Table of contents

Regulation in segment polarity (part 2/2)

Engrailed and Invected repress decapentaplegic and patched in posterior compartments of imaginal discs. Mutant clones completely lacking both en and invected activity ectopically express dpp-lacZ reporter genes in the posterior compartment, where dpp activity ordinarily is repressed. Similarly, patched is also ectopically expressed in such posterior compartment en-inv- null clones. These en-inv- clones also exhibit loss of hedgehog expression. Absence of dpp expression in the posterior compartment is due to direct repression by EN. Ubiquitious expression of en in imaginal discs, eliminates the expression of dpp in its normal A/P boundary stripe. Thus it is probable that an en-hh-ptc regulatory loop responsible for segmental expression of wingless in the embryo is reutilized in imaginal discs to create a stripe of dpp expression along the A/P compartment boundary (Sanicola, 1995).

dpp> is a target of the hh signal acting through Fused. fu mutations rescue the phenotype due to ectopic expression of hh or to the lack of patched activity. fu is also required for the activation of engrailed caused when hh is ectopically activated in the wing disc. Although fu, cos-2 and ci probably form part of the same pathway that controls dpp expression, Protein kinase A probably controls dpp expression by a different pathway (Sánchez-Herrero, 1996).

The Suppressor of fused [Su(fu)] encodes a protein with a PEST sequence involved in rapid protein turn-over. Fused is phosphorylated in response to the Hh signal. A large protein complex that includes Cubitus interruptus, Costal-2 and Fused binds to microtubules and has been implicated in the regulation of Ci cleavage and accumulation, and may be involved in mediating the Hh signal. Although Su(fu) activity is apparently dispensable in a wild-type background, its absence fully suppresses all the fused mutant phenotypes. These data suggest that the activation of Fused in cells receiving the Hh signal relieves the negative effect of Su(fu) on the pathway (Alves, 1998 and references).

The roles of Fused and Su(fu) proteins were examined in the regulation of Hh target gene expression in wing imaginal discs, by using different classes of fu alleles and an amorphic Su(fu) mutation. The fused phenotype consists of a vein 3 thickening and vein 4 disappearance with reduction of the intervein region. At the wing margin, the anterior double row bristles reach the fourth vein. Fused protein is present throughout the entire wing level, but its level is much higher in the anteior compartment. In contrast, fused transcripts are uniformly distributed, suggesting that fused is regulated post-transcriptionally. Observations using fused clones indicate that only fused minus clones located in the region extending between veins 3 and 4 generate a mutant phenotype, consisting of extra-veins, which often bear campaniform sensillae characteristic of vein 3. Thus Fused kinase activity is required at the anterior/posterior (AP) boundary in the anterior compartment. At the AP boundary, Fu kinase activity is involved in the maintenance of high ptc expression and in the induction of late anterior engrailed expression. These combined effects can account for the modulation of Ci accumulation and for the precise localization of the Dpp morphogen stripe. Here, at the AP boundary, Hh signal activates the Fu kinase, leading to a modified active form of Ci required for anterior en expression and high ptc expression. Su(fu) suppresses all fused phenotypes associated with the AP boundary, suggesting that Su(fu) normally functions to antagonize the effects of Fused (Alves, 1998).

Two classes of fused mutants are described with respect to more anterior cells, which are so distant from the AP boundary that they do not receive Hh signal. Class I and class II fused alleles encode structurally different proteins; fused class I alleles encode mutant proteins altered in the catalytic domain but containing at least the 300 C-terminal amino acids, where class II alleles encode proteins truncated in the C-terminal, non-catalytic domain. In class II fused mutant discs, but not in class I mutants, abnormal dpp-lacZ expression is detected at the anterior-dorsal part of the disc in the presumptive hinge region of the wing. This ectopic expression is not correlated with any phenotype, but an interaction of fused with Su(fu) is observed. This interaction consists of an overgrowth of the anterior compartment accompanied by ectopic dpp-lacZ. Taken together, these results demonstrate that whereas at the AP boundary Fu and Su(fu) have opposite effects on the level of ptc and dpp expression, in the anterior compartment class II fused mutant products activate dpp expression and this effect is enhanced when Su(fu) is absent. Thus Fu plays a role independent of its kinase function (but dependent on its C-terminal domain) in the regulation of Ci accumulation in the anterior compartment. In these cells, Fu may be involved in the stabilization of a large protein complex that is probably responsible for the regulation of Ci cleavage and/or targeting to nucleus. In the anterior compartment, no Hh signal is received and Ci cleavage give rise to a short Ci form that represses dpp expression (Alves, 1998).

The Cubitus interruptus controls the transcription of Hedgehog (Hh) target genes. A repressor form of Ci arises in the absence of Hh signalling by proteolytic cleavage of intact Ci, whereas an activator form of Ci is generated in response to the Hh signal. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. To investigate the mechanisms by which the two activities of Ci exert their opposite transcriptional effect, the imaginal disc enhancer of the dpp gene, which responds to both activities of Ci, has been dissected. Within a minimal disc enhancer, the DNA sequences have been identified that are necessary and sufficient for the control by Ci. The same sequences respond to the activator and repressor forms of Ci; their activities can be replaced by a single synthetic Gli-binding site. The enhancer sequences of patched, a gene responding only to the activator form of Ci, effectively integrate also the repressor activity of Ci if placed into a dpp context. These results provide in vivo evidence against the employment of distinct binding sites for the different forms of Ci and suggest that target genes responding to only one form must have acquired distant cis-regulatory elements for their selective behavior (Muller, 2000).

A minimal dpp imaginal disc enhancer has been isolated, which, like the endogenous dpp gene, is regulated by Ci[act] and Ci[rep] activities. In the absence of the internal 100 bp fragment G, sensitivity to both Ci[act] and Ci[rep] is lost. Two Ci-binding sites are necessary for the activity of fragment G and a single synthetic Gli consensus site is sufficient to replace their function, restoring sensitivity to both Ci[act] and Ci[rep]. Furthermore, the ability of a single synthetic Gli-binding site to respond both to Ci[act] and Ci[rep] is preserved in the context of a different, naive enhancer, indicating that a single Gli/Ci-binding site is intrinsically able to mediate both inputs (Muller, 2000).

A model is presented for the activity of Ci along the anteroposterior axis of imaginal discs. The opposing transcriptional activities (Ci[rep] and Ci[act]) are exerted by the distinct molecular forms Ci-75 and activated Ci-155, respectively. The distribution and activity of these different forms of Ci is controlled by Hh signaling. Close to the compartment boundary where Hh signaling activity is high, Ci[act] is prevailant. More anterior, in cells that do not receive Hh, Ci[rep] predominates. The expression of dpp and of the Ci target construct responds both to Ci[act] and Ci[rep]. This responsiveness to both forms of Ci is mediated by common Gli/Ci-binding sites. In A cells close to the AP boundary, these sites would be occupied by Ci[act] and in more anterior cells by Ci[rep]. Both forms of Ci would alter the activity of a ubiquitously present activator which on its own might enable low basal transcription by opening the chromatin structure. Ci[act] would recruit the Pol II-associated transcriptional machinery and hence synergistically enhance this basal activity, whereas Ci[rep] might repel this same complex and suppress the basal activity (Muller, 2000).

dpp may be autoregulating in the eye imaginal disc. The observation that dpp is not expressed in mutant >Mad eye margin clones raises the possibility that DPP signaling is required for the maintenance of dpp expression. Supporting this, mutation in dpp itself reduces expression of a dpp reporter gene in the eye margin (Wiersdorff, 1996).

