Iroquois complex (Iro-C) homeobox genes in the most proximal part of the disc defines the notum, since Iro-C- cells within this territory acquire the identity of the adjacent distal region, the wing hinge. How is the expression of Iro-C confined to the notum territory? Neither Wingless signaling, which is essential for wing development, nor Vein-dependent EGFR signaling, which is needed to activate Iro-C, appears to delimit Iro-C expression. A main effector of this confinement is the TGFß homolog Dpp, a molecule known to pattern the disc along its anterior-posterior axis. At early second larval instar, the Dpp signaling pathway functions only in the wing and hinge territories, represses Iro-C and confines its expression to the notum territory. Later, Dpp becomes expressed in the most proximal part of the notum and turns off Iro-C in this region. This downregulation is associated with the subdivision of the notum into medial and lateral regions (Cavodeassi, 2002).

In third instar wing discs, the expression of dpp in both proximal and distal territories does not suggest a function in regulating the domain of Iro-C. However, in the second instar disc dpp is expressed in distal regions but it is absent from the Iro-C domain. Dpp is a diffusible molecule and, therefore, its range of activity was determined by monitoring the phosphorylated form of the Mad protein (pMad), an intermediate of the Dpp transduction pathway. pMad accumulates in the cells near the source of Dpp, but it is reduced or absent within the Iro-C domain. Another useful indicator of Dpp activity is the type I TGFß receptor Thick veins (Tkv), since its expression is negatively regulated by Dpp signaling. In addition, high levels of Tkv can limit Dpp diffusion and help to confine the region in which the pathway will be activated. The Iro-C domain is located within a region of high accumulation of Tkv, a result compatible with Dpp activity being strongly reduced or absent from that domain (Cavodeassi, 2002).

The complementary territories of Iro-C and dpp signaling activity (pMad) have suggested that the Dpp pathway might repress Iro-C at the early stages of wing disc development. The levels of Dpp signaling were therefore manipulated and the expression of Iro-C was monitored. In the strong hypomorphic dppd12/dppd14 combination, the Iro-C domain comprises most cells of the early wing disc and its distal border is very close to a small area that corresponds to the wing pouch, as identified by the Nubbin (Nub) marker. Since the Iro-C and the Nub domains are well separated in wild-type discs of similar age, this suggested that the Iro-C domain is distally expanded in the dppd12/dppd14 discs and covers at least part of the hinge/proximal-wing territory. However, it might be argued that the expansion of the Iro-C territory is an illusion caused by the apposition of an essentially normal notum to a hinge/wing territory dwarfed by reduced Dpp signaling. This was not the case. By following the development of these discs, it was observed that Iro-C proteins were gradually removed from part of the putative ectopic domain. The region in which Iro-C was gradually switched off was identified as hinge territory by two criteria: (1) it accumulates the Tsh protein very strongly, and (2) it develops a group of several sensory organ precursor cells; such characteristic groups develop in the hinge, but never in the notum. However, ectopic Iro-C expression is maintained in other distal regions. Consistent with the distal expansion of Iro-C in second instar dppd12/dppd14 discs, the Iro-C domain is coextensive with that of Tsh, which includes the territory fated to become hinge. This coexpression is never observed in wild-type discs. Note that the gradual removal of Iro-C protein from the prospective hinge in dppd12/dppd14 discs indicates that, even under conditions of strong Dpp insufficiency, the distal border of the Iro-C domain can be generated, at least in part. This could be due to residual Dpp signaling and/or to additional uncharacterized factors, which would normally contribute to maintain and refine this border. To help distinguish between these alternatives, the effect of the complete loss of reception of the Dpp signal was examined by generating, during the first instar, clones mutant for the null tkva12 allele. Owing to the difficulty of detecting cell clones in second instar discs, they were examined in third instar discs. In these tkva12 clones, the domain of Iro-C expression appears distally expanded, as detected by comparison with the domain of Tsh expression. This, however, is not the case for clones located in the more anterior part of the disc. Note again that this region coincides with that in which Iro-C is first expressed and later removed in dppd12/dppd14 discs. This suggests that after the initial restriction of Iro-C by Dpp signaling, additional factors contribute to maintain the anterior part of the Iro-C border (Cavodeassi, 2002).