The secreted protein Hedgehog has been identified as the signal transmitted along retinal axons which serves as the inductive signal triggering neurogenesis in the lamina. The target of HH in the developing visual system is wingless, which in turn targets decapentaplegic and Distal-less. The lamina neurons and the cortical neurons that contribute axons to the medulla neuropil derive from a neuroblast population (OPC or outer proliferation center) that divides throughout most of larval development. Although cells expressing wg constitute only a small fraction of the OPC, the inactivation of wg at early times results in the later absence of nearly the entire target structure (Kaphingst, 1994).

Wingless regulates the onset and maintenance of dpp expression. Approximately 14 hr after the onset of wg expression, dpp expression begins in single cell domains immediately adjacent to the wg-expressing cells, and is maintained throughout larval development as these cell populations divide up to and including the period of retinal axon ingrowth. In dpp mutants many OPC progeny fail to down-regulate the expression of the cell adhesion molecule fasII, fail to express neuron markers, and fail to contribute axons to the medulla neuropil (Kaphingst, 1994).

Distal-less expression is found in wg-expressing cells adjacent to the dpp domains. dll expression is significantly greater in the dorsal domain. The involvement of dll in neurogenesis in Drosophila has yet to be documented (Kaphingst, 1994). Hedgehog acts upstream of glass, scabrous, hairy and decapentaplegic in the developing eye (Ma, 1993).

The regulation and function of the Hedgehog pathway activity has been compared in eye and wing discs, and there are significant differences. Whereas in the wing disc, engrailed function is required for hedgehog expression, in the eye disc activation and maintenance of hedgehog expression is achieved independently of engrailed. Nevertheless, engrailed functions in the eye disc, as elevated engrailed expression represses dpp, patched and cubitus interruptus in the eye disc, but does not disrupt morphogenesis. Regulation of decapentaplegic expression also differs: in the wing disc it is repressed in the anterior compartment by patched and in the posterior compartment by engrailed. In the eye disc, however, it is repressed posterior to the morphogenetic furrow in the absence of either patched or engrailed activity (Strutt, 1996).

Patterning of the compound eye begins at the posterior edge of the eye imaginal disc and progresses anteriorly toward the disc margin. The advancing front of ommatidial differentiation is marked by the morphogenetic furrow (MF). Hedgehog (Hh), secreted from two distinct populations of cells has two distinct functions: (1) MF propagation and (2) MF initiation. There is in addition a third function of Hh neural patterning. Hh has an early essential role in the initiation of the MF, in addition to its role in MF propagation. Loss of hh from the disc margin, where it is expressed prior to the onset of eye patterning, impedes growth of the disc and prevents all aspects of MF initiation. These results are in conflict with previous reports suggesting that Hh is not required for MF initiation; these reports showed that MF initiation is normal in the hypomorphic hh1 allele. However, it is likely that MF initiation in hh1 initiates normally because the early expression of hh in the margin is normal in the hh1 mutant. In contrast to the direct role of Hh in MF initiation, it appears that the control of MF initiation by Dpp is indirect; it acts by repressing wg. The primary function of Dpp in MF initiation is the repression of wg, which prevents ommatidial differentiation. As in the leg disc, the early expression of dpp and wg may be induced by Hh. It is likely that Hh directly induces early expression of wg and dpp by antagonizing pka activity at the eye disc margin. In addition to a requirement for Hh in MF initiation and propagation, Hh, secreted from cells at the posterior disc margin, is absolutely required for the initiation of patterning and predisposes ommatidial precursor cells to enter ommatidial assembly later. As the MF progresses in a mosiac disc carrying a marginal hh cone, only a single ommatidial unit adjacent to Hh-secreting ommatidia is rescued. Hh induces ommatidial development in the absence of its secondary signals Wg and Dpp. These two functions of Hh in eye patterning (initiation of patterning followed later by differentiation of neurons) are similar to the biphasic requirement for Sonic Hh in patterning of the ventral neural tube in vertebrates (signaling from notochord to ventralize the neural tube followed later by specification of motor neurons by Sonic hedgehog secreted from the floor plate). These results show that the regulatory relationships between Hh, Dpp, and Wg in the eye are similar to those found in other imaginal discs, such as the leg disc, despite obvious differences in their modes of development (Dominguez, 1997).

Homeosis and Homeotic Complex (Hox) regulatory hierarchies have been evaluated in the somatic and visceral mesoderm. Both Hox control of signal transduction and cell autonomous regulation are critical for establishing normal Hox expression patterns and the specification of segmental identity and morphology. Novel regulatory interactions have been identified associated with the segmental register shift in Hox expression domains between the epidermis/somatic mesoderm and visceral mesoderm. A proposed mechanism for the gap between the expression domains of Sex combs reduced (Scr) and Antennapedia (Antp) in the visceral mesoderm is provided. Previously, Hox gene interactions have been shown to occur on multiple levels: direct cross-regulation, competition for binding sites at downstream targets and through indirect feedback involving signal transduction. Extrinsic specification of cell fate by signaling can be overridden by Hox protein expression in mesodermal cells and the term autonomic dominance is proposed for this phenomenon. The endoderm was used to monitor target gene regulation by the Hox proteins (specifically wg, dpp and lab) through signal transduction (Miller, 2001b).

During an investigation of Hox cross-regulation in the midgut visceral mesoderm it was demonstrated that both Antp and Ubx are responsible for the proper maintenance of the posterior boundary of Scr expression in ps4. It is proposed that Ubx represses Scr at this position extrinsically from nearby tissues. The segmental register shift in Hox expression domains found between the epidermis/somatic mesoderm/CNS and visceral mesoderm juxtaposes Ubx expression (ps5) to a position where it can influence Scr expression in the visceral mesoderm (ps4). Since Ubx activates dpp, which represses Scr in the visceral mesoderm, it seems reasonable to conclude that the interaction seen involves the action of dpp. Hox cross-regulation studies demonstrate that ectodermal Gal4 drivers producing ectopic Ubx repress Scr in the visceral mesoderm while stimulating dpp-LacZ expression. Ubx expression in the somatic mesoderm, which is between the epidermis and visceral mesoderm, may be the tissue that actually contributes the signaling influence demonstrated in this interaction. However, the responder only contains the visceral mesoderm regulatory elements and does not demonstrate that dpp gene activation is the signaling source in these outer tissues. Interestingly, ectopic expression of Abd-A outside the visceral mesoderm also demonstrates a posterior expansion of Scr expression in the visceral mesoderm, presumably since it represses Ubx there. Similarly, Antp repression of Scr in ps5 of the visceral mesoderm appears to be through signaling. Scr and Antp expression does not entirely fill the gap when Ubx expression is removed. Additionally, in Antp null mutants, Scr accumulation is seen in cells that normally express Antp in the presence of normal Ubx expression. By counting Scr expressing cells in the visceral mesoderm, it was found that Antp represses Scr in this tissue, contrary to previous reports. Interestingly, ectopic Antp in ectodermal tissues has no effect on ectodermal Scr expression. Thus, both Ubx and Antp contribute to define the Scr domain at its posterior visceral mesoderm boundary apparently through signal transduction (Miller, 2001b).

Scr inductively stimulates growth of the gastric caecae in the visceral mesoderm but seems to block it cell autonomously. Evidence was found for ectopic gastric caecae primordia in ps7 of the visceral mesoderm associated with ectopic Scr. Interestingly, the presumptive gastric caecae primordia at ps7 is generated in the same signaling environment as the native gastric caecae in ps3. During normal gastric caecae development at ps3, wg expression is observed at ps2 and dpp expression is observed at ps3. In ectopic Scr embryos polyps (ectopic gastric caecae?) are found at ps7. The signaling environment for this region of the midgut has dpp at ps7 and wg at ps8. Ectopic expression of Scr alters neither wg expression in ps8 nor dpp expression at ps7. Ectopic Hox expression (including Scr) suppresses complete gastric caecae development at ps3. This suggests that the signaling environment at ps3 may be responsible for development of the gastric caecae while Hox proteins block this morphogenesis cell autonomously (autonomic dominance). A complete map of signal transduction gene expression and Hox influences is critical for understanding this morphogenic process since other signaling agonists are likely also involved (Miller, 2001b).

Ectopic Hox protein expression in the mesoderm can induce lab, lab-LacZ and dpp-LacZ expression in the midgut. Typically, the anterior ectopic endodermal lab expression parallels the observed expression pattern in the visceral mesoderm. The lack of ectopic lab expression posterior to ps7 is probably due to the unaltered high levels of wg expression, that repress lab. Normal lab induction in the endoderm requires wg, dpp and vein; however, dFos dependent (wg independent) lab transcription can be accomplished with high Dpp levels. Typically, lab induction by dpp, wg and vein is coordinated by sgg (GSK3) which may be responsible for the ps4 gap in lab, lab-LacZ, and expression patterns seen in experiments involving ectopic Antp visceral mesoderm expression. Moreover, the lack of expanded lab-LacZ expression (unlike native lab) by ectopic Antp indicates the existence of presently undefined cis-regulatory elements at the lab locus that are not contained in genomic fragments of the identified enhancers. Antp protein may be regulating other influential signaling pathways while the corresponding cis-acting elements are not located in the genomic lab enhancers tested. Antp expression is functionally linked to another TGF-beta agonist 60A (glass bottom boat), as well as the Wnt pathway agonist DWnt4 (Miller, 2001b).

It is concluded that Hox gene interactions in the mesoderm are not always consistent with previous governing hierarchies: posterior dominance and phenotypic suppression. In the visceral mesoderm it is found that posterior dominance (Hox direct cross-regulation) seems legitimate but may be mediated by signal transduction. Phenotypic suppression is violated by morphological changes and target gene regulation. In the somatic mesoderm, more anterior Hox genes alter the identity of the ventral T2 segment, but this tissue is largely extrinsically regulated in the absence of direct Hox expression. In light of this result, the notion of autonomic dominance is proposed: Hox genes cell-autonomously dominate tissues regulated by signal transduction (Miller, 2001b).

The predominant paradigm depends on whether cells are extrinsically or autonomously specified by Hox gene expression. It is argued that non-typical homeosis caused by ectopically expressed Hox proteins (i.e. not following the dictates of posterior prevalence) can be taken to indicate inductively specified tissues and hence, confer autonomic dominance. Interestingly, ectopic expression of the Hox proteins also exhibit non-typical homeosis in the chordotonal organs of the PNS and the thoracic cuticle, suggesting that inductive specification and autonomic dominance may not be restricted to the mesoderm. However, Hox regulatory hierarchies seem to be of limited value in other tissues as well. The mechanism responsible for autonomic dominance has not been determined in this study; only the correlation between autonomous Hox dominance over inductively specified tissue. Signal transduction pathway cross-talk could be the predominant cause of autonomic dominance phenotypes (homeosis) due to Hox regulation of signaling agonists. These agonists could then contribute to the signaling environment to alter the tissue, since these morphogens are potent factors in differentiation. Meanwhile, Hox genes cross-regulate each other cell autonomously and in nearby tissues through signal transduction. This occurs in a tissue specific manner that likely depends on both the signaling environment, transcriptional co-factors, and perhaps any of an estimated 100 target genes for a given Hox protein. The signaling environment of any given tissue is dictated primarily by Hox genes, which is critical for maintenance of Hox expression domains and subsequent differentiation, determination and morphogenesis. This complex set of intrinsic and extrinsic Hox controls are likely responsible for the means by which Hox genes were genetically identified for their abilities to dominate segmental identities as homeotic selector genes (Miller, 2001b).

STAT is an essential activator of the zygotic genome in the early Drosophila embryo

In many organisms, transcription of the zygotic genome begins during the maternal-to-zygotic transition (MZT), which is characterized by a dramatic increase in global transcriptional activities and coincides with embryonic stem cell differentiation. In Drosophila, it has been shown that maternal morphogen gradients and ubiquitously distributed general transcription factors may cooperate to upregulate zygotic genes that are essential for pattern formation in the early embryo. This study shows that Drosophila STAT (STAT92E) functions as a general transcription factor that, together with the transcription factor Zelda, induces transcription of a large number of early-transcribed zygotic genes during the MZT. STAT92E is present in the early embryo as a maternal product and is active around the MZT. DNA-binding motifs for STAT and Zelda are highly enriched in promoters of early zygotic genes but not in housekeeping genes. Loss of Stat92E in the early embryo, similarly to loss of zelda, preferentially down-regulates early zygotic genes important for pattern formation. STAT92E and Zelda synergistically regulate transcription. It is concluded that STAT92E, in conjunction with Zelda, plays an important role in transcription of the zygotic genome at the onset of embryonic development (Tsurumi, 2011).

This study describes a bioinformatics approach to investigating the mechanisms controlling transcription of the zygotic genome that occurs during the MZT; STAT92E was identified as an important general transcription factor essential for up-regulation of a large number of early 'zygotic genes'. The role of STAT92E was described in controlling transcription of a few representative early zygotic genes, such as dpp, Kr, and tll, that are important for pattern formation and/or cell fate specification in the early embryo. These studies suggest that STAT92E cooperates with Zelda to control transcription of many 'zygotic genes' expressed during the MZT. While STAT mainly regulates transcription levels, but not spatial patterns, of dpp, tll, and Kr, and possibly also other 'zygotic genes', Zelda is essential for both levels and expression patterns of these genes (Tsurumi, 2011).

The transcriptional network that controls the onset of zygotic gene expression during the MZT has remained incompletely understood. It has been proposed that transcription of the zygotic genome depends on the combined input from maternally derived morphogens and general transcription factors. The former are distributed in broad gradients in the early embryo and directly control positional information (e.g., Bicoid, Caudal, and Dorsal), whereas the latter are presumably uniformly distributed regulators that augment the upregulation of a large number of zygotic genes. Other than Zelda, which plays a key role as a general regulator of early zygotic expression, the identities of these general transcriptional activators have remained largely elusive. It has been shown that combining Dorsal with Zelda- or STAT-binding sites supports transcription in a broad domain in the embryo. The demonstration of STAT92E as another general transcription factor sheds light on the components and mechanisms of the controlling network in the early embryo. Moreover, STAT92E and Zelda may cooperate to synergistically regulate zygotic genes. The results thus validate the bioinformatics approach as useful in identifying ubiquitously expressed transcription factors that may play redundant roles with other factors and thus might otherwise be difficult to identify (Tsurumi, 2011).