Dpp signaling was next increased by misexpressing UAS-dpp in the proximal region of the disc (MS248-Gal4 driver); it downregulates Iro-C in a large part of the notum territory. Misexpression in cell clones of a constitutively activated form of Tkv (UAS-tkvQD) also suppresses Iro-C expression autonomously, although not completely in some regions. It is concluded that Dpp signaling must be absent (or strongly reduced) from the notum territory for Iro-C expression. Consistently, misexpression of the Dpp pathway antagonists UAS-brinker or UAS-daughters against dpp within this territory (MS248-Gal4) does not detectably affect the expression of Iro-C in second instar discs (Cavodeassi, 2002).

During the third instar, after Iro-C has specified the prospective notum, dpp is turned on in this territory and helps effect its patterning. The activation of dpp in the proximal-most region of the prospective notum is accompanied by a gradual removal of Iro-C, a repression essential to specify the medial versus the lateral notum. Dpp is responsible for this downregulation, since it is prevented by decreasing (dppd12/dppd14 mutant) or abolishing (clones mutant for a null tkv allele) Dpp signaling. In contrast, constitutive activity of the Dpp pathway in cell clones autonomously inhibits Iro-C in the lateral notum, except in a region overlapping or very close to an endogenous source of Dpp. Thus, while in the medial notum there is a correspondence between Dpp expression and Iro-C repression, this correlation does not hold everywhere in the lateral notum, where the appearance of Dpp expression may not result in turning off Iro-C. Interestingly, vn is also maximally expressed in the region of overlap of dpp and Iro-C expressions, and might antagonize, through the activation of EGFR signaling, the repression of the Iro-C genes by the Dpp pathway. It is concluded that, in the third instar disc, the levels of Dpp signaling are critical to establish the medial-lateral subdivision of the notum by its negative regulation of Iro-C in the medial region. This negative regulation should be mediated by pannier, which is activated by dpp in the medial notum (Cavodeassi, 2002).

The eyegone (eyg) gene is involved in the development of the eye structures of Drosophila. eyg and its related gene, twin of eyegone (toe), are also expressed in part of the anterior compartment of the adult mesothorax (notum). The anterior compartment is termed the scutum and consists of the part of the notum from the anterior border to the suture with the scutellum. In the absence of eyg function the anterior-central region of the notum does not develop, whereas ectopic activity of either eyg or toe induces the formation of the anterior-central pattern in the posterior or lateral region of the notum. These results demonstrate that eyg and toe play a role in the genetic subdivision of the notum, although the experiments indicate that eyg exerts the principal function. However, by itself the Eyg product cannot induce the formation of notum patterns; its thoracic function requires co-expression with the Iroquois (Iro) genes. The restriction of eyg activity to the anterior-central region of the wing disc is achieved by the antagonistic regulatory activities of the Iro and pnr genes, which promote eyg expression, and those of the Hh and Dpp pathways, which act as repressors. It is argued that eyg is a subordinate gene of the Iro genes, and that pnr mediates their thoracic patterning function. The activity of eyg gives rise to a new notum subdivision that acts upon the pre-extant one generated by the Iro genes and pnr. As a result the notum becomes subdivided into four distinct genetic domains (Aldaz, 2003).

Localized expression of eyg/toe is achieved by the activity of two antagonistic factors: the promoting activity induced by the Iro and pnr genes, and the repressing activities exerted by the Hh and the Dpp pathways. The latter are probably mediated by Hh and Dpp target genes that are yet to be identified (Aldaz, 2003).

The elimination of eyg activity from the scutellum and lateral notum is caused by the Hh and Dpp pathways. Because the AP compartment border is displaced posteriorly in the notum, these two pathways are active at high levels in the posterior region of the mesothorax. Assuming that the two signals behave as in the wing, Hh activity will be higher in the region close to the AP border, whereas the effect of Dpp will extend further anteriorly. Thus the repressive role of Hh will be greater in the proximity of the AP border and that of Dpp will be greater in more anterior positions. This is precisely what the results indicate. In the region close to the AP border the high Hh levels alone are sufficient to repress eyg. However. in more anterior positions, close to the border of the eyg domain, Hh levels are lower and, although Hh is still necessary, it is not sufficient to repress eyg. Here there is an additional requirement for Dpp activity (Aldaz, 2003).