The conclusion that STAT92E is important for the levels but not the spatial domains of target gene expression in the early embryo is consistent with several previous reports. It has been shown that in Stat92E or hop mutant embryos, expression of eve stripes 3 and 5 are significantly reduced but not completely abolished. In addition, JAK/STAT activation is required for the maintenance of high levels, but not initiation, of Sxl expression during the MZT. Moreover, it has previously been shown that STAT92E is particularly important for TorsoGOF-induced ectopic tll expression but not essential for the spatial domains of tll expression in wild-type embryos under normal conditions. On the other hand, Zelda may be important for both levels and spatial patterns of gene expression. This idea is consistent with the finding that Zelda-binding sites are enriched in both promoter and promoter-distal enhancers regions, whereas STAT-binding sites are enriched in promoter regions only. It has been reported that pausing of RNA polymerase II is prominently detected at promoters of highly regulated genes, but not in those of housekeeping genes. In light of these results that STAT and Zelda sites are highly enriched in the early zygotic gene promoters, it is suggested that these transcription factors might contribute to chromatin remodeling that favors RNA polymerase II pausing at these promoters (Tsurumi, 2011).

Finally, the MZT marks the transition from a totipotent state to that of differentiation of the early embryo. As a general transcription factor at this transition, STAT, together with additional factors (such as Zelda), is important for embryonic stem cell differentiation. Further investigation is required to understand the molecular mechanism by which STAT and Zelda cooperate in controlling zygotic transcription in the early Drosophila embryo. Moreover, it would be interesting to investigate whether STAT plays similar roles in embryonic stem cell differentiation in other animals (Tsurumi, 2011).

Regulation in mesoderm

The Ultrabithorax (Ubx) homeodomain protein directly activates dpp expression in parasegment 7 (PS7) of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle . The EXD protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp. A second mechanism contributing to PS7 expression of dpp appears not to require UBX protein directly, and involves a general visceral mesoderm enhancer coupled to a spatially specific repression element (Sun, 1995).

Expression patterns of wingless, teashirt and dpp are altered in the embryonic midgut of embryos lacking extradenticle, while the expression of their respective regulators (abd-A, Antp and Ubx) remains normal. Two functions of exd in the regulation of dpp can be identified. Exd acts with Ubx to activate dpp expression in parasegment 7 (PS7), via a minimal visceral mesoderm enhancer, and exd represses dpp expression anterior to PS7. Even when Ubx is ubiquitously expressed at high levels in exd mutant embryos, Ubx is incapable of activating dpp enhancer expression (Rauskolb, 1994).

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

The extracellular signals encoded by the Wnt family of genes regulate growth and differentiation in several developmental processes in both vertebrates and invertebrates. Genetic studies of the signaling pathway of the Drosophila Wnt homolog, Wingless, have identified a number of genes, including zeste white 3, that function to transduce the Wingless signal. zeste white 3 encodes a serine/threonine kinase. zw3 is expressed maternally and uniformally in the early embryo. It has been proposed that the Wingless signal is mediated by repression of this kinase activity. This hypothesis was tested by overexpressing zeste white 3 in a tissue-specific fashion using the UAS/GAL4 binary expression system. The wild-type zw3 cDNA was placed under transcriptional control of the yeast GAL4 upstream activating sequence (UAS). UAS-zw3 flies were mated to flies that express the yeast transcriptional activator GAL4 in either a cell- or tissue-specific fashion to drive chronic expression of zw3. Elevated levels of zeste white 3 in the ectoderm and mesoderm result in phenotypes that resemble a loss of wingless. Overexpression of zeste white 3 in the mesoderm disrupts several Wingless-dependent processes, including the specification of a unique cell type in the larval midgut (the copper cell), the formation of the second midgut constriction, and the expression of Wingless target genes Ultrabithorax and decapentaplegic in the mesoderm, and labial in the endoderm. Interstitial cells normally found interspersed with the copper cells are still present. This loss of copper cells is similar to the phenotypes observed due to a loss of labial expression or wg expression, both required for the specification of the copper cells. The second midgut constriction is dependent on Wg signaling; in wg, dishevelled, or armadillo mutant embryos, this constriction does not form. Interestingly, in zw3 mutant embryos the second midgut constriction does form, but it is abnomal, appearing to have multiple folds. Zeste white 3 regulates the stability of Armadillo, which is essential for transducing the Wingless signal to the nucleus. zeste white 3 overexpression blocks Wingless signaling through the modulation of Armadillo since expression of a constitutively active form of Armadillo, which is independent of Zeste white 3 regulation, is epistatic to overexpression of zeste white 3 (Seitz,1998).

Regulation in gut ectoderm

forkhead is required for the activation of wingless, hedgehog and decapentaplegic in both the foregut and hindgut, considered to be ectodermal tissues. wingless is expressed initially in the whole hindgut primordium, but becomes restricted to a ring in the small intestine anterior to the outgrowing Malpighian tubules, and to a ring in the posterior region of the rectum. hedgehog is also expressed in the hindgut primordium but becomes restricted to a ring of cells posterior to the outgrowing Malpighian tubules in the future small intestine of the hindgut. A second hh expression domain is located in the anterior portion of the rectum. These two expression domains are adjacent to the wg expression domains. dpp is expressed in the hindgut primordium and later on one side in the large intestine of the hindgut tube, in between the small intestine and the rectum. Thus the expression domains of wg, hh and dpp subdivide the hindgut tube into a central portion (the large intestine) where dpp is expressed, and two flanking regions (the small intestine and the rectum) where wg and hh are expressed. In fkh mutant embryos, the foregut, the midgut and the hindgut epithelia are disrupted, and fkh is required for the activation of each of these genes in the fore- and hindgut primordia. fkh is expressed in the entire foregut and hindgut, whereas wg, hh and dpp are expressed only in restricted domains. Since the expression of these genes appear not to be established through cross-regulatory interactions, there must be other factors which act to spatially regulate wg, hh and dpp expression along the hindgut (Hoch, 1996).

Alternative splicing modulates Ubx protein function in Drosophila melanogaster

The Drosophila Hox gene Ultrabithorax (Ubx) produces a family of protein isoforms through alternative splicing. Isoforms differ from one another by the presence of optional segments-encoded by individual exons-that modify the distance between the homeodomain and a cofactor-interaction module termed the 'YPWM' motif. To investigate the functional implications of Ubx alternative splicing, this study analyzed the in vivo effects of the individual Ubx isoforms on the activation of a natural Ubx molecular target, the decapentaplegic (dpp) gene, within the embryonic mesoderm. These experiments show that the Ubx isoforms differ in their abilities to activate dpp in mesodermal tissues during embryogenesis. Furthermore, using a Ubx mutant that reduces the full Ubx protein repertoire to just one single isoform, specific anomalies were obtained affecting the patterning of anterior abdominal muscles, demonstrating that Ubx isoforms are not functionally interchangeable during embryonic mesoderm development. Finally, a series of experiments in vitro reveals that Ubx isoforms also vary in their capacity to bind DNA in presence of the cofactor Extradenticle (Exd). Altogether, results indicate that the structural changes produced by alternative splicing have functional implications for Ubx protein function in vivo and in vitro. Since other Hox genes also produce splicing isoforms affecting similar protein domains, it is suggested that alternative splicing may represent an underestimated regulatory system modulating Hox gene specificity during fly development (Reed, 2010).

The experiments described in this study indicate that the generation of structural differences among Ubx proteins by alternative splicing is relevant for the functional specificity of Ubx in vivo. These structural features modulate essential biochemical properties of Ubx proteins such as their DNA-binding profiles in the presence of a cofactor (Reed, 2010).

The differential effects of Ubx in vivo are apparent in the posterior visceral mesoderm, but not in the anterior. To understand this it must first be noted that the mechanism of dpp674 visceral mesoderm enhancer repression is different anterior and posterior to PS7. In the anterior visceral mesoderm, repression requires Exd, since in Exd null mutants, dpp674 is ectopically expressed anterior to PS7. But Hox genes appear to play no role in the normal repression of dpp in this region. The same Exd-dependent mechanism may also be acting in the posterior, but it is not necessary, for no posterior ectopic expression is observed in Exd null mutants. The posterior repression depends instead on the Hox protein Abd-A, which is presumably able to repress dpp674 in the absence of Exd as a cofactor (Reed, 2010).

All Ubx protein isoforms are able to induce dpp ectopically in the anterior, suggesting that they can all override the normal repression mediated by Exd. However, they exert differential effects in the posterior, where Abd-A is the controlling repressor. Abd-A has a very similar DNA-binding specificity to that of Ubx. It binds to multiple sites in the dpp674 enhancer, including those to which Ubx binds. Through some of these sites it serves as an activator, but through others it acts as a repressor. With Ubx form Ia, the repressing effect by Abd-A is dominant. The results suggest that Ubx form IVa is either able to compete more effectively as an activator with the repressing action of Abd-A bound at other sites or able to displace the binding of Abd-A at the sites where it mediates repression (Reed, 2010).

Ubx form IVa also overrides the normal specificity of the dpp-lacZ enhancer for the visceral mesoderm, activating it ectopically in the somatic mesoderm of most trunk segments. It is not known whether the activity of this enhancer is normally restricted to the visceral mesoderm by a required cofactor that is present only in the visceral mesoderm, by repressors present in other tissues, or by both mechanisms. However, UbxIVa at high levels seems able to override this normal tissue specificity. The more efficient DNA binding of this isoform (in the presence of Exd) may bypass the requirement for a cofactor or displace a repressor more effectively (Reed, 2010).

A quantitatively controlled study showed that the levels of Ubx protein are very important to determine the functional outcome of Ubx in vivo. In this context, the results show that in spite of significant variation across expression, levels of UbxIVa protein in different transgenic lines, this isoform is consistently able to produce a similar output in terms of dpp target activation in posterior regions. This suggests that for UbxIVa dpp target activation and protein concentration may relate to one another in the form of a sigmoidal function with a narrow protein concentration interval acting as a threshold that is crossed by all UbxIVa lines tested in this study. Given that UbxIa lines achieving comparable protein expression levels to UbxIVa lines, it was possible to activate dpp in posterior regions of the embryo, it is concluded that qualitative differences in Ubx protein structure as determined by alternative splicing are causal to the observed differential behavior in target activation (Reed, 2010).

The tissue-specific effects of Ubx isoform ectopic expression emphasize the likely role that the splice isoforms play in mediating specific Ubx functions in different tissues. Indeed, the isoforms have different tissue distributions in embryogenesis with UbxIa expressed predominantly in epidermis, mesoderm, and peripheral nervous system whereas UbxIVa appears to be exclusively expressed in the central nervous system. The analysis of the UbxMX17 mutation, which retains the full Ubx expression pattern but generates only isoform UbxIVa, supports the endogenous relevance of splice isoforms for tissue-specific Hox function. Although in UbxMX17 embryos UbxIVa can replace the function of Ubx isoforms I and II in the epidermis with the generation of a normal cuticle pattern, the peripheral nervous system is affected, and in this study clear defects were found in the segmental specification of somatic muscles. Tissue-specific isoform functions may be mediated by effects on cofactor interaction or may also involve effects on collaborative regulatory interactions between Hox proteins and tissue-specific regulators (Reed, 2010).

The results of in vitro binding studies of the Ubx/Exd element show that Ubx isoforms differ in their capacity to interact with DNA in the presence of the cofactor Exd. A possibility that emerges from these results is that different Ubx isoforms display differential levels of interaction with Exd (in the presence of target DNA) and, accordingly, could perform specific functions in vivo as a consequence to the distinct levels of nuclear Exd available in different regions of the embryo (Reed, 2010).

in vitro studies also show that ablation of the YPWM motif has little effect on the ability of Ubx form Ia to form a complex with Exd, but it significantly reduces complex formation by Ubx form IVa (Reed, 2010).

The observation that mutated forms of the Ubx protein lacking the hexapeptide interact with Exd at all is at first sight surprising, especially in view of the structural studies showing that the YPWM motif provides the major contact between Hox and Exd proteins bound to DNA. However, this finding is not inconsistent with earlier work. The article that originally described cooperative interactions between Ubx and Exd used Ubx proteins deleted of all sequences located amino-terminal to the homeodomain. These proteins therefore lacked the hexapeptide motif entirely. Interactions with Exd that do not require the hexapeptide have also been reported in a study focused on Ubx functions independent from Exd and Hth proteins. In addition, alanine replacement of the hexapeptide does not affect the way another Hox protein (i.e., Abd-A) interacts with Exd. Furthermore, a recent study suggests that Ubx-Exd recruitment may rely not on a single, but on several different mechanisms, some of which require a short evolutionarily conserved motif originally termed UbdA. Thus, the hexapeptide is unlikely to be the only Hox protein motif that interacts with Exd (Reed, 2010).

Crystallographic studies show that the Ubx and Exd homeodomains are closely adjacent, almost touching each other, the extent of this proximity being revealed by a significant reduction in solvent-accessible surface area within the area of putative contact. In addition, the distance between the Ubx recognition helix C terminus and the Exd recognition helix N terminus is just 9 Å, with one particular residue of Ubx (Lys58) well positioned to form hydrogen bonds with a residue of Exd (Ser48). Thus, regions within the Ubx homeodomain may be responsible for additional interactions with Exd. Sequences elsewhere in the proteins may also contribute to these interactions. A fine combination of protein mapping and crystallographic studies may be required to reveal the structural details of such interactions (Reed, 2010).

These experiments would be consistent with the possibility that the Ubx linker region itself could be a previously uncharacterized region of Ubx that makes contacts with Exd. This would be supported by experiments of protein-protein interaction in yeast, which also suggest that the Ubx linker region could affect the interaction between Ubx and Exd, and by the high evolutionary conservation of these sequences across phylogenetically distant species of flies. The results could also be accommodated in a model in which the Ubx linker region induces a conformational change elsewhere in the Ubx protein, such that the degree of interaction with Exd is affected. Alternatively, the regulatory potential of the Ubx protein could be affected. In particular, linker-dependent conformational changes may affect the behavior of critical regulatory motifs of Ubx, such as the recently studied QA motif involved in the modulation of Ubx activities in a tissue-specific manner (Hittinger, 2005) and the SSYF motif (Tour, 2005), an evolutionarily conserved motif close to the N terminus of the Ubx protein that is involved in transcriptional activation (Reed, 2010).