Thus, the part of the notum that does not express eyg can be subdivided into two distinct zones according to the mode of eyg regulation: a region close to the AP border that requires only Hh, and a more anterior region that requires both Hh and Dpp. In the posterior compartment, the repression of eyg has to be achieved by a different mechanism because neither the inactivation of the Hh pathway in smo- clones, nor the inactivation of the Dpp pathway, induces ectopic eyg activity. A probable possibility is that en itself may act as repressor (Aldaz, 2003).

The result of the antagonistic activities of the Iro genes and pnr in one case and of the Hh and Dpp signalling pathways in the other, subdivides the notum into an eyg/toe expressing domain and a non-expressing domain. The localized expression of eyg/toe contributes to the morphological diversity of the thorax, as it distinguishes between an anterior-central region and a posterior-lateral one. It provides another example of a genetic subdivision of the body that is not based on lineage. It also provides an example of a patterning gene acting downstream of the combinatorial code of selector and selector-like genes. Its mode of action supports a model in which the genetic specification of complex patterns, such as the notum, is achieved by a stepwise process involving the activation of a cascade of regulatory genes (Aldaz, 2003).

DPP in the genital disc

The integration of multiple developmental cues is crucial to the combinatorial strategies for cell specification that underlie metazoan development. In the Drosophila genital imaginal disc, which gives rise to the sexually dimorphic genitalia and analia, sexual identity must be integrated with positional cues, in order to direct the appropriate sexually dimorphic developmental program. Sex determination in Drosophila is controlled by a hierarchy of regulatory genes. The last known gene in the somatic branch of this hierarchy is the transcription factor doublesex (dsx); however, targets of the hierarchy that play a role in sexually dimorphic development have remained elusive. The gene dachshund (dac) is differentially expressed in the male and female genital discs, and plays sex-specific roles in the development of the genitalia. Furthermore, the sex determination hierarchy mediates this sex-specific deployment of dac by modulating the regulation of dac by the pattern formation genes wingless (wg) and decapentaplegic (dpp). The sex determination pathway acts cell-autonomously to determine whether dac is activated by wg signaling, as in females, or by dpp signaling, as in males (Keisman, 2001).

A number of obstacles make it difficult to demonstrate that the sex determination pathway is responsible for the sex-specific regulation of a gene in the genital disc. These obstacles stem from the fact that the male and female primordia, which are the primary constituents of their respective discs, differ in their segmental origin. This raises the possibility that 'sex-specific' gene regulation is really just segment-specific gene regulation, made to look sex specific by the fact that only one primordium develops in each sex. Attempts were made to address this concern by creating clones of the opposite genetic sex in chromosomally male and female genital discs. Thus, for example, dac regulation could be examined in the male (A9) primordium, in both male and female cells. By varying the genetic sex of cells in a context where segmental identity is uniform, it was hoped that the contributions of sex and segmental identity to dac regulation could be disentangled (Keisman, 2001).

In the male primordium of both male and female discs, the regulation of dac varies according to the genetic sex of the cell. Genetically female clones in the male (A9 derived) primordium of the male genital disc are unable to express dac in the lateral male (dpp-dependent) domain, but are able to express dac when they extended medially, towards the source of Wg. Conversely, in the female genital disc, genetically male clones in the repressed male primordium (A9) lose their ability to express dac in the medial, wg-dependent domain, and begin to express dac laterally, presumably in response to Dpp. Finally, dac expression is abnormal in intersexual genital discs from dsx mutant larvae: the male primordium of dsx genital discs expresses dac in both the endogenous, lateral male domains, and in a slightly weaker medial domain that corresponds roughly to the region where tra + clones are able to activate dac. Thus, it is concluded that in the male primordium, the sex determination pathway determines how a cell will regulate dac (Keisman, 2001).

In the female primordium the results fail to show a role for the sex determination pathway in dac regulation. If such a role exists, it would be expected that genetically male clones in the female primordia of a female genital disc would activate dac laterally, like their counterparts in the male primordia. They do not, even when they take up much of the presumptive dpp-expressing domain. It would also be expected that such clones would repress dac medially. Only a few clones were observed to extend into the medial wg-expressing domain, and as expected these appear to repress dac. Interpretation of these results is complicated by the fact that changing the genetic sex of a cell in the genital disc can cause it to enter the 'repressed' state. Thus, for example, if a genetically male clone represses dac when it intersects the medial dac domain in the female primordia, it can be concluded either that the sex determination pathway regulates dac expression or that the cells, which are now male, have adopted a repressed state and are generally unresponsive. A similar caveat prevents interpreting the failure of tra2IR clones to activate dac ectopically in the female primordium. That tra + clones in the male primordium of male genital discs enter such a generally non-responsive state was not of concern, because these clones both repress and activate dac expression. The expression pattern of dac in the female primordium of a dsx mutant genital disc is also difficult to interpret. dac is not activated ectopically in the lateral domains of the dsx female primordium, which is consistent with the failure of tra2IR clones to cause such activation. However, even the medial, wg-dependent dac domain is frequently absent or severely reduced in the dsx female primordium, and thus the authors are reluctant to draw any conclusions from the absence of ectopic dac laterally (Keisman, 2001).

A model is proposed for dac regulation in the male primordium, in which the different isoforms of Dsx protein modulate dac regulation by wg and dpp. In the absence of dsx, both wg and dpp can activate dac, producing the two domains of dac expression observed in the male primordium of a dsx disc. In the female, Dsxf modulates dpp activity so that dpp becomes a repressor of dac; Dsxf may also potentiate the activation of dac by wg. In the male, Dsxm modulates wg activity so that it becomes a repressor of dac, leaving dpp alone to activate dac. In support of this model, it is noted that the Dsx proteins act in a similar manner to positively or negatively modulate the effect of tissue-specific regulators on the yp genes (Keisman, 2001 and references therein).

The behavior of tra + and tra2IR clones provides insight into the mechanism of repression in the undeveloped genital primordium. It was anticipated that such clones would be difficult to recover when they occurred in the male and female primordium, respectively, because they should adopt the repressed state. Instead, large tra + (female) clones were recovered in the male primordium of a male disc, and large tra2IR (male) clones were recovered in the female primordium of a female disc. Some of these clones constitute a substantial fraction of the primordium in question. Though tra + or tra2IR clones were not scored in adults, previous studies strongly suggest that such clones would fail to differentiate adult genital structures (Keisman, 2001).

It has been shown that tra - (male) clones cause large deletions in the female genitalia, indicating that genetically male cells like those in a tra2IR clone divide but cannot differentiate female genital structures. Further, it has also been shown that male structures are deleted when the mosaic border passes through the male genitalia, suggesting that female tissue cannot differentiate male structures. To reconcile these data, it is proposed that repression of the inappropriate genital primordium involves two separable processes: repression of growth and the prevention of differentiation. Thus, clones of cells of the inappropriate genetic sex cannot differentiate, but they can grow and contribute to a morphologically normal genital primordium. This poses yet another question. Cells in a tra + clone in the male primordium of a male genital disc are analogous to the cells in the repressed male primordium of a wild-type female genital disc: both are genetically female, and both have A9 segmental identity. Why do tra + clones in the male primordium grow, while the repressed male primordium in a female disc does not? One possibility is that the decision of the male primordium to grow in a male disc is made before tra + clones were induced and cannot be over-ridden by a later switch of genetic sex. However, temperature-shift experiments with tra-2 ts alleles suggest that the decision of a genital primordium to develop can be reversed later in development. Furthermore, occasional, large tra + clones can cause severe reductions in male genital discs. This observation leads to the suggestion of a model in which growth in the genital disc is regulated from within organizing zones, such as the domains of wg and dpp expression. According to this model, the sex of the cells in the organizing regions would determine how the disc grows, while cells in other regions would respond accordingly, regardless of their sex. The tra + clones that cause reduction could result when such a clone intersects with one of the postulated organizing centers within the disc. The implication is that the sex determination pathway acts in yet undiscovered ways to modulate the function of the genes that establish pattern in the genital disc. One such interaction was found in the regulation of dac; further study is needed to determine if others exist, and what role they play in producing the sexual dimorphism of the genital disc and its derivatives (Keisman, 2001).

Table of contents

decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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