Other reports have also emphasized that the Hox linker regions are not just passive spacers. One such study shows that mutation of the short linker region in the Hox protein Abd-A specifically disables its capacity to activate wingless, a natural target gene, without affecting its ability to repress dpp. When comparing cofactor and DNA-binding properties of this Abd-A mutant protein with those of the wild-type Abd-A on a repressor element of the Distalless gene (DllR), no differences were seen in the interaction between the mutant form of Abd-A and Exd protein. (It is perhaps worth noting that these in vitro experiments required the presence of Homothorax protein; on this particular DNA target (DllR), the formation of complexes with Abd-A and Exd alone was below the limit of detection of the assay. Another report described Ubx isoform-specific functions for the repression of the Dll gene. These studies suggest that sequences within the Hox linker regions may modify the trans-regulatory potential of Hox proteins without necessarily affecting the DNA-binding properties of these proteins. The results extend these studies, showing that linker regions may at times affect target gene regulation and the DNA binding/cofactor interaction abilities of a Hox protein. The binding results are consistent with a recent study that integrates DNA-binding tests with computational predictions of ordered and disordered segments of the Ubx protein (Liu, 2008), proposing that sequences outside the homeodomain can reduce the DNA-binding activities of the Ubx protein by twofold (Reed, 2010).

Beyond the effects that alternative splicing may have on modulating Hox interactions with cofactors, other possibilities must also be considered. Given that in these experiments a very unstable binding of the Ubx proteins to target DNA was observed in the absence of Exd, it is difficult to advance arguments regarding the affinities of the individual Ubx isoforms for this molecular target on firm grounds. In spite of this, given that in various physiological contexts the binding of Hox proteins to target sites in the absence of cofactors has been demonstrated, it is pertinent to suggest that alternative splicing might also be influencing Hox DNA binding independently from cofactor interactions, at the level of changing DNA-binding affinities of the individual isoforms for their target DNAs. This possibility would be suitable for experimental investigation in the physiological context of Drosophila adult appendage development, as Ubx is known to act independently from cofactors during the development of these structures (Reed, 2010).

Examples from other Drosophila Hox genes further support a more general role of differential splicing in the diversification of Hox function during development, affecting protein modules outside the Ubx linker region discussed above. For instance, genetic dissection of the Abd-B gene demonstrated the existence of two distinct gene functions, originally termed morphogenetic (m) and regulatory (r). The spectrum of mRNAs derived from the Abd-B locus have been cloned, and a family of Abd-B transcripts generated by differential promoter use has been revealed , that, in turn, leads to different splicing variants affecting 5' sequences of the gene. Two proteins products, called m and r, are produced from the Abd-B transcripts; m differs from r in that it encodes an additional large glutamine-rich amino-terminal domain. Furthermore, ectopic expression of each Abd-B protein class leads to specific effects on the larval cuticle, suggesting that the isoforms have specific developmental functions (Reed, 2010).

Given that in Drosophila several Hox proteins possess alternatively spliced modules modifying the distance between the hexapeptide and the homeodomain, while others produce functionally different isoforms via differential promoter use coupled to alternative splicing, alternative splicing may truly represent a very important, yet underexplored regulatory mechanism modulating the functional specificity of Hox proteins during development (Reed, 2010).

Regulation in the midgut

Cross-regulation of Homeotic Complex (Hox) genes by ectopic Hox proteins during the embryonic development of Drosophila was examined using Gal4 directed transcriptional regulation. The expression patterns of the endogenous Hox genes were analyzed to identify cross-regulation while ectopic expression patterns and timing were altered using different Gal4 drivers. Evidence is provided for tissue specific interactions between various Hox genes and the induction of endodermal labial (lab) by ectopically expressed Ultrabithorax outside the visceral mesoderm (VMS). Similarly, activation and repression of Hox genes in the VMS from outside tissues seems to be mediated by decapentaplegic gene activation. Additionally, it has been found that proboscipedia (pb) is activated in the epidermis by ectopically driven Sex combs reduced (Scr) and Deformed (Dfd); however, mesodermal pb expression is repressed by ectopic Scr in this tissue. Mutant analyses demonstrate that Scr and Dfd regulate pb in their normal domains of expression during embryogenesis. Ectopic Ultrabithorax and Abdominal-A repress only lab and Scr in the central nervous system (CNS) in a timing dependent manner; otherwise, overlapping expression in the CNS in tolerated. A summary of Hox gene cross-regulation by ectopically driven Hox proteins is tabulated for embryogenesis (Miller, 2001a).

The expression of the lab gene is regulated in a time and tissue specific manner by ectopic Ubx accumulation. Lab accumulation in the CNS and epidermis are differentially affected by ectopic Ubx. lab repression in the CNS only occurs in prd=>Ubx (prdGal4;UASUbx driver=>responder) animals even though both the 31-1=>Ubx and 69B=>Ubx demonstrate expression in this tissue. However, 31-1=>Ubx animals do not show significant accumulation of Ubx in the CNS prior to stage 9 while 69B=>Ubx genotypes do. This suggests that timing may be the critical factor in setting up regulatory interactions between these genes. If timing is the difference between the 31-1=>Ubx and 69B=>Ubx repression of lab in the CNS, then after stage 9, the locus becomes immune to Ubx's negative influence. This could be due to the absence of an important cofactor, the masking of a lab cis-regulatory site used by Ubx, or changes in the signaling environment (Miller, 2001a).

Driver expression levels in the nearby VMS may be a factor since the prd=>LacZ confocal images shows significantly more LacZ accumulation there. Expanded endodermal lab induction is demonstrated in all three driver=>Ubx combinations. Normally, endodermal lab expression is activated by Ubx in the VMS through a signaling mechanism involving dpp. However, only the prd=>LacZ combination shows significant VMS accumulation which indicates that the VMS may not be the only tissue contributing to this process. It is possible that the observed 31-1 driver expression in the endoderm could autonomously activate the Ubx/Dpp/Lab cascade and provide the observed expansion of Lab accumulation (Miller, 2001a).

Additionally, the sparse VMS accumulation seen using the 69B driver could reflect sufficient ectopic Ubx accumulation to activate the Dpp signal and subsequent lab induction in the endoderm. Consistent with this latter possibility is the fact that the dpp gene demonstrates auto-catalytic regulation in this tissue and could amplify low level stimulation by ectopic Ubx. The activation of Ubx in the VMS by 31-1=>Ubx and 69B=>Ubx, suggests that this signaling pathway is involved. Reduced first midgut constrictions are found in a few 69B=>Ubx and 31-1=>Ubx embryos, likely due to high levels of Dpp protein and subsequent Antp repression. Typically, however there is not enough ectopic Ubx expression in the VMS by any driver=>Ubx combination to repress the first midgut constriction. Despite this, expansion of the Lab endodermal domain is seen in these animals indicating that either the threshold response for lab induction is lower than that for Antp repression or that there is a signaling source other than the VMS for the Ubx generated signal. This signal could originate from the CNS for 31-1=>Ubx or the CNS, somatic mesoderm and epidermis for 69B=>Ubx. Additionally, the amnioserosa normally exhibits dpp expression where 31-1=>LacZ and 69B=>LacZ expression is observed. Gal4 activation of the Ubx responder in the amnioserosa could contribute to dpp activation levels and hence the pool of the diffusible agonist through stage 14 (Miller, 2001a).

The down regulation of Scr and Antp in the VMS by ectopic Ubx accumulation cannot be clearly explained by direct transcriptional repression (posterior dominance). Based on the N.LacZ responder results, 31-1=>Ubx animals should not accumulate significant amounts of Ubx in the VMS while 69B=>Ubx should only produce scattered expression in this tissue. However, direct detection of Ubx protein in these embryos indicates that low level expression of Ubx is activated in VMS cells. If the effect of the drivers is not direct, then what causes the accumulation of Ubx in the VMS? It is proposed that this occurs by the induction of ectopic dpp in this tissue. In this model, ectopic Ubx accumulation in other tissues (outside the VMS) generates a signaling cascade (presumably dpp) that reaches the VMS to stimulate the dpp auto-catalytic feedback. The appearance of Ubx accumulation in the VMS indicates that Ubx is involved in this dpp positive regulatory loop (Miller, 2001a).

Other data support this model for Ubx/dpp signaling influences from nearby tissues. For example, the dpp-LacZ reporter is activated in the VMS in the 31-1=>Ubx and 69B=>Ubx genotypes: coupled with other regional signaling influences, dpp has been shown to activate Ubx in the VMS. Additionally, the activation of ectopic lab expression in the adjacent endoderm indicates that dpp expression has been induced from these drivers. Moreover, occasional repression of Antp, the first midgut constriction and Scr, with both of these Gal4 drivers, suggests that ectopic dpp expression occurs; since dpp has been shown to repress both Scr and Antp. Additionally, 69B=>Dpp has been shown to regulate bagpipe and pox meso in the VMS (Miller, 2001a).

Hence, activation of dpp-LacZ suggests that dpp is responsible for the repression of Scr and Antp in the VMS. There is no apparent activation of dpp-LacZ in the CNS, epidermis nor somatic mesoderm in either 31-1=>Ubx or 69B=>Ubx animals; however, additional studies suggest that Ubx expression and dpp signaling from other tissues may naturally function to regulate Scr expression in the VMS. The dpp-LacZ construct does not entirely mimic the dpp gene expression, only the VMS expression, as it does not contain all the necessary regulatory elements. Since no dpp-LacZ expression is detected in other tissues, it cannot be ruled out that driver=>Ubx experiments actually signal through other pathways. However, it does appear that Ubx directed signaling from the CNS, epidermis or the SM is capable of influencing Hox expression in the VMS and subsequently lab expression in the endoderm. Interestingly, 69B=>Abd-A represses Ubx in ectodermal tissues and appears to produce a slight posterior expansion of Scr expression in the VMS. Notably, the posterior boundary of Scr expression in the VMS is regulated in part, by Ubx expression (Miller, 2001a).

Regulation in Spiracles and Trachea

dpp expression has been examined in two groups of dorsal ectoderm cells at the posterior end of the embryo, in abdominal segment 8 and the telson. These dpp-expressing cells become tracheal cells in the posterior-most branches of the tracheal system (Dorsal Branch10, Spiracular Branch10, and the Posterior Spiracle). These branches are not identified by reagents typically used in analyses of tracheal development, suggesting that dpp expression confers a distinct identity upon posterior tracheal cells. dpp posterior ectoderm expression begins during germ band extension and continues throughout development. The sequences responsible for these aspects of dpp expression have been isolated in a reporter gene. An unconventional form of Wingless (Wg) signaling, Dpp signaling, and the transcriptional coactivator Nejire (CBP/p300) are required for the initiation and maintenance of dpp expression in the posterior-most branches of the tracheal system. These data suggest a model for the integration of Wg and Dpp signals that may be applicable to branching morphogenesis in other developmental systems (Takaesu, 2002).

During early stages of embryogenesis, wg and dpp are expressed in undifferentiated dorsal ectoderm. wg mRNA expression, in 15 stripes along the entire dorsal-ventral axis of the embryo (including the dorsal ectoderm), begins at stage 8. wg expression persists in this striped pattern through stage 17. dpp mRNA is expressed on the dorsal side of the embryo along the entire anterior-posterior axis, beginning at stage 4. dpp mRNA expression persists in a large portion of the dorsal ectoderm through stage 8 and resolves into leading edge cell-specific expression in stage 12 embryos. At this time, the embryonic expression pattern of nej has not been reported. However, some information can be obtained from nej mutant phenotypes. nej zygotic mutant embryos show visible defects in the tracheal system at stage 12. The tracheal system is derived from the dorsal ectoderm, suggesting that nej is expressed in this tissue prior to stage 12 (Takaesu, 2002).

dpp expression in posterior tracheal branch anlagen appears to be initiated by prior episodes of wg and dpp expression in the undifferentiated dorsal ectoderm. The maintenance of dpp expression in posterior tracheal branches appears to require continuous input from wg and from a dpp feedback loop. The initiation and maintenance of dpp expression in posterior tracheal branches also requires continuous nej activity. Overall, the data are consistent with the following combinatorial signaling model. The transcriptional activator Medea (Med, signaling for the Dpp pathway) interacts with the transcriptional activator Arm (signaling for the Wg pathway) via the transcriptional coactivator Nej. This multimeric complex initiates and, with continuous signaling, maintains dpp expression in posterior tracheal branches with the help of Zw3. These data extend previous studies of dpp expression and Dpp signaling in several ways. nej has been reported to participate in Dpp signaling. Expression from Dpp responsive enhancers is reduced in nej zygotic mutant embryos. While they show that nej3 enhances dpp wing phenotypes, this study shows that Med1 enhances nej3 embryonic phenotypes. The Dorsal Trunk Branch forms normally in Mad12 zygotic mutant embryos, and the Dorsal Trunk Branch appears normal in Med1 mutants. nej is involved in mediating combinatorial signaling by the Wg and Dpp pathways and the involvement of nej in morphogenesis of Dorsal Branch, Spiracular Branch, and the Posterior Spiracle is demonstrated. A region of the histone acetyltransferase domain of Nej binds to Mad. Further study is needed to reveal the mechanisms used by Nej to interact with Wg and Dpp signaling. Several questions remain about the regulation of dpp expression by Wg, Dpp, and Nej. Two questions arise about the mechanism of signal integration: how is zw3 involved and how is Nej recruited to bridge the two pathways? It is tempting to speculate that, in response to a Wg or a Dpp signal, Zw3 (a serine-threonine kinase) is involved in Nej recruitment. Numerous studies have shown that p300/CBP transcriptional coactivation functions are stimulated by its phosphorylation, but the site of phosphorylation has never been mapped. Other questions concern the molecular nature of the enhancers that direct dpp expression in the posterior tracheal branches. A 54-nucleotide region has been identified that contains two sets of conserved, overlapping consensus binding sites for dTCF and Mad/Med. Analyses of DNA-protein interactions predicted by the data involving this candidate combinatorial enhancer have begun (Takaesu, 2002).

From a broader perspective, mammalian homologs of Dpp and orthologs of Wg are important in branching morphogenesis in a variety of developing tissues. For example, BMP2 is involved in renal branching and Wnt4 plays a role in mammary gland branching. The widespread use of TGF-ß and Wnt signals in branching suggests that a greater understanding of the regulation of dpp tracheal expression and dpps role in specifying the unique identities of posterior tracheal branches will have wide relevance (Takaesu, 2002).

A combinatorial enhancer recognized by Mad, TCF and Brinker first activates then represses dpp expression in the posterior spiracles of Drosophila

Analysis of a reporter gene carrying a 375-bp region from a dpp intron (dppMX-lacZ) revealed that the Wingless and Dpp pathways are required to activate dpp expression in posterior spiracle formation. Within the dppMX region there is an enhancer with binding sites for TCF and Mad that are essential for activating dppMX expression in posterior spiracles. There is also a binding site for Brinker likely employed to repress dppMX expression. This combinatorial enhancer may be the first identified with the ability to integrate temporally distinct positive (TCF and Mad) and negative (Brinker) inputs in the same cells. Cuticle studies on a unique dpp mutant lacking this enhancer showed that it is required for viability and that the Filzkorper are U-shaped rather than straight. Together with gene expression data from these mutants and from brk mutants, the results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is associated with dorsal-ventral patterning and is necessary for designating the posterior spiracle field. The second is governed by the combinatorial enhancer and begins during germ band retraction. The second round appears necessary for proper spiracle internal morphology and fusion with the remainder of the tracheal system. Intriguingly, several aspects of dpp posterior spiracle expression and function are similar to demonstrated roles for Wnt and BMP signaling in proximal-distal outgrowth of the mammalian embryonic lung (Takaesu, 2008).

These data show that within the dppMX region there is a combinatorial enhancer that contains binding sites recognized by TCF and Mad that are essential for activating dpp expression in the spiracular chambers, in the spiracular branches and in the dorsal trunk branches. There is also a binding site recognized by Brinker that is likely employed to repress dpp expression late in spiracle development (Takaesu, 2008).

What makes this enhancer different from other enhancers in Drosophila also capable of integrating three inputs in the same cells. These enhancers integrate only positive signals. In all cases, PointedP2 binding displaces the Yan repressor that is constitutively bound to the enhancer in the absence of PointedP2. The difference is that the dppMX enhancer is actively repressed by Brk binding after being stimulated by positive input from the Dpp and Wg pathways. What makes this enhancer different from other enhancers in Drosophila that integrate positive and negative signals such as the enhancer of Ultrabithorax where positive input from TCF is associated with a competition between Mad (positive) and Brk (negative) inputs. The difference is that in the same cells the dppMX enhancer responds sequentially to positive combinatorial input from TCF and Mad and then to negative input from Brinker. The Ultrabithorax enhancer responds simultaneously to positive input from TCF and Mad in parasegment seven and to negative input from Brinker in the adjacent cells of parasegment 8 (Takaesu, 2008).

If combinatorial signaling by the Dpp and Wg pathways, via TCF and Mad, turn on the dppMX enhancer in posterior spiracle primordia of the dorsal ectoderm at stage 13, then where do the Dpp signals originate? One possibility is that Dpp signals derive from the adjacent region of the dorsal ectoderm -- leading edge cells located just anterior to the posterior spiracle primordia. In leading edge cells of the dorsal ectoderm, dpp expression is activated at stage 8. dpp leading edge expression is activated by enhancers distinct from the dppMX enhancer, and the leading edge enhancers are themselves stimulated, in part, by dpp blastoderm expression that sets up the embryonic dorsal/ventral axis. In this scenario, the activation of the dppMX enhancer in posterior spiracles by Dpp leading edge signaling represents the last step in a cascade, covering nearly all of embryogenesis, of increasingly spatially restricted rounds of Dpp dorsal ectoderm signaling (Takaesu, 2008).

The most likely the source of the Wg signal is a small group of cells in the spiracular chamber. wg expression in the spiracular chamber becomes visible at stage 11 and is present through the remainder of embryogenesis. This group of Wg expressing cells is required for the maintenance of Cut and Spalt expressions, genes shown in this study to be independent of Dpp signaling. The involvement of Wg in spiracle cell fate determination and dpp activation results in more severe spiracle defects in wg mutants than in brkF124 embryos or dpp null embryos with two copies of the dpp-ΔKX rescue construct (Takaesu, 2008).

The source of the signal that activates brk in the posterior spiracles is less easy to identify. However, one possibility is suggested by the mutant phenotype generated by ubiquitous expression of unpaired (a ligand of the Jak/Stat pathway with a role in posterior spiracle formation. These embryos display a U-shaped Filzkorper similar to brkF124 embryos and dpp null embryos with two copies of the dpp-ΔKX rescue construct (Takaesu,2008)

The data advance understanding of posterior spiracle development and the role that Dpp signaling plays in this process in three areas: (1) that dpp activity in dorsal/ventral patterning is genetically separable, in part, from its activity in posterior spiracle development; (2) that dpp signaling does not appear to influence posterior spiracle cell fate or external morphology but instead regulates spiracle internal morphology; and (3) that a functioning posterior spiracle is necessary for viability prior to hatching (Takaesu, 2008).

Regarding the separability of dpp dorsal-ventral patterning and posterior spiracle functions, this view contrasts with the prevailing wisdom that all dpp posterior spiracle defects are downstream consequences of dorsal-ventral patterning defects. Instead, the results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is necessary for setting up the posterior spiracle field in association with dorsal-ventral patterning at the blastoderm stage. The second begins during germ band retraction and appears to regulate the internal morphology of the spiracles. One possible explanation for why these distinct aspects of dpp function have been connected in the conventional wisdom is that the dppMX enhancer is located in an intron alongside dorsal/ventral patterning enhancers and is deleted in several widely studied dppHin alleles (Takaesu, 2008).

This two-round model for dpp signaling in posterior spiracle development fits well with analysis of Dpp signaling in heart development. Here, there is a second round of Dpp dorsal ectoderm to mesoderm signaling late in development that maintains the boundary between pericardial cells and the adjacent dorsal muscle cells. The second round of Dpp signaling in heart development is autoactivated by Dpp signals that also likely derive from dpp leading edge expression. Thus, in heart development, there is also evidence of a multi-step cascade of increasingly spatially restricted rounds of Dpp dorsal ectoderm signaling (Takaesu, 2008).

Regarding the function of the second round of Dpp signaling in posterior spiracle development, the data show that the expression of three transcription factors essential for cell fate determination in the spiracles is independent of Dpp signaling. In addition, pMad data show that the lumen of the spiracular chamber forms normally suggesting that spiracle external morphology and invagination, under the control of Rho signaling, is also independent of Dpp (Takaesu, 2008).

Cuticle data indicate that the primary defect in dpp posterior spiracle mutants is fully differentiated but U-shaped Filzkorper that do not appear to connect to the dorsal trunk branches. This phenotype plus the fact that dpp mRNA and pMad expressions normally span the spiracular chamber, spiracular branches and dorsal trunk branches suggests the hypothesis that Dpp regulates the internal morphology of the spiracles. Given the mutant phenotype and gene expression patterns, it is tempting to speculate that Dpp signaling via pMad directs the anterior outgrowth of the spiracles, the posterior outgrowth of the dorsal trunk branches and their eventual fusion into a coherent tracheal system (Takaesu, 2008).

Regarding posterior spiracle function in embryos, the fact that dpp posterior spiracle mutants do not hatch suggests that gas exchange through the posterior spiracles and the spiracular branches begins and is required to sustain the individual prior to hatching. This is an advance in the understanding of Drosophila embryonic and larval respiration (Takaesu, 2008).

Table of contents

decapentaplegic continued: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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